Human cells transformed in vitro by human cytomegalovirus: tumorigenicity in athymic nude mice.

Athymic nude mice were inoculated with human embryo lung cells transformed in vitro by human cytomegalovirus (CMV). Of the inoculated animals, 62% developed tumors after an average latent period of 19 days. The tumors were composed of small, polygonal cells with large nulei and scanty cytoplasm embedded in an abundant collagenous matrix. The cells were poorly differentiated but may have been of epithelial origin. Adjacent structures were rarely invaded. CMV-related intracellular and membrane antigens were detected by indirect and anticomplement immunofluorescence techniques in cells cultured in vitro from the tumors.

Partial characterization of a herpes-type virus (K9V) derived from Kaposi's sarcoma.

A herpes-type virus that was originally isolated from a cell culture (designated K9V) derived from a tumor biopsy specimen from a patient with Kaposi's sarcoma was partially characterized. The host range of K9V, as determined by the induction of virus-specific cytopathology, synthesis of antigens, and plaque formation, was limited to human cells and particularly to fibroblasts. Immunofluorescence and complement fixation assays confirmed the specificity of the presence of cytomegalovirus (CMV)-type antigens in K9V-infected human fibroblasts. In addition, the density of K9V DNA was consistent with the density of CMV DNA. However, some peculiarities were observed in the K9V strain of CMV. The virus seemed more cell-associated in human fibroblasts than were known laboratory strains: The spread of cytopathology was slow and did not always involve the whole cell sheet, and the total regression of cytopathology with the establishment of a persistent infection was common. Similar characteristics have recently been observed in the Mj strain of CMV, which has been shown to be oncogenic in human fibroblasts.

Properties of mouse embryo fibroblasts transformed in vitro by infectious bovine rhinotracheitis virus.

Infection of CD-1 mouse embryo fibroblast(s) (MEF) with infectious bovine rhinotracheitis virus (IBRV) strain HMC resulted in persistent infection and subsequent transformation of these cells. IBRV-transformed MEF cultures consisted of short fibroblastoid cells, and IBRV-specific membrane and intracellular antigens were detectable in early in vitro passages by indirect imunofluorescence (IF) techniques. The presence of IBRV genetic information was confirmed in IF-positive and IF-negative cells by in situ hybridization. IBRV-transformed MEF induced fibrosarcomas in athymic nude mice given sc transplants. Infectious virus could not be rescued from the transformed cells or from tumor cells by cocultivation with rabbit kidney cells, by treatment with 5-iodo-2'-deoxyuridine, or by UV irradiation. Nontransformed control cells did not survive more than 10 in vitro passages and did not induce tumors when transplanted to athymic nude mice. These observations represent new data concerning the mouse cell-transforming potential of IBRV and confirm the presence of at least part of the virus genome in the transformants.

Properties of human epithelioid cells established in vitro by a herpesvirus [IBRV(HMC)] isolated from cytomegalovirus-transformed human cells.

Infectious bovine rhinotracheitis virus [IBRV(HMC)], a double-enveloped herpesvirus, was isolated from human embryo lung fibroblasts transformed by cytomegalovirus (CMV). This agent was identified as an IBRV strain that was antigenically related to human CMV. Inoculation of a primary human kidney cancer cell culture with IBRV(HMC) resulted in persistent infection and subsequent establishment of a cell line [IBRV(HMC)HKC-1]. Virus-related nuclear, cytoplasmic, and cell membrane antigens were detected in these cells in early in vitro passages by anticomplement and indirect immunofluorescence tests. Infectious virus was rescued from one of the cell sublines after temperature-shock treatment at passage 26. Karyotypic analysis confirmed the human origin of the cells. Control uninfected kidney cancer cells survived only six in vitro passages. The established cells grew to more than 100 in vitro passages 1 year after initiation of the experiments and induced an epithelioid cancer of variable morphology that infiltrated nerves and muscles when inoculated sc into athymic nude mice.

Recognition of virally transformed cells by lymphocytes from patients with prostatic cancer.

Data presented describe the first assay using human peripheral blood lymphocytes (PBL) against two unique virally transformed cell lines in vitro. Human cells transformed by a cytomegalovirus (CMV-Mj) isolated from normal human prostate tissue were used as target cells in microcytoxicity assays with lymphocytes from 100 patients. Three target cell types were used: control human embryonic lung cells (HEL), transformed HEL cells (CMV-Mj-HEL-2), and transformed HEL cells retrieved from tumors induced in athymic nude mice (CMV-Mj-HEL-2, T-1) by injection of CMV-Mj-HEL-2 cells. PBL preparations from 84% of all patients tested significantly killed CMV-Mj-HEL-2, T-1 cells. However, only PBL from patients with prostatic carcinoma were cytotoxic for CMV-Mj-HEL-2 cells significantly more often than for control HEL. The implications of this approach are discussed.

