Site-specific regulation of oestrogen receptor-alpha and -beta by oestradiol in human adipose tissue.

AIM: To examine the expression of oestrogen receptors alpha and beta (ERalpha and ERbeta) and their regulation by 17beta-oestradiol (E2) in stromal cells and adipocytes from human subcutaneous (s.c.) and omental (o.m.) adipose tissue. METHODS: Subcutaneous and o.m. abdominal adipose tissues were obtained from 10 women (mean age 63.5 +/- 4.8 years; mean weight 75.6 +/- 6.7 kg) undergoing elective or cosmetic surgery. Immunohistochemistry and RT-PCR analysis were used to detect the presence of ERalpha and ERbeta. The regulation of ERalpha and ERbeta by E(2) (10(-7) M to 10(-9) M) was examined using Western immunoblotting analysis in both s.c. and o.m. stromal cells and mature adipocytes cultured in serum-free, phenol red-free medium. RESULTS: Immunostaining of s.c. and o.m. adipose tissue showed that the ER subtypes were localized predominantly within the nucleus. Western analysis demonstrated that E2 treatments differentially altered ERalpha and ERbeta expression in s.c. and o.m. adipocytes. In s.c. and o.m. stromal cells, E(2) (10(-8) M) produced a significant up regulation relative to control of 66 kDa ERalpha (s.c.:1.87 +/- 0.22; o.m.:1.97 +/- 0.17; p < 0.05) and 60 kDa ERbeta (s.c.:1.66 +/- 0.3; o.m.: 1.68 +/- 0.16; p < 0.05). In s.c. adipocytes, however, ERalpha expression significantly decreased with E(2) 10(-8) M relative to control while ERbeta expression increased (ERalpha 0.58 +/- 0.06, ERbeta: 1.47 +/- 0.11; p < 0.05). In o.m. adipocytes, the inhibition of ERalpha with E(2) was not observed (ERalpha 1.86 +/- 0.36, ERbeta:1.03 +/- 0.15, p < 0.05) CONCLUSIONS: ERalpha and ERbeta are expressed but differentially regulated by E(2) in s.c. and o.m. adipocytes and stromal cells. The upregulation of ERbeta by E(2) suggests that E(2) maintains the expression of these receptors. The feed-back inhibition of ERalpha expression by E(2) in s.c. but not o.m. adipocytes observed in vitro is consistent with the data from ERalpha knock out mice where s.c. fat is increased. Selective ER modulators may have different effects in different adipose sites.

Herceptest: HER2 expression and gene amplification in non-small cell lung cancer.

HER2 is an erbB/HER type 1 tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3+ cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting.

Immunoreactivity for latent membrane protein 1 of Epstein-Barr virus in nevi and melanomas is not related to the viral infection.

Epstein-Barr virus (EBV) is a human herpes virus with oncogenic potential, associated with several malignancies. The EBV-encoded latent membrane protein 1 (LMP1) is one of nine proteins regularly expressed in virally infected and immortalised B lymphocytes. We now document the consistent immunoreactivity for LMP1 in 90% of 65 nevi and melanomas, using the monoclonal antibody cocktail CS1-4. The immunocytochemical findings, however, were not confirmed using reverse-transcription polymerase chain reaction (RT-PCR) experiments, which failed to demonstrate any actual expression of LMP1 mRNA. In situ hybridisation for EBV-encoded RNAs (EBERs 1 and 2) and PCR amplification of EBV genomic sequences also failed to document any viral infection. Several normal and neoplastic human tissues have also been immunostained for LMP1, without any positive staining, with the exception of a minor percentage of skin melanocytes and of normal blasts of the myeloid and erythroid lineages. We conclude that the vast majority of nevi and melanomas express a still uncharacterised molecule, cross-reacting with anti-LMP1 (CS1-4) antibodies, which may be considered a consistent marker of melanocytic proliferations. The immunoreactivity of normal and neoplastic human tissues for the anti-LMP1 reagent should not be taken as evidence of EBV infection.

Angiostatin generation by human tumor cell lines: involvement of plasminogen activators.

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.

Angiogenic balance in human melanoma: expression of VEGF, bFGF, IL-8, PDGF and angiostatin in relation to vascular density of xenografts in vivo.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. In most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor.

An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma.

hCAP-18 is a newly described protein of human neutrophilic granulocytes which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The present work describes the production of recombinant hCAP-18, the generation of antibodies to the protein and the development of an accurate, sensitive and specific ELISA for the detection of hCAP-18 in cells, plasma and urine with a detection limit of 0.084 ng/ml. The amount of hCAP-18 in neutrophils is 0.627 microgram protein per 10(6) cells. The plasma level is 1.18 micrograms/ml which is several fold higher than for other neutrophil specific granule proteins. hCAP-18 is present in plasma as high molecular weight complexes. In accordance with this, hCAP-18 is barely excreted in the urine. The bone marrow appears to be the major source of plasma hCAP-18. The high level of hCAP-18 in plasma may provide an important defense against microorganisms and endotoxins.

