Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells.

Kaposi's sarcoma (KS), a vascular tumour that contains characteristic spindle cells forming slit-like spaces, may have an infectious aetiology. Recently, sequences of a new human herpesvirus, KSHV/HHV-8, have been identified in both HIV-associated and classical KS. We sought to identify the target cell of this virus in KS tumour tissue. Using PCR in situ hybridization (PCR-ISH) we show that KSHV/HHV-8 is present in the flat endothelial cells lining vascular spaces of KS lesions as well as in typical KS spindle cells. These findings show that KSHV/HHV-8 is present in the cell types thought to represent neoplastic cells in these lesions.

Heat shock cognate 70 mutations in sporadic breast carcinoma.

Heat Shock Cognate 70 (HSC70) is a constitutively expressed molecular chaperone, functions of which regulate the structure, subcellular localization, and turnover of cell proteins. It is also a component of the centrosome facilitating rearrangements during mitotic/meiotic spindle formation and cytoplasmic microtubule organization. We localized HSC70 to 11q23.3, a region deleted in 40% of sporadic breast and other cancers. Sequencing demonstrated mutation in the NH2-terminal ATPase domain of HSC70 in 2 of 15 sporadic breast carcinomas examined. In both cases, mutation was coincident with allelic imbalance, suggesting that HSC70 is a target of somatic mutation and deletion in a fraction of breast carcinomas.

Antiepileptic treatment and risk for hepatobiliary cancer and malignant lymphoma.

The possible influence of phenobarbital and phenytoin treatment on cancer risk was investigated in a case-control study nested in a cohort of 8004 epileptic patients in Denmark. Information on anticonvulsive treatments was abstracted for 95% of 60 patients with cancers of the liver and biliary tract or malignant lymphoma and for 94% of 171 cancer-free control patients. Use of anticonvulsive drugs was correlated with angiographic procedures that used Thorotrast, a well-known human liver carcinogen. After exclusion of study subjects exposed to Thorotrast, no association was seen between treatment with phenobarbital and cancer of the liver (odds ratio, 1.0; 95% confidence interval, 0.1-8.0) or biliary tract (odds ratio, 0.8; 95% confidence interval, 0.1-4.2). Furthermore, a histopathological evaluation of slides from 7 of 9 liver cancer patients not treated with Thorotrast revealed that 3 of the 4 cases of hepatocellular carcinoma involved cirrhosis of the liver, which suggested an etiological role for alcohol or viral hepatitis. A possible link was observed between use of phenytoin and risk for non-Hodgkin's lymphoma (1.8; 0.5-6.6), with a rising trend in risk with increasing dose. Our results suggest that the increased risk for cancers of the liver and biliary tract among Danish epileptic patients is likely to be due to Thorotrast administration and factors associated with cirrhosis of the liver rather than to anticonvulsive treatment.

Allelic deletions at chromosome 11q22-q23.1 and 11q25-qterm are frequent in sporadic breast but not colorectal cancers.

We identified the chromosome 11q23 region as containing a putative tumour suppressor gene(s) frequently deleted in nonfamilial breast and other cancers. To define this region(s) further, we performed a systematic genetic analysis at chromosome 11q14-qterm in sporadic breast and colorectal cancer. Tumour and constitutional DNA from a panel of 81 cases (51 breast and 30 colorectal cancers) were analysed with multiple microsatellite markers distal to 11q13. Of 51 breast cancers, 31 of 49 informative cases (63%) showed LOH at the 11q22-q23.1 region (approximately 8 Mb). Furthermore, 23 of 45 informative cases (51%) had a deletion at 11q25 (approximately 2 Mb). Overall, LOH on 11q occurred in 37 of 51 breast cancers (72%). Colorectal cancers had LOH at 11q22 in two of 18 informative cases (11%), LOH at 11q23.3 in two of 17 informative cases (12%) and LOH at 11q25 in three of 20 informative cases (15%). Overall, LOH at 11q occurred in five of 30 colorectal cancers (16%). This data shows that chromosome 11q contains at least two independent regions (one novel) frequently deleted in breast cancers. Contrary to previous reports, LOH at distal 11q is not frequent in colorectal cancer. Chromosome 11q22-q23.1 and 11q25-qterm contain putative tumour suppressor genes with a significant role in breast but not colorectal carcinogenesis.

Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia.

Twenty per cent of cervical intraepithelial neoplasias-III (CIN-III) progress to invasive cancer. Human papillomavirus (HPV) infection alone does not determine progression. CIN-III lesions were collected from 161 women. Each tissue was microdissected into a maximum of 32 contiguous units and assayed at multiple microsatellite loci on chromosome 11q, a region frequently deleted in invasive cervical and other cancers. Eight of 108 informative cases (7%) had 11q23.3-q25 deletions; focally intra-lesional in six (one with focal loss of alternate alleles), and pan-lesional in two cases. Hence, 11q deletion can occur early in cervical neoplasia, and possibly predisposes to invasion.

Quantification of oncogene dosage in tumours by simultaneous dual-label hybridization.

Gene amplification and allele loss occur in a variety of human tumours and some have prognostic value. Therefore, techniques which facilitate detection and quantification of gene dosage could have wide applicability in cancer research. Using the INT-2 gene as a model system, a quantitative procedure has been developed for measuring gene copy number using dual-label hybridization to DNA dot blots. A probe specific for the INT-2 gene was labelled with [alpha-32P]dCTP and a probe to beta-actin, the control locus, was labelled with [alpha-35S]dATP. Flat-bed scintillation counting was used to detect and separate the emissions resulting from each bound probe, and gene dosage was calculated from the ratio of INT-2 to the beta-actin probe compared with the ratio derived from constitutional DNA. Calculated ratios of greater than 1.22 and less than 0.78 indicated gene amplification and allelic loss respectively, at the 99% confidence limit derived from the population of 35 constitutional DNAs. The results were validated by RFLP analysis. It is expected that this technique will permit precise gene dosage quantification in many areas.

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region. An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells. These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.

Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages.

Studies over the past 20 years have established that the development of new capillaries from an existing vascular network (a process called angiogenesis) is an essential component of tumor growth. Malignant tumors do not grow beyond 2-3 mm3 in size unless they stimulate the formation of new blood vessels and thus provide a route for the increased inflow of nutrients and oxygen and outflow of waste products. Tumor angiogenesis also provides an essential exit route for metastasizing tumor cells from the tumor to the bloodstream. Indeed, extensive neovascularization is a poor prognostic factor in several forms of human cancer. Angiogenesis is a complex, multistep process driven by many local signals within the tumor. This involves the degradation of the extracellular matrix around a local venule after the release of collagenases and proteases, the proliferation and migration of capillary endothelial cells, and their differentiation into functioning capillaries. Cytokines produced by various cell types present within the microenvironment of solid tumors form a complex, dynamic network in which they have multiple effects on tumor progression. Herein we review our work on the presence, and possible regulatory influence on tumor angiogenesis, of a number of these cytokines within invasive breast carcinomas. We have combined immunocytochemistry with a single cell cytokine release assay called the reverse hemolytic plaque assay to investigate the cellular sources of the key angiogenic cytokines, vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor-alpha. Tumor-associated macrophages in the stromal compartment of these tumors and/or malignant epithelial cells were seen to be a major producer cell for these cytokines, whereas tumor necrosis factor-alpha receptors were expressed by leukocytes, malignant cells, and endothelial cells in tumor blood vessels.

Tissue culture, protein and collagen synthesis in antibiotic sterilized canine heart valves.