Impact of frontal lobe lesions on rehabilitation and recovery from acute brain injury.

The frontal lobe has been viewed historically in very different ways, ranging from enigmatic and mystifying to the crucial neural substrate for higher cognition and social behavior. Frontal lobe damage poses a unique and difficult set of challenges to the patient, their family and the neurorehabilitation team. Because of the role of the frontal lobe in adaptation and adjustment, such damage adversely affects a patient's participation in the process and content of rehabilitation. To aid diagnosis and treatment planning, a model of frontal lobe organization is outlined, focusing on the specific cognitive and behavioral symptom clusters associated with superior mesial, inferior mesial, dorsolateral and orbital frontal lesions. A taxonomy of social executive processes is presented for identifying impairments in social behavior and personality, based upon the domains of social self-regulation, social self-awareness, social-sensitivity (empathy), and social-salience. Specific interventions are described that encompass dopamine agonist therapy for blunted affect, mutism and akinesia, cognitive strategies for improving organization and planning deficits, and evolving treatments for social impairments.

Induction of persistent infection in mice and oncogenic transformation of mouse macrophages with infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis virus (IBRV) established long-term persistent infection in intracerebrally inoculated athymic nude mice. After intraperitoneal injection into outbred mice, virus was isolated for only 3 days from spleens and livers. In vitro inoculation of outbred mouse spleen fragments with IBRV resulted in persistent infection and subsequent transformation of spleen macrophages. The IBRV-specific membrane and intracellular antigens were detected by indirect immunofluorescence techniques in transformed cells in early in vitro passages. The presence of IBRV genetic formation was confirmed by in situ hybridization. The IBRV-transformed mouse macrophages induced fibrosarcomas and cystic tumors in athymic nude mice. Infective virus could not be rescued from transformed cells by cocultivation with rabbit kidney cells, treatment with iododeoxyuridine, or ultraviolet irradiation.

Evidence for early nuclear antigens in cytomegalovirus-infected cells.

Human cytomegalovirus (CMV) induces nuclear antigens resembling the Epstein-Barr nuclear antigen (EBNA) as early as 3 h after infection. These early antigens can be detected only with the anti-complement immunofluorescence staining (ACIF) technique. Synthesis of these new antigens is not influenced by cytosine arabinoside (ara-C).

Herpesviruses and prostate carcinogenesis.

Prostatic cancer cell cultures possessed intracellular immunofluorescent antigens specific for human cytomegalovirus (CMV) but produced no infectious virus particles. Norman human prostatic tissue yielded a CMV isolate that transformed primary human embryo lung cells in vitro. These cell transformants were highly oncogenic when transplanted to athymic nude mice. Immunological studies revealed that sera from prostatic cancer patients reacted significantly more frequently with CMV-related antigens than sera from age-, race-, and socioeconomic status-matched benign prostate hyperplasia groups. Specific reactivity against CMV-transformed cells of lymphocytes from prostatic cancer patients was also detected. These complex findings indicate that CMV may be involved in the development of prostatic neoplasia.

Alterations in biological properties of different lines of cytomegalorivus-transformed human embryo lung cells following in vitro cultivation.

Diverse alterations in biological properties of CMV-transformed cell lines were observed during prolonged in vitro cultivation. In the CMV-Mj-HEL-2 parent line there was a gradual decrease in the number of cells expressing CMV-related antigens; at the same time, an increase in oncogenicity was observed. One tumour line, designated CMV-Mj-HEL-2,T-1, however, retained the original ratio of cells expressing CMV-related antigens for over 100 in vitro passages. The cells lost their original moderate oncogenicity during this period. A later increase in the ratio of cells without CMV antigenic markers was accompanied by the return of moderate tumorigenicity and karyotypic changes. Both cell lines were studied to determine sensitivity to superinfection with herpesviruses, induction of immune response in nude mice, and release of infectious virus.

Infection of human amnion cells with cytomegalovirus.

Human cytomegalovirus (CMV), known to replicate in vitro in human fibroblastic cells, was found to replicate in epithelial human amnion (HA) cells. Large syncytia formed in these cells after infection with CMV; inclusion bodies were observed in the nuclei, and CMV antigens were demonstrated in both the cytoplasm and the nucleus by indirect immunofluorescence techniques. The synthesis of virus DNA was also detected, and the production of infectious virus was followed. The titers were lower (from 10(4) to 6 X 10(5) using different isolates of CMV) than those obtained in human embryo fibroblast (HEF) cells, and the replication cycle was slower in HA than in HEF cells.

Replication of herpesviruses in human cells transformed by cytomegalovirus.

