Delayed foreign-body tumorigenesis in mice infected with lactate dehydrogenase-elevating virus: Brief communication.

Female and male CBA/H mice were infected with lactate dehydrogenase virus (LDV). Two weeks later, these mice and noninfected controls received double sc implants of unplasticized vinyl chloride-vinyl acetate copolymer films (0.2 X 15 X 22 mm). Foreign-body (FB) tumorigenesis was delayed in LDV-infected females and males by 2 months. This result could not be explained by an effect of LDV on cellular immunity, inasmuch as cellular immunity does not influence the course of FB tumorigenesis.

Carcinogenesis from polymer implants: new aspects from chromosomal and transplantation studies during premalignancy.

Inbred CBA/H and CBA/H-T6 mice received implants of 15 X 22 mm plastic films. Plastic inserts and tissue capsules were cut in thirds at half monthly and monthly intervals. The first portion of the inserts and capsules was left in the original animal. The second portion was separated and individually transplanted into recipients that differed from the original animals with respect to the T6 marker chromosome. The third portion and all tumors which developed in original and recipient animals were examined by karyological, histological, and cultural methods. Film pieces caused tumors in recipient animals up to 9 months after transfer, capsule tissue only up to about 1 month after transfer. Tumors in original and corresponding recipient animals were identical in their chromosomal stemlines and pace of premalignant maturation. The karyotype of the stemline was never discovered among the film-attached cell population because there seemed to be no cell division. This points to the existence of a single, specific premalignant cell clone residing on the film surface in a dormant state of nondivision many months before tumor appearance. At the end, the (pre)malignant cells detached from the film, invaded the capsule tissue, and propagated to produce the tumor within about 4 weeks. The existence of a specific inhibition phenomenon during the premalignant phase is suggested.

Foreign-body tumorigenesis by vinyl chloride vinyl acetate copolymer: no evidence for chemical cocarcinogenesis.

We investigated whether vinyl chloride monomers, released from implants of vinyl chloride vinyl acetate copolymer (VCA), exerted cocarcinogenic activity and added thereby to the mechanism of foreign-body (FB) tumorigenesis. CBA/H and CBA/H-T6 mice were used. No evidence was found to indicate that chemical carcinogenic activity partakes in tumorigenesis by VCA implants. Hence it was concluded that VCA plastic is not suitable for the study of the combined process of FB/chemical cocarcinogenesis. Furthermore, experimental results obtained with VCA film implants were representative of FB tumorigenesis in the absence of demonstrable chemical carcinogenic activity.

Foreign-body tumorigenesis: in vitro isolation and expansion of preneoplastic clonal cell populations.

Foreign-body reactions were induced in coisogenic CBA/H and CBA/H-T6 mice by sc implantation of 15 times 22 times 0.2-mm unplasticized vinyl chloride vinyl acetate copolymer films. At 6 months' post implantation, implants and unopened tissue capsules were transferred to recipient animals of the T6-different partner strain. After another 3 months, part of the film/capsule complex was transferred to (C57BL/10ScSn times CBA/H-T6)F1 mice for tumor development. Capsule-derived and film-attached cells of the other part were separately cultured. Cultures consisting initially of euploid cells were often gradually replaced by different cells with specific aneuploid karyotypes which were identical with, or closely related to, those of the corresponding tumors. The cultured cells implanted in hybrid recipients at different passage numbers frequently gave rise to homologous tumors. Hence, it was possible to prepare in vitro cells with prefixed specific tumor determinants at different stages of preneoplastic maturation.

Foreign-body tumorigenesis induced by glass and smooth and rough plastic. Comparative study of preneoplastic events.

