Human xenografts. A model system for human papillomavirus infection.

Epithelial chips of human foreskin or cervix infected in vitro with one strain of human papillomavirus type 11 (HPV-11) and subsequently transplanted to the renal capsule of athymic mice will yield, after 3 months of in vivo incubation, epithelial cysts that are morphologically transformed and appear identical in every way to human condylomata. These cysts synthesize virus RNA, DNA, proteins, and infectious virions. The cysts can be utilized as a source of virus for continued passage of infection. The original HPV-11 infecting material was a cell-free saline extract of pooled human vulvar condylomata. DNAs of other HPV types were not detected in this material by either dot blot or Southern blot analyses. The copy number of HPV-11 virus genomes per cell genome in experimental condylomata ranged from about 200 to 1,000, a range expected for episomal papillomavirus DNA. Analyses of cloned HPV-11 DNA (pBT-1) from experimental lesions demonstrated that the size and restriction endonuclease map of the HPV-11-Hershey isolate closely matched that of the prototype. A few nucleotide changes that were detected during analyses of pBT-1 DNA resulted either in no amino acid change or a conservative change of amino acid. Physical characterization of the cloned experimental HPV-11 DNA as well as HPV typing in clinical lesions and experimental cysts are presented.

Analysis of viral proteins in human cytomegalovirus-infected cells during impaired lytic replication of herpes simplex virus.

Herpes simplex virus (HSV) latency can be established in vitro following arrest of virus replication and survival of infected cells in culture. Human cytomegalovirus (HCMV) has been shown to interact with HSV, resulting in reactivation of latent HSV. In addition, impaired replication of superinfecting HSV occurs in HCMV-infected human cells. HCMV-infected human embryonic lung cells inhibit production of infectious HSV despite replication of HSV DNA at levels comparable to those in control cultures infected only with HSV. Using radioimmunoprecipitation techniques, we found that the synthesis of HSV type 1 proteins of the alpha, beta/gamma, and gamma kinetic classes was impaired during the restricted replication of HSV in HCMV-infected HEL cells. However, synthesis of the HSV beta protein ICP-8 and HCMV alpha and beta proteins was not significantly affected in superinfected cell cultures.

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.

Analysis of long-term human cytomegalovirus latency in vitro.

Human leukocyte interferon (IFN-alpha) and acyclovir (ACV) have been used to establish human cytomegalovirus (HCMV) latency in infected human embryo lung fibroblast (HEL-F) cells. HCMV latency was maintained for a short interval (less than 9 days) after removal of inhibitors by increasing the incubation temperature. We now report a model system in which HCMV latency has been dramatically extended. HEL-F cells pretreated with IFN-alpha (200 IU/ml) and ACV (300 microM) were infected with a low MOI of HCMV, and treated for 23 days with the same inhibitor combination at 37 degrees. Infectious HCMV and virus antigens were undetectable at the time of inhibitor removal and remained undetectable during continued incubation at 40.5 degrees. A minimum of 0.4% of the cell population, however, contained a virus genome that could be reactivated at the time of inhibitor removal; this value declined to 0.0005% after 77 days at 40.5 degrees. HCMV reactivation was achieved by maintaining the infected cells at 37 degrees after inhibitor removal or by decreasing the incubation temperature to 37 degrees at any time during maintenance at 40.5 degrees. The HCMV genome was analyzed by blot hybridization in latently infected cells 23 days after inhibitor treatment at 37 degrees or 4 days after inhibitor removal at 40.5 degrees. Although many HCMV-unique DNA genomic sequences were retained in HEL-F cultures after 23 days of inhibitor treatment, a significant reduction in retained HCMV sequences occurred after inhibitor removal and temperature shift to 40.5 degrees. The XbaI HCMV DNA fragments retained in the latently infected HEL-F cultures were present at a copy number of at least 0.5 copies per haploid cell genome equivalent.

Distinct high-performance liquid chromatography pattern of transforming growth factor activity in urine of cancer patients as compared with that of normal individuals.

Reverse-phase high-performance liquid chromatography (HPLC) performed on urine from cancer patients and normal controls revealed the presence of seven chromatographically distinct peaks of transforming growth factor (TGF) activity, as measured by colony formation of normal rat kidney cells in soft agar. Comparison of urines from normal donors and cancer patients showed no differences in EGF (epidermal growth factor)-dependent beta-TGF-like activity but did reveal distinct patterns of EGF-related, EGF-independent alpha-TGF-like activity. All urine samples contained at least two chromatographically distinguishable forms of EGF-dependent TGF activity, eluting from HPLC as broad peaks with 30 and 43% acetonitrile. The remaining five TGFs eluted as sharp peaks with 32, 34, 35, 37, and 38% acetonitrile, demonstrated EGF-competing activity, and thus were functionally related to EGF. Two of the five EGF-related TGFs were consistently elevated only in the urine of cancer patients and eluted with 32% (TGFA) and 37% (TGFD) acetonitrile Two of the other EGF-related TGFs, eluting with 34% (TGFB) and 35% (TGFC) acetonitrile, were commonly found in both normals and cancer patients. The fifth EGF-related TGF, TGFE, eluting with 38% acetonitrile, was found only in normal donor specimens. TGFA corresponded to the unique Mr 30,000 TGF activity previously identified only in the urine of cancer patients. These observations demonstrate that cancer patients produce high levels of EGF-related TGF activities which can be readily distinguished, using reverse-phase HPLC, from EGF-related TGFs produced by normal individuals. Using a solid-phase competitive radioreceptor binding assay for EGF, we demonstrated that quantitation of EGF-competing activity is as sensitive and effective as the soft-agar colony formation assay for distinguishing HPLC profiles of urinary TGF from cancer patients versus that from normal individuals.

High-molecular-weight transforming growth factor activity in the urine of patients with disseminated cancer.

Urine from 22 patients with a variety of disseminated cancers and from an equivalent number of nonmalignant controls of similar age and sex was tested for the presence of transforming growth factor (TGF) activity as measured by the ability to promote the growth in soft agar of nontransformed indicator cells. Cancer patients included those with carcinomas of the lung, breast, colon, and ovary, as well as melanomas and sarcomas. The nonmalignant controls included both normals and individuals with a variety of inflammatory and infectious disorders. Aliquots of unfrozen urine were acid extracted, chromatographed on a Bio-Gel P-30 column, and then tested for TGF activity using normal rat kidney fibroblasts and epidermal growth factor (EGF)-competing activity with human carcinoma A431 cells. These assays revealed that a high-molecular-weight TGF activity (Mr 30,000 to 35,000) which coelutes with EGF-competing activity was present in 18 of 22 cancer patients but present in only five of 22 nonmalignant controls (p less than 0.01). In contrast, a low-molecular-weight TGF activity (Mr 6000 to 8000) which does not coelute with EGF-competing activity was found in all urines tested. These results indicate that an EGF-related, high-molecular-weight TGF activity is found in the urine of cancer patients and may be a useful tumor marker. Unlike other tumor markers described previously, high-molecular-weight TGF activity has a biological activity which is related to the expression of the transformed phenotype.