Damselfish Neurofibromatosis Research Articles

Patterns of transcription of a virus-like agent in tumor and non-tumor tissues in bicolor damselfish

Damselfish neurofibromatosis (DNF) is a transmissible disease characterized by peripheral nerve sheath and pigment cell tumors which occurs in bicolor damselfish (Stegastes partitus) on Florida reefs. The damselfish virus-like agent (DVLA) is associated with the development of DNF and contains a 2.4 kb DNA genome which was found at high levels in tumors and tumor-derived cell lines and at lower levels in non-tumor tissues of both spontaneously diseased fish (TF) and fish with experimentally induced tumors (EF). An analysis of transcription patterns revealed up to five DVLA derived RNAs ranging in size from 300 to 1400 bp in these cell types. DNA was the most commonly distributed DVLA component in TF and EF followed by RNA. Prevalence of transcripts varied by tissue type. The smallest transcripts were the most common in all cell types and the most complete patterns, which included the larger transcripts, were observed primarily in tumors. The presence of viral RNAs in addition to DNA in non-tumor tissues suggested these tissues were infected by DVLA and indicated a wide tissue tropism for this agent. The high levels of DVLA nucleic acids found in tumors suggest that replication is occurring there. However, the potential for DVLA replication in other tissues where only a limited range of transcripts were present is not known. The mechanism of tumorigenesis by this agent is unknown. However, the association of the larger transcripts with most tumor tissues and their absence in most non-tumor tissues suggests that these RNAs may be involved in tumor formation.

Degranulation of eosinophilic granule cells in neurofibromas and gastrointestinal tract in the bicolor damselfish

Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish ( Stegastes partitus Poey) on Florida reefs. Previous studies have demonstrated high densities of eosinophilic granule containing cells (EGC), the proposed equivalent of mast cells in fishes, in neurofibromas and malignant peripheral nerve sheath tumors (mpnst) in DNF. These lesions are similar to those in the disease neurofibromatosis type 1 (NF1) in humans, which contain large numbers of mast cells. In the present study, experiments were conducted to measure the response of EGC in these tumors as well as in the submucosa of the digestive tract to the mast cell degranulating agent compound 48/80. Degranulation of these cells was visible by light microscopy and characterized by conspicuous swelling of granules and often by the presence of free granules adjacent to the EGC. Degranulation occurred by release of intact granules (diacytosis), as reported in other fishes, rather than by fusion of granules with the cell membrane (exocytosis) as reported in mast cells in mammals. Baseline levels of EGC exhibiting degranulation ranged from 20–26% in the submucosa to 30% in tumors. Within 1–2 h of exposure to compound 48/80, significant increases in average levels of degranulation were observed, to 67% in the gut and 72% in tumors. Degranulation was significantly more extensive in the tumors than in the gut. The outermost edges of the tumors contained significantly higher densities of EGC but these cells exhibited lower rates of degranulation than those in the inner regions of tumors. These observations support the hypothesis that the EGC present in neurofibromas and mpnst in DNF are equivalent to the mast cell component in neurofibromas in NF1.

A virus-like agent associated with neurofibromatosis in damselfish.

Damselfish neurofibromatosis (DNF) is a transmissible disease involving neurofibromas and chromatophoromas affecting bicolor damselfish Stegastes partitus on Florida reefs. Analysis of genomic DNA by Southern blotting techniques demonstrated the presence of a group of extrachromosomal DNAs in tumors from fish affected with DNF but not in healthy individuals. Cell lines obtained from tumors contained identical DNAs and were shown to be tumorigenic in vivo, while lines established from healthy fish did not contain such DNA and were not tumorigenic. These DNA patterns were also observed in experimentally induced tumors. A DNase resistant component of this DNA was isolated from both tumor cells and conditioned media of tumor cell lines suggesting that these sequences were encapsulated in viral particles. These data support the hypothesis that one or more of these extrachromosomal DNA forms is the genome of an unusual virus and that this virus is the etiologic agent of DNF. We have tentatively termed this agent the damselfish virus-like agent (DVLA).