Foreign-body (FB) tumorigenesis was induced in female CBH/H and CBA/H-T6 mice and their hybrids by sc implantation of about 0.2-mm thick, large (660-720 mm2) or small (210-400 mm2) pieces of glass, smooth-surfaced plastic, or roughened plastic (rigid unplasticized vinyl chloride vinyl acetate copolymer). The tumorigenic process was analyzed in the various implantation groups by the evaluation of tumor incidences and latencies, and by the determination of 1) frequency of originator ("parent") cells, 2) appearance of preneoplastic cells in FB-reactive capsule tissue, 3) expansion of preneoplastic cell clones throughout the tissue capsule, and 4) pace of cellular preneoplastic maturation in terms of time remaining until neoplastic autonomy. Established methods included transfer of preneoplastic FB-reactive tissue capsules to recipient animals (hybrids of CBA/H and CBA/Br or C57BL/10ScSn). Specific preneoplastic events or stages of FB tumorigenesis were affected differently, depending on the size, material, and surface properties of implants.

Foreign-body tumors of mice: strain and sex differences in latency and incidence.

Sarcomas were induced by sc implantation of unplasticized polyvinylchloride acetate films in female and male mice of strains AKR/J, BALB/cJ, BALB/cWat, CBA/H and CBA/H-T6, C3H/HeJ, C57BL/10ScSn, C57BL/6J-bgj, C57BL/cdJ, DBA/-1J l/LnJ, LP/J, SJL/J, X/Gf, 129/J, and hybrids (CBA/H-T6 X AKR/J)F1, (C57BL/10ScSn x CBA/H or CBA/H-T6)F1, (C57BL/6J-bgj x C57BL/6J)F1. The strains and sexes showed marked differences in incidence and mean latency of resulting tumors. Crucial information was provided for the selection of appropriate mouse strains for the study of interrelationships between genotypes, defined somatic properties, and the multifactorial process of foreign-body tumorigenesis.

Foreign-body tumorigenesis in mice: DNA synthesis in surface-attached cells during preneoplasia.

(CBA/H X CBA/H-T6)F1 mice were given sc implants of unplasticized vinyl chloride acetate 15 X 22-mm copolymer films. The animals were pulsed with [3H]thymidine at various times during the 15 months following implantation. DNA synthesis occurred in the film-attached cell population, predominantly macrophages, throughout the preneoplastic phase in both females and males. Giant cells with fewer than 10 nuclei were labeled synchronously and asynchronously. No DNA synthesis was detected in giant cells with more than 10 nuclei. Previous studies have shown that phagocytic inactivity and ultrastructural signs of functional dormancy are characteristic for the foreign-body-reactive macrophage. However, this investigation demonstrated that the macrophage was not dormant with respect to DNA synthesis.

Multiphasic incidence of foreign body-induced sarcomas.

Single or multiple plastic films (unplasticized vinyl chloride vinyl acetate copolymer) of different sizes and shapes were implanted s.c. in female CBA/H and CBA/H-T6 mice. Tumor incidence increased and accelerated with increased total surface area of multiple implants or with increased size of single implants. Tumor distribution curves over time were generally multiphasic. The profiles changed in proportionate relation to implant size. These findings indicate class differences between tumors according to latency. Since latency is known to be a predetermining characteristic of foreign body-induced tumors, class differences seem to exist at the originator cell level, reflecting diversity of intrinsic carcinogenic factors.

Etiological factors, stages, and the role of the foreign body in foreign body tumorigenesis: a review.

Attempts were made to analyze the process of foreign body (FB) tumorigenesis and to identify etiologically significant factors by correlating information in the literature and recent experimental data from our labroatory. It appears that the process of FB tumorigenesis is dependent on sequence of specific conditions as expressed by the following criteria: (a) cellular proliferation and tissue infiltration during acute FB reaction; (b) fibrosis of the tissue capsule surrounding the FB; (c) quiescence of the tissue reaction, i. e., dormancy and phagocytic inactivity of FB-attached macrophages; and (d) availability of a FB surface for direct contact with clonal preneoplastic cells. There is no indication that the initial acquisition of neoplastic potential and the determination of specific tumor characteristics are based on direct physical or chemical reaction between cells and the FB. These etiological key events occur presumably in mesenchymal stem cells associated with the microvasculature no later than during the acute stage of FB reaction and certainly long before clonal descendants of these cells are first found in contact with the FB surface. In fact, there is no reason to assume that cells with neoplastic determination may be present in normal tissue prior to the introduction of a FB and that the FB would only create the conditions required for stepwise preneoplastic maturation.