The replication of human cytomegalovirus (CMV) and herpes simplex virus (HSV) was studied in three human embryo cell lines (CMV-Mj-HEL-I, CMV-Mj-HEL-2, and CMV-Mj-HEL-2,T-I) transformed in vitro by human CMV. Growth studies revealed that these cells were completely resistant to infection by CMV strains ADI69 and Mj and partially resistant to HSV types I and 2. Neither virus DNA nor virus proteins were synthesized in the transformed cells infected with CMV AD169. The HSV production in CMV-transformed human embryo lung (HEL) cells was delayed when compared to the virus production in normal HEL cells and spread of HSV c.p.e. was slower in the transformed cells. The treatment of normal HEL cells with a crude extract of CMV-transformed HEL cells also resulted in inhibition of the spread of c.p.e. of HSV types I and 2. The inhibitory effect was not due to interferon since vesicular stomatitis virus replication was not affected and several experiments showed that it was not due to mycoplasma. The presence of virus inhibitor molecules in CMV-transformed cells absent in normal HEL cells is postulated.

Immune response of prostatic cancer patients to cytomegalovirus-infected and -transformed human cells.

The indirect immunofluorescent test was used to determine the prevalence of humoral immunity to cytomegalovirus (CMV)-induced antigens in prostatic cancer patients as compared to age-matched controls. Significantly more prostatic cancer patients demonstrated high CMV-antibody titers than did the benign prostatic hyperplasia and nonurogenital cancer groups; however, no significant difference in reactivity was found between paients with prostatic cancer and transitional cell carcinoma of the urinary bladder. When screened against CMV-transformed human cell lines, the reactivity of the sera followed the rate of expression of CMV-related antigens of cell lines used in these tests.

Establishment and characterization of a new endometrial cancer cell line (SCRC-1).

A new human endometrial cell line, SCRC-1, has been established from a uterine adenocarcinoma. During a period of 57 weeks in culture, the SCRC-1 cells have been passaged over 100 times. Cultures have an epitheloid morphology, high mitotic activity, and a tendency to form multiple cell layers. The cells are polygonal and are arranged in pavement-like fashion. Most have multiple nucleoli, some are multinucleated, and all have heavily vacuolated cytoplasm. Pretreatment with iododeoxyuridine did not reveal viral markers by cocultivation or immunofluorescence techniques. Karyologic studies indicate that the SCRC11 cell line is essentially diploid with a small subpopulation of tetraploid cells. Over a period of 20 weeks, 1 of 14 athymic nude mice inoculated with SCRC-1 cells developed a tumor which histologically closely resembled the original human tumor. Cells were subsequently propagated from this tumor and the resulting epitheloid-like cell line was designated SCRC-1, Tu 1.

Evidence for the association of cytomegalovirus with carcinoma of the prostate.

A human genital isolate of cytomegalovirus is shown to have transformed human embryonic lung cells in vitro. These cells produce tumors when injected into athymic nude mice. Two cell lines derived from tissue from human prostatic carcinoma have survived more than 20 passages in vitro and demonstrate cytomegalovirus-specific membrane antigen. Significant humoral antibody titers against cytomegalovirus have been demonstrated. Cell-mediated lymphocytotoxicity against these transformed cells has been demonstrated in patients with urinary tract tumors. This evidence indicates that an association between cytomegalovirus and human prostatic cancer may be more than coincidental.

Lymphocyte reactivity against virally transformed cells in patients with urologic cancer.

Lymphocytes from patients with urologic cancer were tested in microcytotoxicity assays against human cells transformed by cytomegalovirus. Human lymphocytes were significantly cytotoxic against the transformed cell line when compared to a normal human control cell line. Patients with prostatic carcinoma demonstrated greater target cell reduction than those with benign prostatic hyperplasia.

Cytomegalovirus and cancer of the prostate: in vitro transformation of human cells.

Urogenital tissue specimens were maintained in culture for 2 years. Epithelioid growth was enhanced with use of collagenase digestion rather than trypsinization. Twenty of 34 prostate cancer cell cultures survived more than ten in vitro passages, during which time four of 20 demonstrated epithelioid morphology. One epithelioid line (T-157) survived 32 in vitro passages. The cells demonstrated lack of contact inhibition in culture, were slightly positive in acid phosphatase tests, and reacted positively with cytomegalovirus (CMV)-immune sera in indirect immunofluorescence (IF) tests. These cells, which were proven to be of human male origin, failed to yield infectious virus and could be re-isolated from a nodule induced by the cells when injected sc into weanling athymic nude mice. The serum of the patient from which the tumor cells were derived demonstrated high CMV antibody titers and reacted with the virus-specific membrane and intracellular antigens of CMV-transformed human cells in IF tests. A CMV strain isolated from one of the normal prostate cell cultures established an in vitro long-term persistent infection of human embryo lung cells which resulted in the development of two transformed cell lines. The transformed cells possessed CMV antigenic markers and induced non-differentiated tumors when transplanted into athymic nude mice. The results constitute further evidence of the transforming capacity of CMV, and suggest that the virus may be oncogenic in its natural (human) host.