New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.

AIMS: To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS: Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS: A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS: After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.

Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.

Immunohistochemical identification of neuron-specific enolase, synaptophysin, chromogranin and endocrine granule constituent in neuroendocrine tumours.

Immunohistochemical identification of neuroendocrine tumour markers in paraffin embedded material from 22 tumours (5 small cell carcinomas of the lung (SCCL), 12 carcinoids, 2 medullary thyroid carcinomas, 2 pheochromocytomas and one paraganglioma) with electron microscopically verified dense-core granules revealed neuron-specific enolase in all but one tumour, synaptophsin in 15/22 (2 SCCL and 6 carcinoids negative), chromogranin in 16/22 (all SCCL and one carcinoid negative), and endocrine granule constituent in 18/22 (4 SCCL negative). The Grimelius silver methods stained 13/22 (all cases of SCCL and 4 carcinoids negative).

Epidemiological studies of piglet diarrhoea in intensively managed Danish sow herds. IV. Pathogenicity of porcine rotavirus.

Colostrum-deprived piglets inoculated with rotavirus 24 h after birth developed a profuse diarrhoea that spread to non-inoculated, colostrum-deprived litter mates and, occassionally, to colostrum-fed piglets. Case fatality rates in these 3 categories of piglets were 63.2%, 35.7% and 8.3%, respectively. Surviving piglets recovered in 1-2 weeks, but shedded virus via the faeces for up to 3 weeks p.i. The D-xylose test revealed severe malabsorption, with extremely flat absorption curves for up to 3-4 weeks p.i. Malabsorption was more marked in piglets with a long-lasting faecal virus excretion than in piglets where virus disappeared from the faeces within 10 days p.i. Infected piglets (colostrum-fed and colostrum-deprived) had decreased weight gains and were 5 days older at a bodyweight of 25 kg than non-inoculated controls. It is concluded that rotavirus is probably of significance in diarrhoeal syndromes in suckling piglets, alone or in combination with E. coli or other pathogens.

An equine rotavirus (FI-14 strain) which bears both subgroup I and subgroup II specificities on its VP6.

An equinine rotavirus FI-14 strain, originally isolated from a diarrheic foal in New York state, was shown to belong to serotype 3 by neutralization assay. In addition, it was found to react with both subgroup I and subgroup II monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA), thus representing the first rotavirus strain to exhibit both subgroup specificities. By using hybridoma technology, we successfully produced monoclonal antibodies directed against the major inner capsid protein VP6 (the sixth gene product) of FI-14 virus. Such monoclonal antibodies reacted specifically with either subgroup I or subgroup II rotaviruses thus demonstrating that the VP6 of FI-14 virus has both subgroup I- and subgroup II-specific epitopes. Four additional monoclones directed to the VP6 of FI-14 demonstrated distinct reactivities by ELISA with a panel of 49 rotavirus strains derived from 11 different animal and avian species. Thus, at least six distinct antigenic sites were shown to exist on VP6 of FI-14 virus. When these 49 rotavirus strains were arranged based on their reactivity patterns with the six representative monoclones, they fell into one of eight reactivity groups. Analysis of the reactivity patterns of rotaviruses derived from various animal species suggested that human rotaviruses may have two ancestral lineages: one (subgroup II, serotype 1, 3, and 4) with pig-human lineage, and the other (subgroup I, serotype 2) with bovine-simian-human lineage. When analyzed by radioimmunoprecipitation, the molecular weight of the FI-14 virus VP6 (subgroups I and II) appeared to be larger (approx 45K) than those (approx 42K) of rhesus monkey MMU18006 virus VP6 (subgroup I) or human Wa virus VP6 (subgroup II). By RNA-RNA hybridization analysis, the FI-14 virus was shown not to share significant homology with viruses belonging to the four known human rotavirus serotypes.

VP7 serotype-specific glycoprotein of OSU porcine rotavirus: coding assignment and gene sequence.

With a reassortant from a cross of human rotavirus DS-1 (serotype 2) and OSU (serotype 5) it was determined that the OSU major neutralization glycoprotein antigen (VP7) was encoded by gene segment 9. A full-sized cloned cDNA copy of the OSU gene 9 was produced and sequenced. Hybridization of such labelled cDNA with the corresponding segment of a reassortant DS-1 X OSU virus confirmed the coding assignment. Comparison of the deduced amino acid sequence of the VP7 of OSU with those previously determined for five other rotavirus strains, representing four distinct serotypes, revealed some hydrophilic regions that exhibited significant homology and other hydrophilic domains with greater amino acid divergence. In one of the latter hydrophilic domains each of the five serotypes had a distinct amino acid substitution at residue 146, suggesting that it may be involved in serotype specificity.