Distribution of a novel infectious agent in healthy and diseased bicolor damselfish in Florida and the Caribbean

The disease damselfish neurofibromatosis (DNF) affects bicolor damselfish (Stegastes partitus) on reefs in Florida. This disease consists of peripheral nerve sheath and pigment cell tumors that are eventually fatal. The development of DNF is correlated with the appearance of unusual, extrachromosomal DNAs in affected fish that appear to be the genome of an undescribed, virus-like agent. Probes derived from these sequences were used to determine the distribution of this agent in diseased and healthy fish from several reefs in South Florida and from selected locations elsewhere in the range of this species. These analyses demonstrated that naturally diseased fish exhibited high levels of these DNAs in tumors and, to a lesser extent, in unaffected tissues. Fish with experimentally induced DNF exhibited similar levels of this DNA. Healthy adult fish had either no detectable levels of this DNA or very low levels, depending on the sensitivity of the detection technique used. Healthy adults from high disease prevalence reefs were more likely to test positive for this DNA than those from low disease reefs. Juvenile damselfish never contained detectable levels of this agent. Very low levels of this DNA were also detected in healthy fish from the Caribbean and the Bahamas. Dose-response experiments using tumor-derived cell lines indicated that tumor development was directly related to exposure dosages and that very low concentrations of this material did not yield tumor development. Taken together, these data indicate that high levels of this agent were correlated with the appearance of this disease and the very low levels often observed in healthy fish were not a predictor of tumor development.

Single K channel currents in Schwann cells from normal and neurofibromatosis‐affected damselfish

Damselfish neurofibromatosis is a naturally occurring disease of a tropical marine fish species. Affected fish exhibit peripheral nerve sheath tumors which contain morphologically abnormal Schwann cells (SC), similar to tumors encountered in the human disease neurofibromatosis type 1. Unitary A-type K channels in cell-attached membrane patches of SC were studied. Three different K channel conductances of approximately 5, 10, and 15 pS were present in both normal SC (n = 10) and tumored SC (n = 9). The variability in K channel conductance coincided with a large range of both mean open time and open probability in patches from normal and tumored SC. Channel open time histograms were fit by a single exponential. The ranges of time constants for open times irrespective of conductance were 0.26–9.3 msec in patches from normal cells and 0.60–0.73 msec in patches from tumored cells. These ranges were not significantly different. Inactivation time constants from ensemble averages of single channel currents averaged 83 ± 46 msec for normal SC and 44 ± 26 msec for tumored SC, which were not significantly different. These results suggest that A-type K currents from fish SC are composed of channels exhibiting multiple conductances and a variety of inactivation rates, which may account for the range of inactivation observed in whole cell currents but whose activity in membrane patches may not be wholly applicable to the whole cell currents. J. Neurosci. Res. 48:342–351, 1997. © 1997 Wiley-Liss, Inc.

Single K channel currents in Schwann cells from normal and neurofibromatosis-affected damselfish

Damselfish neurofibromatosis is a naturally occurring disease of a tropical marine fish species. Affected fish exhibit peripheral nerve sheath tumors which contain morphologically abnormal Schwann cells (SC), similar to tumors encountered in the human disease neurofibromatosis type 1. Unitary A-type K channels in cell-attached membrane patches of SC were studied. Three different K channel conductances of approximately 5, 10, and 15 pS were present in both normal SC (n = 10) and tumored SC (n = 9). The variability in K channel conductance coincided with a large range of both mean open time and open probability in patches from normal and tumored SC. Channel open time histograms were fit by a single exponential. The ranges of time constants for open times irrespective of conductance were 0.26-9.3 msec in patches from normal cells and 0.60-0.73 msec in patches from tumored cells. These ranges were not significantly different. Inactivation time constants from ensemble averages of single channel currents averaged 83 +/- 46 msec for normal SC and 44 +/- 26 msec for tumored SC, which were not significantly different. These results suggest that A-type K currents from fish SC are composed of channels exhibiting multiple conductances and a variety of inactivation rates, which may account for the range of inactivation observed in whole cell currents but whose activity in membrane patches may not be wholly applicable to the whole cell currents.