Viability of canine heart valve leaflet fibroblasts was assessed after varying periods of sterilization and storage in antibiotic-nutrient solution. Tissue culture and assessment of protein and collagen synthesis showed that tissue obtained under optimal conditions rarely retains viability beyond 3 weeks in antibiotic-nutrient solution and is severely impaired after 2 weeks. This casts serious doubts on viability in current clinical homograft valve practice.

Viability in human heart valves prepared for grafting.

Viability of antibiotic sterilized and stored human heart valves obtained at routine necropsy was assessed by tissue culture and protein and collagen synthesis. Only three of 23 valves examined showed any evidence of viability, in striking contrast to earlier work on canine valves obtained under optimal conditions. These findings justify doubts regarding pre-implantation viability in human heart valves prepared for grafting.

Rodent osteoblast-like cells support osteoclastic differentiation of human cord blood monocytes in the presence of M-CSF and 1,25 dihydroxyvitamin D3.

Fracture repair requires the involvement of osteoclasts (OC), multinucleated cells which are responsible for bone resorption and form by fusion of circulating mononuclear haemopoietic precursors. The nature of these circulating precursor cells, in particular their relationship to blood monocytes, is uncertain. To define further the nature of the circulating human OC precursor, and to determine the role bone stromal cells and humoral factors play in the differentiation of OCs, we co-cultured human umbilical cord blood monocytes with UMR106.01 osteoblast-like cells in the presence and absence of 1,25 dihydroxyvitamin D3 [1,25 (OH)2D3], macrophage-colony stimulating factor (M-CSF) and dexamethasone on both bone slices and coverslips. Isolated cells were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14 and HLA-DR) and negative for OC markers [tartrate resistant acid phosphatase (TRAP), vitronectin receptors (VNR) and calcitonin receptors (CT receptors)] and did not form resorption pits on bone slices after 24 hr in culture. However, after 14 days in co-culture with UMR106.01 cells, in the presence of 1,25 (OH)2D3 and M-CSF, numerous TRAP, CT receptor and VNR positive multinucleated cells capable of extensive lacunar bone resorption were formed in these co-cultures. The presence of 1,25 (OH)2D3, M-CSF and a bone-derived stromal cell population were absolute requirements for OC differentiation. It is concluded that mononuclear phagocytes are capable of differentiating into mature functional OCs when cultured under specific cellular and hormonal conditions. This is vitro model of human OC differentiation should prove useful in further analysing factors controlling OC generation in bone remodelling and repair.

Cellular and hormonal factors influencing monocyte differentiation to osteoclastic bone-resorbing cells.

Osteoclasts are multinucleated cells which form by fusion of circulating mononuclear hemopoietic precursors. The nature of these precursor cells and the roles bone stromal cells and hormonal factors play in their differentiation to osteoclasts are unknown. We cocultured adherent murine blood monocytes (nonspecific esterase and F4/80 positive; tartrate-resistant acid phosphatase negative) with osteoblastic and fibroblastic stromal cell lines in the presence of 2 x 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Tartrate-resistant acid phosphatase and calcitonin (CT) receptor-positive osteoclastic cells, which formed numerous resorption pits in vitro, were noted after only 4 days in coculture with UMR106 osteoblast-like cells. Resorption was seen in cocultures to which as few as 100 peripheral blood mononuclear cells had been added. 1,25-(OH)2D3 and contact with live bone stromal cells were absolute requirements for monocyte differentiation into bone-resorbing cells. Both salmon CT (5 IU/ml) and prostaglandin E2 (10(-6) M) significantly inhibited bone resorption. Thus, a significant proportion of the peripheral blood mononuclear cells in the monocyte fraction are capable of differentiating into cells showing the cytochemical and functional characteristics of osteoclasts. The presence of specific hormonal [1,25-(OH)2D3] and bone stromal cell elements is necessary for this process to occur; the resultant resorption can be modulated by known inhibitors of bone resorption, CT and prostaglandin E2.

The human osteoclast precursor circulates in the monocyte fraction.