Localization of a 11.5 kDa Zn(2+)-binding protein (parathymosin) in different rat tissues. Cell type-specific distribution between cytosolic and nuclear compartment.

The distribution of a small Zn(2+)-binding protein (11.5 kDa ZnBP), which we have shown to be identical with parathymosin, was studied in various rat tissues by immunocytochemistry, immunoblot analysis and quantified by enzyme-linked immunosorbent assay (ELISA), using monospecific polyclonal antibodies. The content in liver was 105 micrograms/g wet weight. Similar amounts were found in brain, adrenal gland and smooth muscle, whereas in testis, spleen, lung, and kidney about half the amount was detected. Very low levels were found in skeletal muscle (2 micrograms/g) and adipose tissue, while erythrocytes did not contain measurable amounts. The specificity of the antibodies was established by immunoblotting. Purified 11.5 kDa ZnBP as well as 11.5 kDa ZnBP detected in crude soluble fractions from various tissues appeared always as a doublet of protein bands of about equal intensity, indicative for two isoforms of the 11.5 kDa ZnBP. By immunocytochemistry, in brain, high concentrations of 11.5 kDa ZnBP were found in the deep cerebellar nuclei, in soma and dendrites of Purkinje cells, and in the large neurons of the pons/medulla. In most cell types reacting with the antibody, exclusively the cytoplasm was stained. In contrast, in duodenal and jejunal crypt cells immunostaining was restricted to the nuclei, whereas the more mature cells at the top of the villi contained most of the antigen in the cytosol. Immunostaining of the nuclei was also observed in pancreatic duct cells, whereas in the duct cells of the parotid gland immunostaining was detected exclusively in the cytoplasm. In both tissues immunostaining of the acinar cells was negative.

Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria.

A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed. The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand. After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed. The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria. With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed. For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined. All strains colonized the root tips when inoculated as single strains. When inoculated in competition with the efficient root colonizer P. fluorescens strain WCS365, many strains were outcompeted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also. The gnotobiotic system was used to screen a collection of 300 random P. fluorescens WCS365::Tn5 mutants for colonization-impaired mutants. Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS. One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain. The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor. As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.

Metabolite-controlled phosphorylation of hepatic phosphofructokinase proceeds by cAMP-dependent protein kinase.

In hepatocytes 32P-incorporation into rat liver phosphofructokinase is stimulated by glucose as well as by glucagon, the effects of both stimuli being prevented by L-alanine [Eur. J. Biochem. (1982) 122, 175]. The phosphopeptides of the enzyme derived from limited proteolysis by subtilisin and from exhaustive tryptic digestion were analyzed either by one-dimensional mapping on sodium dodecyl sulphate-polyacrylamide slab gels and by fingerprint mapping, respectively. It is shown that in vivo stimulation of 32P-incorporation by glucose or by glucose plus glucagon results in identical phosphopeptide maps, and that these maps were identical with those obtained from phosphofructokinase phosphorylated in vitro with catalytic subunit of cAMP-dependent protein kinase. It is concluded that in the intact liver cell phosphofructokinase is phosphorylated by cAMP-dependent protein kinase but that the state of phosphorylation is modified by metabolite control.

The primary sequence of the PFK-1 inactivating zinc-binding protein as deduced from cDNA sequencing. Identity of the zinc-binding protein with rat parathymosin.