Damselfish with neurofibromatosis exhibit cytotoxicity towards retrovirus infected cells

Lymphocytes from tumor-bearing damselfish are cytotoxic towards target cell lines derived from damselfish neurofibromatosis. These cell lines contain at least one retrovirus which appears to be related to the etiology of the disease. The current studies were designed to characterize the effectors of this cytotoxic reaction. Data presented here show that cells separated using an antibody (5C6.10.4) directed towards non-specific cytotoxic cells of catfish sequesters all antitumor activity in the 5C6.10.4 negative population. Thus, damselfish 5C6.10.4 positive cells bind to tumor targets, but do not contribute to target cell death. In contrast, SC6.10.4 positive cells are cytotoxic towards xenogeneic erythrocytes. Cytotoxicity of splenocytes from animals inoculated with virus purified from the 88–503 cell line suggested that prior exposure to the retrovirus enhanced reactivity, especially towards 88–503. In addition, cytotoxicity was significantly greater in tumor homogenate injected animals that resisted tumor development for more than 5 months as compared to those that developed tumors quickly. Lastly, cytotoxic responsiveness towards primary cultures of mock and virus infected autologous and allogeneic cells implies that the cytotoxic effector is directed towards retrovirus infected cells.

A retrovirus isolated from cell lines derived from neurofibromas in bicolor damselfish (Pomacentrus partitus)

Damselfish neurofibromatosis (DNF) is a naturally occurring, neoplastic disease affecting bicolor damselfish (Pomacentrus partitus) living on coral reefs in southern Florida, USA. The disease consists of multiple neurofibromas, neurofibrosarcomas and chromatophoromas and has been proposed as an animal model for neurofibromatosis type 1 in humans. DNF is transmissible by injection of crude tumour homogenates, cell-free filtrates of homogenates or cells from tumour cell lines. An analysis of tumorigenic cell lines derived from fish with spontaneous or experimentally induced DNF revealed virus particles budding from cells and present in conditioned media. The 90-110 nm particles resembled type C retroviruses. This virus exhibited a buoyant density of 1.14-1.17 g/cm2 in sucrose, at least six virus proteins of 15 to 80 kDa and reverse transcriptase (RT) activity. RT activity was maximized with a poly(rC).oligo(dG) template.primer combination and Mn2+ at a concentration of 0.5-1.0 mM. The optimum temperature for RT was determined to be 20 degrees C, a finding consistent with the ambient temperatures encountered by this species. This retrovirus, tentatively named damselfish neurofibromatosis virus (DNFV) may be the aetiological agent of DNF. Whether DNFV or another, as yet unidentified, virus is the cause of DNF, this agent may be unique in virus oncogenesis; neoplastic transformation of the cell types involved in DNF, Schwann cells and chromatophores, has not been documented in any other transmissible tumour.

Melanophores in Damselfish Neurofibromatosis: Alterations in Morphology and Melanosome Aggregation Responses

Abstract Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish Pornacentrus partitus on south Florida reefs. This disease exhibits many traits in common with neurofibromatosis type-1 in humans, including multiple plexiform neurofibromas and areas of hyperpigmentation. An important issue in understanding the pathogenesis of DNF is the relation between chromatophores and other cell types that give rise to tumors in this disease. Melanophores cultured from tumors of fish with DNF were compared in vitro with cells from normal skin of healthy fish to determine if these cells were morphologically or physiologically altered in this disease. Melanophores from tumors were significantly larger in cross-sectional perimeter and area than normal counterparts. Pigment translocation responses were measured by bath application of norepinephrine. At drug concentrations of 5 × 10-9 to 5 × 10-5 M, significantly fewer tumorderived cells than normal cells exhibited complete melanosome aggrega...