The osteoclast is known to be formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. The precise nature of these circulating cells and, in particular, their relation to monocytes is unknown. We have developed an in vitro system of human osteoclast formation whereby human monocytes [CD14, CD11a, CD11b and HLA-DR positive, and tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), vitronectin receptor (VNR) negative] were isolated and cocultured for up to 21 days with UMR106 rat osteoblast-like cells or ST2 mouse preadipocytic bone marrow stromal cells in the presence of 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) and macrophage colony stimulating factor (M-CSF). Numerous TRAP, VNR and CTR positive multinucleated cells, capable of extensive lacunar bone resorption, formed in these cocultures; the absolute requirements for this to occur were contact with the above bone stromal cells, 1,25(OH)2D3, and M-CSF. These results show that the human mononuclear osteoclast precursor circulates in the monocyte fraction and exhibits a monocyte phenotype, acquiring osteoclast phenotypic features in the process of differentiation into mature functional osteoclasts.

An assay for C1q biosynthesis in cultured human fibroblasts.

With a standard C1 haemolytic assay, cultured human fibroblasts were shown to synthesize and secrete haemolytically active C1 and C1q. An assay for detection and quantitation of intra-cellular biosynthesis and secretion of C1q was then developed, using Sepharose beads covalently coated with goat monospecific anti-C1q IgG. The molecular structure of fibroblast C1q was found to be unusual in 2 ways: firstly, it probably represented an enlarged pro-C1q, and secondly, the secreted molecule had a different structure from the cell associated molecule.

Non-isotopic in situ detection of mRNA for interleukin-4 in archival human tissue.

Non-isotopic in situ hybridisation (NISH) is a rapid and sensitive method currently used in molecular pathology to identify DNA in fresh and archival human biopsies. In this study we have extended NISH technology for mRNA detection to include the use of digoxigenin-labelled riboprobes to localise low abundance mRNA for interleukin-4 in paraffin embedded material (from active inflammatory bowel disease). As this methodological approach makes the retrospective study of cytokine gene expression in archival material possible for the first time, it could act as a prototype for studies of cytokine involvement in various human diseases.

Measurement of cytokine release by human cells. A quantitative analysis at the single cell level using the reverse haemolytic plaque assay.

The reverse haemolytic plaque assay has been adapted to detect and measure the release of such cytokines as interleukin-1, -2 and -6, GM colony-stimulating factor or interferon-gamma by individual human cells derived from either peripheral blood or enzymatically dispersed breast carcinomas. Since each of these peptides is released by more than one cell type, this in vitro assay has been coupled with immunocytochemistry to identify the particular cell type(s) contributing to the release of each cytokine. This technique is useful in (i) obviating the need for purification of a given cell type prior to estimating cytokine release, and (ii) evaluating quantitative differences in secretion amongst cells of a particular type. Such a method has the additional advantage over most alternative methods applied at the single cell level in that the cells remain viable at the end of the assay and can be used in further studies. This assay thus provides a powerful new tool in the investigation of the role of cytokines in both the normal modulation of the immune system and the development of such diseases as neoplasia.

Differential recovery of phenotypically and functionally distinct circulating antigen-presenting cells after allogeneic marrow transplantation.