The role of rotaviruses in pediatric diarrhea.

PIP: Although rotavirus has been recognized as the most common etiologic agent of gastroenteritis in infants requiring hospitalization, there are several important gaps in the understanding of rotavirus infection. Obstacles to such an understanding have included difficulties in cultivating the virus from human stools, the lack of a simple animal model to examine the immune response, and differences in the epidemiology of the infection in developed vs. developing countries. There is a strong need for community-based longitudinal studies of rotavirus infections over a period of time, especially in developing countries. This article summarizes current knowledge of the rotavirus structure, genetic variations, antigenic components, epidemiologic features, clinical aspects and treatment, and prevention of rotavirus diarrhea. The typical clinical picture of rotavirus gastroenteritis is indistinguishable from diarrheas of other etiologies; however, dehydration occurs more frequently in children with rotavirus infection. The enzyme-linked immunosorbent assay is generally considered the most efficient and simple method of identifying rotavirus in the stool. Several approaches to rotavirus vaccines are currently being tested, and among the vaccine candidates are strains of rotavirus naturally attenuated for humans, cold-adapted strains, and laboratory-derived reassortants. Also under exploration is the potential of genetic engineering techniques to achieve in vitro production of rotavirus antigen.^ieng

Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus: comparison with homologous genes from four strains of human and animal rotaviruses.

A full-size cloned cDNA copy of the rotavirus gene encoding the structural neutralization glycoprotein (VP7) of Nebraska calf diarrhea virus (NCDV), a strain recently shown to be effective as a vaccine in children, has been sequenced. Comparison of the deduced amino acid sequence of NCDV (serotype 6) VP7 with that of four other rotavirus strains (human WA serotype 1, human HU-5 serotype 2, simian SA-11 serotype 3, and bovine UK serotype 6) indicates that the degree of amino acid homology among VP7 neutralization proteins of these serotypes ranges from 75 to 86%. Four hydrophilic regions at amino acid residues 174-183, 248-256, 287-294, and 310-317 exhibit significant homology and hence may represent common antigenic determinants, while one hydrophilic area at amino acid residues 83-102 exhibits sufficient divergence to suggest it may be involved in serotype specificity.

Infection in piglets with a porcine rotavirus-like virus. Experimental inoculation and ultrastructural examination.

A rotavirus-like virus has been isolated from cases of neonatal diarrhoea in piglets. No antigenic relationship with known rotaviruses or with an American strain of a rotavirus-like virus has been demonstrated. Morphologically the virus is similar to known rotaviruses, but it differs in the ability to form syncytia of the enterocytes in the small intestine.

Rotavirus associated diarrhoea in nursing piglets and detection of antibody against rotavirus in colostrum, milk and serum.

A blocking method of ELISA for the detection and quantification of antibody against porcine rotavirus in serum, colostrum and milk has been compared with a plaque reduction test. The results obtained with the two techniques correlated (Fig. 1). Antibody against rotavirus was demonstrated in 384 serum samples representing 25 swine farms, indicating a widely spread and dense distribution of the infection with porcine rotavirus among Danish swine. The antibody contents in milk samples from 7 gilts and sows from 2 farms with a previously diagnosed problem with rotavirus associated diarrhoe showed a rapid decline during the first few days of the lactation periods (Fig. 1). An increase in the contents of rotavirus specific antibody was observed from day 15 in the milk samples from one of the gilts. The excretion of rotavirus with feces from the 7 litters of piglets were followed through the suckling period. Rotavirus war found in all litters but one and the virus was excreted in periods ranging from 4 to 9 days.

Congenital tremor type AI: light and electron microscopical observations on the spinal cords of affected piglets.

The histology and ultrastructure of the spinal white matter from the dorsolateral funiculus of the third cervical segment was studied in normal control pigs and pigs whose dams were inoculated with the Weybridge congenital tremor strain of swine fever virus in early pregnancy. Only inoculated sows produced abnormal piglets. These showed congenital tremors and ataxia. The severity of clinical signs was related to the degree of spinal myelin deficiency. Morphologically this was quantified by determination of the thickness of myelin investing axons classed according to their diameter. In clinically affected pigs fewer axons were myelinated than normal. Though the myelin sheath thickness increased with increasing axon diameter in all pigs whether clinically normal or not, the increase was less in moderately affected and much less in severely affected pigs. The deficiency of spinal myelin was probably due to delayed or sub-normal myelination accompanied by paranodal myelin abnormalities, myelin degeneration and remyelination.