Leon Dmochowski

Studies on Human Leukemia.

Summary 1) Electron microscope studies have been carried out on ultrathin sections of lymph nodes from 6 patients with acute lymphatic, 1 with acute and 1 with chronic monocytic, 1 with acute myeloid leukemia, 2 with lymphosarcoma and 1 with Hodgkins disease. Virus particles of approximately 900 Å in diameter have been observed in cells of lymph nodes from 3 cases of acute lymphatic, 1 of acute myeloid leukemia and 1 of lymphosarcoma. Similar studies of sections of lymph nodes from 5 non-leukemic patients have failed to reveal presence of virus particles. 2) Cytopathogenic changes have been observed in cells of lymph nodes grown in vitro from 15 out of 24 patients with leukemia and lymphosarcoma. Cells of lymph nodes grown in vitro from 4 non-leukemic patients have not shown changes observed in lymph nodes from leukemic patients during similar number of passages. Cell-free extracts of lymph nodes from 8 patients tested have induced changes in monkey kidney cell cultures which could be serially transmitted. These changes have not been seen in similar cultures treated with extract of a non-leukemic lymph node. Extracts of leukemic lymph nodes which induced changes in monkey kidney cell cultures did not have a similar effect in human embryo cultures. 3) Cell-free extracts of biopsy material from leukemic and lymphomatous lymph nodes and fluids from monkey kidney cell cultures treated with these extracts have not shown specific leukemia-inducing activity in Swiss (Holtzman), C3HZb/Bi and C3H/Bi strain mice. 4) It is of particular interest that in one patient with lymphosarcoma, not only were virus particles observed in ultrathin sections of the biopsy specimens, but also similar virus particles have been found in cells from the same specimen, grown in vitro for 6 sequential passages during which cytopathogenic changes had been observed.

Studies on the presence of particles resembling RNA virus particles in human breast tumors, pleural effusions, their tissue cultures, and milk

Ultrastructural studies have been carried out on biopsies of 84 breast cancers, 13 metastatic lymph nodes, 3 fibroadenomas, and on cells from 33 pleural effusions from breast cancer patients. Breast tumor tissues of 157 patients and pleural effusion cells of 36 patients have been put in tissue culture. Growth has been obtained of cells derived from 47% of the breast tumor biopsies and from 90% of the pleural effusions. However, no cell line could be established. Milk specimens of 4 women with breast cancer and of 17 apparently healthy women also have been examined for the presence of virus particles. Particles resembling murine type B and/or type C virus particles and small virus‐like particles have been observed in a number of specimens. Particles resembling type B and/or type C virus particles have been found in 34 of the 100 breast tumor, metastatic lymph node, and fibroadenoma biopsies. They also have been observed in 2 of 33 specimens of pleural effusions, in 4 of 72 tissue culture specimens, and in 4 of 21 milk specimens. Small virus‐like particles have been observed in 29 of the 100 biopsy specimens and in 3 of 38 tissue cultures derived from breast tumors. They also have been shown in 4 of 21 milk specimens. The relationship of these virus‐like particles to the origin of human breast neoplasia remains to be clarified.

Immunofluorescence studies on sera of patients with breast carcinoma

Indirect immunofluorescence tests were carried out on sera of 42 patients with breast carcinoma, 4 with fibrocystic disease, and 45 blood bank donors. Thirty‐one of the 46 sera showed positive reaction which consisted of faint nucleolar and strong cytoplasmic fluorescence in cells of tissue cultures derived from either 1 breast carcinoma, 1 case of fibrocystic disease, or 2 osteosarcomas. Sera of 3 breast cancer patients reacted with breast cancer cells only; sera of 6 breast cancer patients and of 1 with fibrocystic disease reacted with fibrocystic disease cells only; sera of 7 breast cancer patients reacted with breast cancer and fibrocystic disease cells; and sera of 3 breast cancer patients reacted with cells of breast cancer, fibrocystic disease, and osteosarcoma. Sera of 2 breast cancer patients reacted with cells of breast cancer and osteosarcoma, and sera of 5 breast cancer patients and of 2 with fibrocystic disease reacted with cells of fibrocystic disease and osteosarcoma. Sera of a small number of patients with osteosarcoma reacted with fibrocystic disease, and osteosarcoma cells and sera of these patients reacted also with breast cancer cells. None of the sera tested reacted with cells of the following cultures: giant cell tumor, rhabdomyosarcoma, Levine‐3 cell line, HEK‐1‐HRLV (human embryo kidney culture producing Rauscher leukemia virus), normal human embryo, and human adult skin. All sera from blood bank donors were negative with cells of all cultures tested. Absorption experiments with some of the positive sera indicate that the fluorescence reaction may be due to a tumor antigen.