Low-density cells (LDC) prepared from peripheral blood by fractionation over hypertonic metrizamide contain 95% of cells with veiled morphology, almost all of which are HLA-DR-positive and have characteristics of antigen-presenting cells. In normal individuals the monoclonal antibodies RFD1 and RFD2 divide these cells into three phenotypically distinct populations, D1+D2-, D1-D2+ and D1-D2-. The RFD1-positive population is nonphagocytic. We have investigated the recovery of LDC in peripheral blood after (T cell-depleted) marrow transplantation, to assess whether defects in antigen-presenting cell (APC) subpopulations could contribute to the prolonged immune-paresis of marrow graft recipients. We find that APC of donor origin and with apparently normal morphology, phenotype, and function appear within 6 weeks of BMT. By three months the donor-derived nonphagocytic RFD1-positive subset has disappeared, although phagocytic RFD2-positive cells remain. The disappearance of the RFD1-positive subset is associated with a loss of antigen presentation by patients' LDC of the soluble protein antigen tetanus toxoid, though the capacity to present alloantigen and stimulate in a mixed lymphocyte reaction is retained. Donor-derived RFD1-positive cells and soluble antigen-presenting capacity do not reappear for one year or more. This biphasic recovery of RFD1-positive cells contrasted with the continued production of RFD2-positive APC, implies that the phenotypic and functional distinction between APC subpopulations in peripheral blood also reflects a separate ontogeny. Since these marrow graft recipients retain the phagocytic (RFD2-positive) APC but lose the nonphagocytic (RFD1-positive) APC subset, there is now an opportunity to explore the role of each subset in antigen processing and presentation.

Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots.

A dual-labeling technique was developed for direct quantification of specific mRNA using a flatbed liquid scintillation counter. This method simultaneously measures cpm of 32P- and 35S-labeled probes bound to RNA dot blots and subtracts counts due to nonspecific background radioactivity bound to the filter. Probes for T-cell receptor and beta-actin (as the internal standard) were hybridized both separately and simultaneously to RNA isolated from five different sources. There was concordance between the radioactivity measured from single- and dual-hybridizations for each combination of 35S- and 32P-labeled probes. This methodology directly quantifies specific mRNA sequences bound to membranes and has potential for measuring gene dosage, without the need for re-probing or densitometric analysis.

Morphological correlation of human papillomavirus infection of matched cervical smears and biopsies from patients with persistent mild cervical cytological abnormalities.

Human papillomavirus (HPV) analysis of cytological material has been advocated for determining those patients with low-grade cervical cytological abnormalities who have current high-grade squamous intraepithelial lesions (SILs). In this study, we analyzed concurrent cervical smears and biopsies from 167 patients with Papanicolaou (Pap) smears showing persistent atypical squamous cells of uncertain significance (ASCUS) or low grade SILs (1) to compare the detection of HPV by nonisotopic in situ hybridization (NISH) on matched smears and biopsies; (2) to analyze the type and distribution of NISH signal within lesions; and (3) to define further the ability of NISH techniques to distinguish between patients with low- and high-grade SIL. Whether present in cervical smears or biopsies, high-risk HPV types (16, 18, 31, 33, and related types) were significantly associated with high-grade SILs (P < .001) but were found in 15% of low-grade SILs. Ninety percent of high grade lesions were directly infected by these HPV types, and good concordance (92.2%) was found between NISH analysis of matched cervical smears and biopsies, indicating accurate colposcopic targetting of biopsies and excision specimens. Punctate signal morphology, which correlates with viral integration, was associated with high-grade SILs but was also observed in two low-grade SILs. Although the presence of high-risk HPV types in low-grade SILs limits the predictive ability of HPV testing by this means in this group of patients, those patients with high-risk HPV infection of low-grade SILs may be a greater risk of progression to high-grade SIL or invasive carcinoma. If this were the case, HPV testing would be of potential value in the management of patients with low-grade cytological abnormalities.

Squamous intraepithelial neoplasia in an ovarian cyst, cervical intraepithelial neoplasia, and human papillomavirus.

A case of squamous intraepithelial neoplasia in an ovarian cyst in association with cervical intraepithelial neoplasia (CIN) III is described. In view of the association of human papillomavirus (HPV) and CIN, the possibility that HPV infection could be associated with similar changes in the ovary was postulated. The HPV genome was shown in formalin-fixed tissue of the cervical lesion by nonisotopic in situ hybridization (NISH) and by the polymerase chain reaction (PCR). However, HPV could not be shown in the ovarian lesion by NISH or PCR. On the basis of these findings there appears to be no association between HPV infection and squamous intraepithelial neoplasia in an ovarian cyst.