The virion host shutoff function of herpes simplex virus degrades the 5' end of a target mRNA before the 3' end.

During lytic infections, the virion host shutoff (vhs) function of herpes simplex virus (HSV) disaggregates host polysomes and induces rapid turnover of both cellular and viral mRNAs. To examine the steps in vhs-induced mRNA degradation, an RNase protection assay was used to compare the relative decay rates of sequences from the 5' and 3' ends of a selected target mRNA. In cells infected with wild-type HSV-1, sequences at the 5' end of the HSV-1 thymidine kinase mRNA were degraded more rapidly than those at the 3' end of the transcript. In contrast, in cells infected with a vhs mutant, the decay rates of sequences at the 5' and 3' termini of the transcript were much slower and were essentially indistinguishable from each other. Vhs-induced degradation of the transcribed portion of the mRNA was not preceded by detectable shortening of the poly(A) tail in vivo; nor was a poly(A) tail required to make an RNA a target for the vhs activity in vitro. The results suggest that degradation of sequences at or near the 5' end of an mRNA is an early step in vhs-induced decay.

Ovine lentivirus-infected macrophages mediate productive infection in cell types that are not susceptible to infection with cell-free virus.

Ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) are prototypic lentiviruses that replicate predominantly in macrophages of infected animals. In situ hybridization of pathologically affected tissues from diseased animals has shown that viral RNA exists in permissive macrophages as well as in non-macrophage cell types that do not support productive virus replication. These findings raise questions about the cellular tropism of these viruses in vivo and how this may relate to their pathogenesis and the establishment of persistent infections. In this study, the susceptibility of macrophages and fibro-epithelial cells derived from goat synovial membrane (GSM) to infection by 14 North American ovine lentivirus strains was examined. All 14 strains were macrophage-tropic, as indicated by expression of viral proteins and by fusion and development of syncytial cytopathic effects following co-culture of infected macrophages with GSM cells. In contrast, neither viral DNA nor viral proteins was detected in GSM cells inoculated with cell-free virus from nine of the 14 strains. Specific virus proteins were immunoprecipitated from restrictive GSM cells following culture with infected macrophages and serial passage of GSM cells to remove the macrophages. The lack of infection of GSM cells by cell-free virus from some ovine lentivirus field strains was circumvented by cell-associated virus infection from infected macrophages to GSM cells following cell-to-cell contact. This strategy could be one of the mechanisms involved in the escape from immune surveillance and establishment of persistent infection in infected animals.

Male breast cancer in the hereditary nonpolyposis colorectal cancer syndrome.

A male member of a large HNPCC kindred, affected by primary malignancies of the breast and colon, was identified. This individual was found to harbor a germline mutation of the MLH1 mismatch repair gene previously shown to segregate with disease in this kindred. The breast tumor exhibited somatic reduction to homozygosity for the MLH1 mutation, and microsatellite instability was evident in the breast tumor. We conclude that hereditary male breast cancer can occur as an integral tumor in the HNPCC syndrome.

Cumulative incidence of colorectal and extracolonic cancers in MLH1 and MSH2 mutation carriers of hereditary nonpolyposis colorectal cancer.

The extracolonic tumor spectrum of hereditary nonpolyposis colorectal cancer (HNPCC) includes cancer of the endometrium, ovaries, stomach, biliary tract, and urinary tract. This study was designed to determine the penetrance of colorectal and extracolonic tumors in HNPCC mutation carriers. Forty-nine patients (22 females and 27 males) were identified with an MSH2 germline mutation, and 56 patients (28 females and 28 males) were identified with an MLH1 I mutation. Cumulative incidence by age 60 (lifetime risk) and mean age of cancer diagnosis were compared. The lifetime risk of extracolonic cancers in MSH2 and MLH1 carriers was 48% and 11%, respectively (P = 0.016). Extracolonic cancer risk in MSH2 females and males was 69% and 34%, respectively (P = 0.042). Mean age of extracolonic cancer diagnosis was significantly older for MSH2 males than females (55.4 vs. 39.0, P = 0.013). No difference was observed in colorectal cancer risk between MLH1 and MSH2 carriers (84% vs. 71%). Colorectal cancer risk was 96% in MSH2 males compared to 39% in MSH2 females (P = 0.034). No differences in colorectal and extracolonic cancer risks between MLH1 females and males were identified. The risk of extracolonic cancer by age 60 was greater in MSH2 mutation carriers than in MLH1 carriers. Gender differences in colorectal and extracolonic cancer risk were observed for MSH2 carriers only. These phenotypic features of HNPCC genotypes may have clinical significance in the design of genotype-specific screening, surveillance, and follow-up for affected individuals.

Colorectal carcinoma survival among hereditary nonpolyposis colorectal carcinoma family members.