Studies on ultrastructure of Ewing's sarcoma of bone

Seven cases of Ewings sarcoma of bone were examined by electron microscopy. The presence of atypical junction complexes represents a new finding. The presence of a considerable amount of glycogen confirms previous observations. The ultrastructure of the tumor cells supports the conclusion that Ewings sarcoma is a separate neoplastic entity different from reticulum cell sarcoma and hemangiomatous neoplasms. Cells in tissue cultures derived from biopsies of Ewings sarcoma showed no growth pattern nor morphological appearance different from cells of other malignant mesenchymal tumors. No virus particles could be detected in the primary surgical specimens nor in the tissue cultures. Filamentous structures were observed in one tumor biopsy specimen. These structures could be interpreted as structures resembling developmental stages of some known viruses.

Studies on the acid mucopolysaccharide coat of viruses and transformed cells

Ruthenium red staining method was applied to study the properties of acid mucopolysaccharides (AMPS) in cells of cultures of normal, RNA virus‐transformed, and RNA virus‐producing tumor cells. The AMPS layer of plasma membranes of virus‐transformed cells was found to be thicker than that of the plasma membrane of normal cells and more resistant to ovine hyaluronidase digestion than that of control normal cells. Hyaluronidase digestion of the AMPS layer of virus‐producing tumor cells followed by several centrifugation runs resulted in a considerable concentration of RNA virus particles. This procedure may therefore prove helpful in virus concentration studies. Ruthenium red staining revealed three distinct layers within the unit membrane of the plasma membrane of virus‐producing tumor cells as well as in the envelopes of type C and type BRNA virus particles. Study of a mouse mammary tumor (D‐W) cell line producing both type B and type C RNA virus particles after labeling by conjugated ferritin with appropriate anti‐sera and staining by ruthenium red revealed the presence of ferritin granules in the AMPS layer of these particles. As demonstrated by ruthenium red staining, the functional and immunologic properties of the AMPS layer on virus particles are as follows: it plays at least a part in the adherance of RNA virus particles to the plasma membrane of cells; acts as a protective layer of virus particles against enzymatic damage, and it is a site of viral antigen‐antibody reactions. This study has also demonstrated similarities in the ultrastructure of plasma membrane of RNA virus‐transformed and virus‐producing tumor cells and of the envelope of RNA type B and type C virus particles.

Ultrastructural studies of human prostatic neoplasia.

Forty‐two specimens of human prostatic neoplasia (32 carcinomas, eight benign hyperplasia, two bladder tumors infiltrating prostatic tissue, and 15 tissue cultures derived from prostatic neoplasia) were examined by electron microscopy. Intracisternal viruslike particles, 150–200 nm in diameter and budding, were found in epithelial cells of four carcinomas. In some of these particles, an electron‐dense central core or two concentric layers were discernible. In addition, particles resembling type C virus particles, 90–130 nm in diameter, were observed in intracytoplasmic vacuoles in five cases of carcinomas and in one case of benign prostate hyperplasia. Thus, viruslike particles were found in 9 of 32 cases of prostate carcinoma and in one of eight cases of benign prostate hyperplasia. Virus particles have, so far, not been found in any of the tissue culture specimens. Further studies are required to determine the nature of these particles and their relationship to the origin of human prostatic neoplasia. Additional observations in both benign hyperplasia and carcinoma include intranuclear mitochondria, multilayered nuclear inclusions, bundles of intranuclear fibrils, intracytoplasmic tubules, extracellular tubulo‐filamentous structures, and cilia.

Studies on a virus ("polyoma") inducing multiple tumors in animals.