We have recently described the sequence of the Zn2+-binding domain (43 amino acid residues) of a newly detected Zn2+-binding protein which reversibly inactivates phosphofructokinase-1 in a Zn2+-dependent manner [(1986) J. Biol. Chem. 269, 5895-5900; (1988) Eur. J. Biochem. 177, 561-568]. Here, we describe the primary sequence of this protein based on a full-length cDNA. A sequence comparison reveals the identity of the Zn2+-binding protein with a protein called parathymosin-alpha.

Rapid changes in intracellular Zn2+ in rat hepatocytes.

Changes in the concentration of free Zn2+ were monitored in isolated rat hepatocytes using the fluorescent indicator zinquin (ethyl[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetat e). The concentration of Zn2+ in freshly isolated hepatocytes was 1.3 x 10(-6) M (range 0.61-2.7 x 10[-6] M). This value decreased by about 10%-15% during incubation in the absence of zinc and increased in a time- and concentration-dependent manner in the presence of exogenous zinc (Km approximately 10 microM). IIb group metal ions led to a concentration-dependent increase in zinquin fluorescence. The rank of efficacy was Hg approximately Cd > Pb (IVa) >> Cu (Ib) >>> Ni (VIII). This rank resembles their ability to mobilize zinc from metallothioneins. 8-Br-3',5'-cAMP (10[-4]M) caused a rapid decrease in Zn2+ epifluorescence which was apparent within 10 min and was sustained throughout the experiment. This effect was gradually obliterated in the presence of external ZnCl2. The effect was specific for cAMP (or cAMP generating hormones) as the calcium-dependent hormone [arg8]vasopressin (5 x 10[-8] M) did not affect intracellular Zn2+. An integrated role of zinc as a possible mediator in signal transduction is discussed.

Expression of MAGE, gp100 and tyrosinase genes in uveal melanoma cell lines.

In order to determine the possible use of uveal melanoma cell lines as stimulators in immunotherapy, we evaluated the expression of the human genes for MAGE-1, -2 and -3, gp100 and tyrosinase in uveal melanoma cell lines. mRNA expression of the MAGE-1, -2 and -3, gp100 and tyrosinase genes and the HLA class I specificity were determined in five primary and three metastatic uveal melanoma cell lines. Expression of the examined genes was heterogeneous in the primary and metastatic cell lines. The cell lines OCM-1 and OMM-1 expressed MAGE-1, -2 and -3, whereas EOM-3, MEL202, 92-1 and OMM-3 were negative for these antigens. gp100 was expressed in all cell lines, and tyrosinase in all but three (EOM-29, OMM-2 and OMM-3). Except for EOM-3, the HLA-A type of all the cell lines could be determined by complement-dependent microlymphocytotoxicity assay. Since at least two melanoma-associated antigens can be found in uveal melanoma cell lines, as well as the HLA class I molecules, these cell lines may be applicable as immunogens for specific immunotherapy against metastatic uveal melanoma.

Packed bed column fermenter and kinetic modeling for upgrading the nutritional quality of coffee husk in solid-state fermentation.

Studies were carried out to evaluate solid-state fermentation (SSF) for the upgradation of the nutritional quality of coffee husk by degrading the caffeine and tannins present in it. SSF was carried out by Aspergillus niger LPBx in a glass column fermenter using factorial design experiments and surface response methodology to optimize bioprocess parameters such as the substrate pH and moisture content and aeration rate. The first factorial design showed that the moisture content of the substrate and aeration rate were significant factors for the degradation of toxic compounds, which was confirmed by the second factorial design too. The kinetic study showed that the degradation of toxic compounds was related to the development of the mold and its respiration and also to the consumption of the reducing sugars present in coffee husk. From the values obtained experimentally for the oxygen uptake rate and CO(2) evolved, the system determined a biomass yield (Y(x/o)) of 3.811 (g of biomass).(g of consumed O(2))(-1) and a maintenance coefficient (m) of 0.0031 (g of consumed O(2)).(g biomass of biomass)(-1).h(-1). The best results on the degradation of caffeine (90%) and tannins (57%) were achieved when SSF was carried out with a 30 mL.min(-1) aeration rate using coffee husk having a 55% initial moisture content. The inoculation rate did not affect the metabolization of the toxic compounds by the fungal culture. After SSF, the protein content of the husk was increased to 10.6%, which was more than double that of the unfermented husk (5.2%).