BACKGROUND: Patients with hereditary nonpolyposis colorectal carcinoma (HNPCC) reportedly have better prognoses than sporadic colorectal carcinoma (CRC) patients, but it has been unclear whether this could be due to differences in stage at diagnosis. The current study compared stage and survival in a retrospective cohort of HNPCC family members who developed CRC with the same factors in an unselected hospital series of patients with sporadic CRC. METHODS: This retrospective cohort study compared HNPCC cases (274 cases from 98 HNPCC families) with an unselected hospital series comprising 820 consecutive CRC cases. All patients were staged according to the TNM system of the American Joint Committee on Cancer and the International Union Against Cancer. Median follow-up among living patients was > 10 years and 8.5 years, respectively, for the two cohorts. Cox regression was used to compare survival in stage-stratified analyses of time from diagnosis to death. RESULTS: Compared with the unselected series, the HNPCC cases had lower stage disease (P < 0.001), and fewer had distant metastases at diagnosis (P < 0.001 in an analysis stratified by T classification). In stage-stratified survival analysis, the HNPCC cases had a significant overall survival advantage regardless of adjustment for their younger age. A conservative estimate of the hazard ratio (of HNPCC cases to the unselected series) was 0.67 (P < 0.0012). CONCLUSIONS: HNPCC patients had lower stage disease at diagnosis than the unselected CRC cases, mainly due to rarer distant metastases at diagnosis. They survived longer than unselected CRC patients with tumors of the same stage. The estimated death rate for the HNPCC cases, adjusted for stage and age differences, was at most two-thirds of the rate for the hospital series.

Neuroinvasion by ovine lentivirus in infected sheep mediated by inflammatory cells associated with experimental allergic encephalomyelitis.

Maedi Visna Virus (MVV) is a prototypic lentivirus that causes infection only in cells of macrophage lineage, unlike the primate lentiviruses which infect both CD4+ T lymphocytes and macrophages. In primates, the earliest viral invasion is associated with the ability of the virus to infect and activate T cells which convey virus to the brain. Infected monocytes in blood rarely cause CNS infection in absence of activation of CD4+ T cells. In the face of lack of infection or activation of T cells by MVV in sheep, the question arises, how does MVV gain access to the brain to cause the classical lesions of visna? In previous studies on experimental induction of visna, sheep were inoculated with virus directly in the brain. In this study, we asked whether neuroinvasion by MVV would occur if sheep were inoculated with virus in a non-neural site. Nine sheep were inoculated intratracheally and all developed systemic infection when examined 3 weeks later. At this time, five were injected intramuscularly with brain white matter homogenized in Freund's complete adjuvant to induce EAE. None of the four animals inoculated with virus alone developed CNS infection despite typical lentiviral infection in lungs, lymphoid tissues and blood-borne mononuclear cells. In contrast, all five of the sheep injected with brain homogenate developed infection in the brain. Virus was produced by macrophages associated with the EAE lesions. This study illustrated that both activated T cells specific for antigen in the CNS and infected macrophages are essential for lentivirus neuropathogenesis.

Etiology, natural history, management and molecular genetics of hereditary nonpolyposis colorectal cancer (Lynch syndromes): genetic counseling implications.

We estimate that 5-10% of virtually all forms of cancer are due to a primary hereditary etiology. However, a hereditary cancer diagnosis is often missed because the family history of cancer is given short shrift in medical practice. Hereditary nonpolyposis colorectal cancer (HNPCC) certainly fits this estimate, although some studies suggest that a minimum of 2% with a range as high as 10% of the total colorectal cancer burden is due to HNPCC. Mutations in one of the four mismatch repair genes, i.e., hMSH2, hMLH1, hPMS1, and hPMS2, account for about 70% of HNPCC kindreds. Other germ-line mutations are likely to be identified to account for the remainder of HNPCC patients. By far the most common HNPCC mutations involve hMSH2 and hMLH1, with hPMS1 and hPMS2 accounting for only about 3% of such families. Prior to these molecular genetic discoveries, the genetic counselor could only provide the patient with an estimate of a 50% likelihood of manifesting HNPCC based on the counselee having one or more first-degree relatives manifesting syndrome cancers in their direct genetic lineage. Because DNA testing has become available in families with known mutations, we have provided pretest group education in the form of a family information service with intensive education about the natural history, genetic risk, surveillance, and options for management of HNPCC, as well as discussion of the potential for fear, anxiety, apprehension, and insurance or employer discrimination that might impact on this DNA testing. Following informed consent, these relatives were then counseled on a one-to-one basis. Using DNA-based genetic counseling involving hMSH2 or hMLH1, we have provided this service to four extended HNPCC kindreds. Details of this genetic counseling experience on these four kindreds will be discussed.

Genetic counseling in hereditary nonpolyposis colorectal cancer: an extended family with MSH2 mutation.