Experimental induction of neurofibromatosis in bicolor damselfish

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 23:201-212 (1995) - doi:10.3354/dao023201 Experimental induction of neurofibromatosis in bicolor damselfish Schmale MC Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting the peripheral nervous system and chromatophores of bicolor damselfish Pomacentrus partitus on Florida (USA) reefs. This report presents the results of transmission experiments using intramuscular and intraperitoneal injection of a variety of tumor preparations to induce DNF. Neurofibromas developed within 1 yr of injection in 76% of fish injected with homogenates of spontaneously occurring tumors with earliest tumor development occurring at 5 to 6 wk post-injection. Cell-free transmission using 0.2 um filtrates of DNF tumor homogenates was observed, demonstrating that the agent responsible for DNF is subcellular. Four out of five tumor cell lines established from neurofibromas were found to be tumorigenic when injected, while cells cultured from normal fish had no effect, indicating that the etiologic agent of DNF was present and infectious in tumor cell lines but not in cell lines or primary cultures derived from healthy fish. Serial passages of tumors were conducted through 19 passage generations in vivo. A comparison of the cumulative time to tumor distributions showed that most experiments beyond the third serial passage yielded significantly faster tumor development than was obtained by passaging from spontaneous tumors. Induced tumors exhibited more localized growth patterns and were typically more rapidly growing and more rapidly lethal than spontaneous tumors. In contrast to the differences in growth patterns observed, the histological phenotype of the spontaneous, wild-type DNF lesions was indistinguishable from that of the induced tumors obtained from serial passages as well as in experiments involving cell-free filtrates and cultured tumor cells. These observations indicate that the process of neoplastic transformation is relatively stable in this system, with little or no phenotypic drift caused by these manipulations. Naturally occurring, transmissible cancers that arise from cells of the central or peripheral nervous system or from chromatophores have not been previously identified. Thus, DNF is a unique disease and may provide an important model for investigating this rather unique form of viral oncogenesis. Tumor . Neurofibromatosis . Damselfish . Transmission Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 23, No. 3. Publication date: November 23, 1995 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1995 Inter-Research.

Characterization of Schwann cells from normal nerves and from neurofibromas in the bicolour damselfish

Schwann cells are an important component of neurofibromas, one of the primary lesions encountered in neurofibromatosis type 1 in man. A central question in studies of neurofibromatosis type 1 has been whether the Schwann cells present in these tumours are intrinsically abnormal or exhibit abnormal phenotypes in response to stimuli from other cell types in these tumours. Damselfish neurofibromatosis is a naturally occurring disease in a species of marine fish, the bicolour damselfish, that is being developed as an animal model of neurofibromatosis type 1. Affected fish exhibit multiple neurofibromas and neurofibrosarcomas (malignant schwannomas). The present study compares the morphology, antigen expression and proliferative capacity in vitro of Schwann cells derived from peripheral nerves of normal, healthy fish with cells isolated from both spontaneously occurring and experimentally induced neurofibromas. Schwann cells from normal nerves expressed S100 antigens but not fibronectin or glial fibrillary acidic protein antigens and were similar in morphology and proliferative capacity to Schwann cells isolated from mammalian peripheral nerves. Tumour-derived cultures contained variable proportions (27-79%) of S100-positive cells that were identified as Schwann cells based on this feature. These tumour-derived Schwann cells exhibited a different morphology than normal Schwann cells, usually exhibited an increased reactivity to anti-S100 antibodies and were able to proliferate in vitro without added mitogens. Repeated subculturing of tumour-derived cultures led to the production of six cell lines all of which were composed exclusively of Schwann cells as indicated by S100 expression. These findings show that Schwann cells are an important component of tumours in Damselfish neurofibromatosis and that these cells are morphologically and physiologically altered in this disease. Observations of cell lines also suggest that tumour-derived Schwann cells are intrinsically abnormal and that this phenotype is not a result of stimuli from other cell types in the tumours.