Breast imaging in women under 35 with symptomatic breast disease.

With the increasing utilization of mammography, young women under the age of 35 are being referred for mammographic examination more frequently. A review of the mammograms of 159 consecutive patients aged under 35 was conducted to evaluate the clinical value of the examination in the age group for whom the probability of malignancy is low. 74% of patients referred had no discrete palpable mass and presented predominantly with lumpy or tender breasts, the remaining 24% had a discrete palpable mass. In neither group did radiographic examination beneficially influence clinical management. We propose protocol where no imaging is performed in women under 35 in the absence of a palpable mass unless there is a localized bloody discharge or a strong family history or previous personal history of breast cancer. In patients with a palpable mass, ultrasound should be performed initially to identify simple cysts and if negative only then progressing to mammography.

Early detection of relapse after treatment for metastatic germ cell tumour of the testis: an exercise in medical audit.

The relapse patterns of 29 patients who recurred following treatment for metastatic germ cell tumours of the testis (seminoma n = 7, non-seminomatous germ cell tumour n = 22) have been analysed and the relative effectiveness of clinical follow-up and routine investigations in detecting relapse at an early stage have been examined. The analysis shows that routine estimation of the serum tumour markers human chorionic gonadotrophin and alpha-foetoprotein (HCG and AFP) is the single most important follow-up procedure. This is so, even in patients who were previously marker negative; it was the first indicator of relapse in 55% of the patients. Regular clinical examination and chest radiograph in asymptomatic patients was of little value. Chest radiograph gave the first evidence of relapse in only 2 cases (7%). The optimum frequency for follow-up computed tomographic scanning of the chest and abdomen remains debatable. In this series, it was the first abnormal investigation in 7 patients (24%) and proved to be particularly important in patients who had residual radiological abnormalities at the end of initial therapy. Cost analysis shows that intensive follow-up produces a total expenditure on investigations of approximately 4,500 pounds per relapse detected. Regular computed tomographic scanning is especially demanding on resources and costs approximately 12,880 pounds per relapse detected if the recommended protocol is followed.

Risk assessment of carcinogenesis at implantation sites.

Of the 98 foreign-body or scar-related cancers reported in the literature, over 25 percent have developed within 15 years, and over 50 percent within 25 years. Substantial numbers of various implants have now been in place for 10 to 20 years. Since at least 25 percent of cancer cases should already have occurred, the low number actually observed permits the prediction that the incidence of cancers at implantation sites will remain low. This conclusion is supported by studies on 27 specimens of chronic foreign-body reactions against a variety of implants that had been in situ for 1 to 19 years. Employing a cell-culture technique previously developed for experimental mice, an attempt was made to identify specific precancer cells in the tissues. None were detected, in contrast to foreign-body reactions of mice, in which the incidence of foreign-body tumors is high.

Long-term follow up of trans-sphenoidal hypophysectomy for Cushing's disease.

Fourteen patients with Cushing's disease treated by trans-sphenoidal hypophysectomy between 1962 and 1975 were reviewed in 1983. Complete ablation had been attempted. There were no surgical deaths and one episode of bacterial meningitis. Two patients required a second operation for a cerebrospinal fluid leak. There have been three late deaths from unrelated causes. All patients had a biochemical remission of their Cushing's disease postoperatively and no relapse has been recorded. Most patients need some hormone replacement but residual pituitary function and sella radiography have remained stable. This treatment seems satisfactory and the evidence implies a pituitary aetiology of the syndrome.