OBJECTIVES: Molecular genetic advances have increased the demand for DNA testing. We describe DNA based genetic counseling in a hereditary nonpolyposis colorectal cancer (HNPCC) family. METHODS: This extended HNPCC family was found to harbor the MSH2 germline mutation. Family history, medical, and pathology documents enabled us to secure a high degree of verification that the kindred qualified as HNPCC. DNA testing revealed the MSH2 germline mutation that was verified independently in two laboratories. Genetic counseling was provided before DNA testing and disclosure of MSH2 findings. RESULTS: Genetic counseling revealed a variety of findings characterized by emotional stress in MSH2 germline mutation carriers. Concerns centered around reproductive issues, potential transmission of the deleterious gene to their progeny, and discrimination by insurance carriers and employers. More than one-half of the patients found to harbor the MSH2 mutation considered the option of prophylactic subtotal colectomy. CONCLUSION: DNA testing should be restricted to well-verified candidate families in which genetic counseling should be mandatory. HNPCC family members sought genetic risk assessment for their own health and that of their children. Contrasting emotional responses took place when told of their gene testing status and this required a sensitive empathetic listening ear. Patients have many concerns about their lifetime cancer destiny when told that they harbor the culprit MSH2 germline mutation.

Genetic characterization of two phenotypically distinct North American ovine lentiviruses and their possible origin from caprine arthritis-encephalitis virus.

Ovine and caprine lentiviruses are closely related genetically and antigenically although the diseases that these viruses cause in their respective host animals can vary greatly. In sheep, syndromes consist primarily of interstitial pneumonia with rare occurrences of arthritis and encephalitis, whereas in goats, the disease expresses mainly as arthritis in adult animals with rare cases of encephalitis in newborns. Experimentally, viruses from either sheep or goats can infect animals of the reciprocal species and many field strains of ovine lentivirus have biological properties similar to those of caprine viruses. However, a molecular correlation for the phenotypic differences between ovine and caprine lentivirus strains is unknown. To investigate this, we examined genetic characteristics of two phenotypically distinct North American ovine lentiviruses. Nucleotide sequence analysis of the envelope regions from virus strains 85/34 and 84/28 showed that despite significant biological differences, these viruses are closely related to each other and are genotypically more homologous to caprine arthritis-encephalitis virus (CAEV) than to visna virus of sheep. Furthermore, analysis of the nucleotide substitutions in their env regions indicated that when differences between the two ovine viruses and CAEV were found, the changes often resulted in nucleotides homologous with visna virus. These results suggest that the two field strains of ovine lentivirus may have originated from a cross-species infection of sheep by a CAEV-like virus and, evolution of their genomes toward that of ovine lentivirus may be reflective of adaptation of these viruses to the new ovine host.

Problems and practical considerations in assessing accuracy with NIST SRM 909a: report of defective vials.

During an experimental period of 12 months in 1992-1993, while we were comparing the effectiveness of monthly vs quarterly use of the National Institute for Standards and Technology Standard Reference Material (NIST SRM) 909a as an accuracy material for the projected 30-year Fernald Medical Monitoring Program, we encountered three random defective vials with a glucose recovery of less than 30% of the NIST-assigned value. Analysis with five different multichannel instruments confirmed the original finding. Concomitant glucose recovery from adjacent vials was 97%-104%, as determined by using the same instruments, reagents, calibrators, and quality-control criteria on the same days. Recoveries of uric acid and cholesterol were also low (53-75% and 75-80%, respectively) in the three defective vials. Other analytes were unaffected. Studies to identify the cause of the defective vials were carried out with microbiological, electron microscopic, and biochemical techniques. When used for accuracy studies, each vial of NIST SRM 909a should have a concomitant check for glucose recovery to detect whether the vial is defective.

Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep.

Seventy-nine 1-year-old lambs from three individual farms and a feedlot were examined for natural lentivirus infection. We used three different methods to detect infection and to identify the stage of the ovine lentivirus life cycle in blood-derived macrophages. Cytopathic infectious virus was obtained from 14/14 Border Leicester animals obtained from a naturally infected flock. Neither virus particles, virus proteins, virus specific antibodies nor viral DNA were detected in samples from 34 lambs from two South Kansas City farms. However, among 31 feedlot lambs, we identified 11 infected animals. Specific viral proteins were immunoprecipitated from macrophages of one animal, but no infectious cytopathic virus was isolated from these cells. Cells from ten of the other feedlot animals harboured viral DNA but neither viral particles nor proteins could be detected by our techniques. Thus, in these naturally infected animals, the virus life cycle either proceeded to completion, subject to differentiation of infected precursor cells in blood, or remained arrested at the DNA stage despite maturation of monocytes to macrophages. Sequence analysis of the env gene of viral genomes from two of the ten feedlot sheep showed sequences distinct from those of known ovine and caprine lentiviruses. Surprisingly, these sequences have a higher identity (of nucleotide and derived amino acid sequences) to caprine arthritis-encephalitis virus than to the ovine prototype, maedi-visna virus. These data suggest that the ovine and caprine lentiviruses found in North American sheep may have a common ancestral genotype that is closely related to the caprine virus.

Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures.

Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication.

Platelet-derived growth factor and wound contraction in the rat.

This experiment was undertaken for three purposes: (1) to determine a dose-response curve of acute steroid inhibition of wound contraction in the rat; (2) to confirm the results of our preliminary study that platelet-derived growth factor (PDGF) enhanced wound contraction in acutely steroid impaired rats; and (3) to examine the histology of the PDGF-treated wounds. To determine the dose-response of acute steroid inhibition of wound contraction, the rats were suppressed with daily doses of methylprednisolone and wound contraction was measured. Results demonstrated that significant glucocorticoid-induced inhibition of wound contraction begins with daily methylprednisolone doses of 2.0 mg/wound/day or 6.7 mg/kg/day. In an effort to confirm the results of our previous study of the effect of PDGF on wound contraction in acutely steroid-impaired rats and to study the histology of the PDGF-treated wounds, rats were suppressed with methylprednisolone or hydrocortisone and administered daily topical doses of rPDGF-BB. Wound contraction measurements revealed no improvement in the amount or rate of wound contraction. Histologically, the wounds were all very similar in the patterns of cellularity, granulation tissue maturity, collagen content, and epithelial migration. We have clarified the dose response of acute steroid inhibition of wound contraction in rats, data previously unavailable, and have concluded that PDGF in reasonable doses does not improve wound contraction in steroid-impaired rats nor does it alter the histology of the wounds.

Isolation of a herpes simplex virus type 1 mutant with a deletion in the virion host shutoff gene and identification of multiple forms of the vhs (UL41) polypeptide.

The virion host shutoff (vhs) gene (UL41) of herpes simplex virus type 1 (HSV-1) encodes a virion component that induces degradation of host mRNAs and the shutoff of most host protein synthesis. Subsequently, the vhs protein accelerates the turnover of all kinetic classes of viral mRNA. To identify the vhs (UL41) polypeptide within infected cells and virions, antisera raised against a UL41-lacZ fusion protein were used to characterize the polypeptides encoded by wild-type HSV-1 and two mutants: vhs1, a previously characterized mutant that lacks detectable virion host shutoff activity, and vhs-delta Sma, a newly constructed mutant containing a deletion of 196 codons from UL41. Two forms of the vhs (UL41) polypeptide were identified in cells infected with the wild-type virus or vhs1. Wild-type HSV-1 produced a major 58-kDa polypeptide, as well as a less abundant 59.5-kDa form of the protein, while vhs1 produced 57- and 59-kDa polypeptides that were approximately equally abundant. Although for either virus, both forms of the protein were phosphorylated, they differed in the extent of phosphorylation. While both vhs polypeptides were found in infected cells, only the faster migrating, less phosphorylated form was incorporated into virions. vhs-delta Sma encoded a smaller, 31-kDa polypeptide which, although present in infected cells, was not incorporated into virions. The results identify multiple forms of the vhs (UL41) polypeptide and suggest that posttranslational processing affects its packaging into virions, as well as its ability to induce mRNA degradation.

A correlation study of bone scanning with clinical and laboratory findings in the staging of nonsmall-cell lung cancer.

Fifty consecutive patients who had a bone scan and a diagnosis of nonsmall-cell lung carcinoma were studied, retrospectively, to determine the usefulness of bone scans in the presurgical workup. Bone scans were interpreted as positive for bone metastases if the scanning abnormality could not be explained by other causes (e.g., trauma or arthritis) or by additional studies (e.g., radiography or CT scanning). Seventeen percent of the patients whose initial clinical and laboratory findings suggested only localized resectable tumor had positive bone scans, changing treatment from surgery to more conservative therapy. Thirty-six percent of patients with no evidence of lung cancer extending to the mediastinum or beyond by CT of the chest and upper abdomen had bone scans indicating bone metastases (positive bone scan). These results suggest that bone scan adds useful information to the presurgical evaluation of patients with nonsmall-cell lung cancer. Clinical findings and/or CT of the chest and upper abdomen are not sensitive or specific enough to exclude bone metastases. Bone scan is recommended before thoracotomy for patients considered for surgery for localized, potentially resectable nonsmall-cell lung cancer.

Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum.

Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.

DNA-repair in mammary tumor cell lines with different X-ray sensitivities.

2 murine mammary tumor cell lines with different radiosensitivities (D0 = 65 and 92 rad) showed no significant differences in levels of unscheduled DNA-synthesis following X-ray or UV-exposure.