Damselfish with neurofibromatosis exhibit cytotoxicity toward tumor targets

Damselfish neurofibromatosis (DNF) is a malignant transmissible disease affecting Schwann cells, and is the only naturally occurring animal model of human neurofibromatosis type 1. The current study was designed to determine whether fish in the early stages of disease have measurable immune responses toward DNF tumor cells. Three DNF tumor cell lines were used as targets in standard 51Cr cytotoxic assays. In addition, Lutjanus griseus erythrocytes served as nonspecific targets, and concanavalon A (Con A) blasts from healthy animals served as normal target cells. Results of this study show that tumor-bearing damselfish have cells capable of destroying tumor targets but healthy animals display minimal, if any, reactivity toward the DNF tumor lines. The majority of antitumor activity resides in the spleen; the pronephros appears to contain the majority of nonspecific activity. Data also show that some of the effector cells are analogous to the nonspecific cytotoxic cells of catfish. No lysis of healthy damselfish targets was observed. Thus damselfish have cytotoxic cells capable of interacting with tumor targets, but in the majority of animals this response is not adequate to circumvent the process of tumorigenesis.

Differences in a K current in Schwann cells from normal and neurofibromatosis-infected damselfish.

Patch clamp techniques were used to study whole cell ionic currents in Schwann cells (SC) from a tropical marine fish, the bicolor damselfish, Pomacentrus partitus. The bicolor damselfish is affected by a disease termed damselfish neurofibromatosis (DNF), being developed as an animal model of neurofibromatosis-type 1 (NF1) in humans. NF1 affects SC, fibroblasts, and perineurial cells. The sole depolarization-activated ionic current present in cultured SC from normal fish peripheral nerve and from neurofibromas of fish with induced or spontaneously occurring DNF was an inactivating K+ current (K current), with a strong dependence on the Nernst potential for K+. This K current activated at depolarizations to -40 mV and above and inactivated during a maintained test pulse (0.2-1 s), but inactivation was significantly greater in tumored SC. Both currents were inhibited by 4-aminopyridine (Kd approximately 1 mM) and by dendrotoxin (15 microM) but were insensitive to extracellular tetraethyammonium (< or = 150 mM), indicating that the whole cell currents were similar pharmacologically. The currents could be distinguished on the basis of their sensitivity to depolarized holding potential, with normal cells less sensitive. Half-inactivation of the current was -32 mV in normal cells and -38 mV in tumored cells. Inactivation curves constructed from the average normalized current for many SC were significantly different in normal and tumored cells. When the depolarized holding potential was maintained between test depolarizations, greater voltage-dependent inactivation in tumored cells was apparent. Normal cells maintained an average of 36% of peak current at a holding voltage of -40 mV, while in tumored cells this average was 12%, a significant difference.

Prevalence and distribution patterns of tumors in bicolor damselfish (Pomacentrus partitus) on South Florida reefs

Damselfish neurofibromatosis (DNF) is a cancer affecting bicolor damselfish (Pomacentrus partitus) on reefs in South Florida, USA. Bicolor damselfish exhibiting DNF were observed on 18 of 19 reefs surveyed, with prevalence rates varying from 0.4 to 23.8% of the adult fish in these populations. These rates were very stable over 5 to 9 yr periods (1981 to 1989) on the two reefs monitored. High disease prevalence was associated with a high population density of large size-class fish and a population structure dominated by large fish. These patterns were observed both between and within reef populations. The distribution of cases within reefs was clustered rather than uniform or random. These findings are consistent with the hypothesis that DNF is distributed primarily via fish to fish (horizontal) transmission of an infectious agent. Disease patterns are not consistent with what would be expected to result from inherited (or vertically transmitted) or water-borne agents.

Immune responses in the bicolor damselfish, Pomacentrus partitus, and their potential role in the development of neurogenic tumours

Bicolor damselfish on southern Florida reefs are commonly affected by a disease consisting of Schwann cell tumours. Several features of this disease, termed damselfish neurofibromatosis (DNF), indicate that the immune system of these fish may play an important role in the expression and rate of progression of the tumours. The majority of fish in the advanced stages of DNF showed abnormal, presumably degenerative, histological changes in both the spleen and kidney, suggesting that a general, non‐specific suppression of immune responses might be occurring. This hypothesis was supported by results of mitogen‐induced proliferation and mixed leucocyte responses in fish with DNF which indicated a near total failure of several functions of the immune system involving the pronephros and the spleen. A sparse infiltrate of granulocytes and monocyte/macrophages was seen in these tumours.