Summary Electron microscope examination of ultrathin sections of mouse (NIH-Swiss and Af/Dm strain) embryo cells grown in vitro and showing cytopathogenic changes following passage of polyoma virus preparations has been carried out. Spherical particles of 270 Å in diameter and of low electron density have been observed chiefly in the nucleus, also in the cytoplasm and outside the cells. In some particles an internal structure could be discerned, composed of a more dense center of 50 Å in diameter, surrounded by an electron translucent outer zone with outlines of a limiting membrane. Similar particles have also been found in parotid gland neoplasms and mammary tumors induced in NIH (Swiss) mice by polyoma infected tissue culture fluids. Particles of this type have not been observed in uninfected control cultures.‡

Virus-Like Particles in a Case of Human Prostate Carcinoma

Two morphologically different types of intracisternal virus-like particles were observed electron microscopically in a biopsy specimen of human prostate cancer. Particles of one type were 150-200 nm in diameter and contained either an electron-dense core or two concentric inner layers. Particles of the other type were smaller, 80-100 nm in diameter, and appeared mostly in filamentous or chainlike formation. Both types of particles and budding were observed in endoplasmic cavities of epithelial tumor cells. The particles had ultrastructural characteristics that suggested a viral nature but were different from the known type B, type C, or type H (hamster type R) virus particles. This was the first election microscopic observation in prostate cancer of virus-like particles similar to those previously reported in a case of human breast carcinoma.

Isolation of a virus from infectious bovine kerato-conjunctivitis.

Discussion and summary A virus has been isolated from biopsy material from eyes of cattle with acute infectious bovine keratoconjunctivitis. No serum samples from these animals were available for testing against the virus. The virus is cytopathogenic for cells of bovine and human origin; ether resistant; relatively heat labile; does not hemagglutinate red blood cells from chickens, mice, hamsters, guinea pigs, rabbits, bovines, sheep, or man. Fluorescence microscopy of infected cells stained with Coriphosphine O and studies on the influence of 5-BUDR, 5-FUDR, and 5-FU on virus multiplication indicate that it is a DNA virus. Antisera (bovine or rabbit) against enteric, respiratory and other bovine viruses have failed to neutralize cytopathogenic activity of the virus. Per-nasal instillation of virus-containing tissue culture fluids has induced classical infectious bovine kerato-conjunctivitis of varying degrees of severity in 8 out of 18 inoculated cattle. It is of interest that 7 out of 12 heifers have failed to respond clinically and immunologically to inoculation of the virus which may be due to failure of inoculation rather than to immunity as pre-inoculation serum samples had no neutralizing antibodies. Sera from animals with experimentally induced acute kerato-conjunctivitis have neutralized the cytopathic effect of the virus. Electron microscope studies have demonstrated that the virus induces nuclear changes of a type not previously described in tissue culture cells infected with any known virus. Recent electron microscope studies(9) of infectious pustular vulvovaginitis virus (IPV) and IBR virus in tissue culture have confirmed previously published studies on IBR virus (10,11). There appears to be a similarity in particle size and structure between these viruses and the agent isolated from eyes of cattle with IBKC. However, there appears to be a difference between nuclear changes observed in cells infected with the agent isolated in the present study (formation of whorls, see Fig. 7) and those observed in cells infected with IBR (10,11) and IPV (9). The findings indicate that this virus might be causatively related to acute infectious bovine kerato-conjunctivitis (“pinkeye”), should further animal inoculation studies confirm these preliminary observations.

HODGKIN'S DISEASE AND VIRUSES: AN ELECTRON MICROSCOPE STUDY*

The viral etiology of leukemia in mice is now well established15). Electron microscopic studies combined with biological studies have led to our knowledge of the submicroscopic morphology and mode of development of m h e leukemia virus~P1~1~*). In 1967, DMOCHOWSKI and GREY~O) first reported the presence of virus-like particles in the intercellular spaces of cervical lymph node from a patient with acute lymphocytic leukemia. Following this observation, extensive electron microscope studies on human material were carried out in our and in other laboratories. Virus-like particles were found in various types of tissue in patients with different types of leukemia and lymphoma by many investigators. Electron microscopic studies on tissues in cases of human leukemia and lymphoma have been described in a number of reports by DMOCHOWSKI and his associates*-18). The results of examination of material from patients with different types of leukemia revealed the presence of particles resembling murine leukemia virus in 23 cases of a total of 122 patients examined and of mycoplasmalike structures in 29 of the 122 studied. Similarly, the results of examination of material from patients with lymphoma revealed the presence of particles resembling murine leukemia virus in 18 cases of a total of 76 patients examined and mycoplasma-like structures in 18 of the 76 studied. DMOCHOWSKI pointed out that it is very difEicult to demonstrate the presence of the virus-like particles and many sections have to be examined, frequently as many as a hundred or more before a few, if any, can be found8). Only in several cases have murine leukemia virus-like particles been observed in numbers similar to those found in murine leukemia (Fig. 1).