Mycobacteria, but not mercury, induces metallothionein (MT) protein in striped bass, Morone saxitilis, phagocytes, while both stimuli induce MT in channel catfish, Ictalurus punctatus, phagocytes.

Recent advances in molecular immunology indicate that the expression of inducible pro-inflammatory proteins is increased in vertebrates in response to both infectious disease agents and various xenobiotics. For example, iNOS, COX-2, and CYP1A are induced by both inflammation and AhR ligands. Moreover, the expression of these proteins in response to stimuli varies among individuals within populations. Little is known of the differences among fish in the inducibility of proinflammatory proteins in response to both infectious agents and xenobiotics. Through random screening of a striped bass, Morone saxitilis, peritoneal macrophage cDNA library, a full length metallothionein (MT) gene was cloned and sequenced. MT is a low-molecular weight (6-8 kDa), cysteine-rich metal binding protein. Metals are required by pathogenic bacteria for growth, and by the host defense system by serving as a catalyst for the generation of reactive oxygen intermediates (ROIs) by phagocytes. A recombinant striped bass MT (rMT) was expressed and purified, then used to generate a specific mAb (MT-16). MT protein expression was followed in freshly isolated striped bass and channel catfish, Ictalurus punctatus, phagocytes after in vitro exposure to the naturally occurring intracellular pathogen Mycobacteria fortuitum or to 0.1 and 1 microM mercury (Hg), as HgCl(2). MT expression was increased by 24 h in both channel catfish and striped bass phagocytes as a result of exposure to M. fortuitum cells. On the other hand, MT was induced by Hg in channel catfish cells, but not those of striped bass. These results indicate that metal homeostasis in phagocytes is different between catfish and striped bass. In addition, these data suggest that care should be taken to distinguish between inflammation-induced vs. metal-induced MT when using MT expression as a biomarker of metal exposure.

Expression of a hepatocyte growth-factor activator protein in turkey (Meleagris gallopavo) deferent duct epithelial cells.

In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease. Our experimental objective was to identify the gene that encodes this protein. A lambda phage cDNA library from duct cell mRNA was constructed. The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease. Phage containing the protease cDNA was excised and re-circularized into plasmids. E. coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing. NCBI BLAST searches within the GenBank database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively. The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns. Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease. Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct. HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor. Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor.

Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry.

Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nalpha-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.

Purification of vitellin from grass shrimp Palaemonetes pugio, generation of monoclonal antibodies, and validation for the detection of lipovitellin in Crustacea.

Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68-85 kD and two high molecular mass bands in the range of 190-221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii ) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.

Survivability and immune responses after challenge with Edwardsiella ictaluri in susceptible and resistant families of channel catfish, Ictalurus punctatus.

Diseases in catfish farming are prevalent and costly, particularly the bacterial disease Enteric Septicemia of Catfish. Considerable research has focused on different aspects of this disease, including the biology of the causative agent, Edwardsiella ictaluri. However, no satisfactory treatment or preventive has resulted from these efforts. One solution is to increase the natural disease resistance of the fish through genetic selection. Recent research has demonstrated that genetic factors influence resistance to infection in mammals as well as fish. Selective breeding for disease resistance in channel catfish is ongoing, however differences in defence mechanisms among E. ictaluri challenged strains and families are only now being investigated. Antigen-specific as well as non-specific immune responses of full-sib families of channel catfish to laboratory challenge with E. ictaluri have been investigated. Both resistant and sensitive families produce a humoral response as specific antibody, but there were no differences found in the level of specific antibody produced. The sensitive family produced a slightly higher percentage of B lymphocytes in mononuclear cell preparations from peripheral blood, while the resistant family had a higher percentage of T lymphocytes in those preparations. The most significant observation was that the resistant family produced more macrophage aggregations in the spleen and posterior kidney throughout the infection than the sensitive family. Neither family produced stress-associated amounts of cortisol.

Macrophage secretory function is enhanced by low doses of tributyltin-oxide (TBTO), but not tributyltin-chloride (TBTCl).

Numerous studies suggest that tributyltin (TBT) is a potent immunotoxicant in nontarget organisms with lymphoid atrophy being a hallmark response. Two of the most common formulations of TBT are bis (tri-n-butyl)-tin oxide (TBTO) and tri-n-butyl-tin chloride (TBTCl). Most of studies investigating TBT-related immunotoxicity have used relatively high doses of both compounds, but little is known about the effects of very low doses. In addition, no studies have directly compared the effects of both formulations on immune function(s). We exposed female B6C3F1 mice to a single dose of TBTO or TBTCl at 0.3, 3.0, 30 mM/kg or corn oil as a carrier control. Forty-eight h later mice received a 4% solution of thioglycolate intraperitoneally to elicit peritoneal macrophages. Ninety-six h later macrophages were harvested and stimulated with a mixture of gamma-interferon (IFN-gamma) and lipopolysaccharide (LPS). Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1), and phorbol ester-stimulated oxidative burst activity were then measured. Nitric oxide and TNF-alpha production were significantly elevated in the 0.3 and 3.0 mM TBTO/kg-treated groups but not in those treated by TBTCl. Background TNF-alpha production (without stimulation) was also elevated at these two doses but suppressed in TBTCl-treated animals. Oxidative burst activity was elevated at 0.3 mM TBTO/kg but not by TBTCl. TGF-beta1 production was not altered by either treatment, nor were body wts and organ-body wt ratios. To further evaluate the difference between the effects of TBTO and TBTCl on macrophage function, the in vitro toxicity of the two was determined using elicited peritoneal macrophages from untreated mice. Following a 24-h exposure to increasing concentrations of TBTO or TBTCl, functional viability was evaluated using the MTT assay. There were no differences between the two compounds in terms of treatment-related viability except that at the very highest concentrations (10(-6) M) TBTO was more toxic than TBTCl.

Adoptive immunotherapy of malignant glioma using tumor-sensitized T lymphocytes.

Malignant glioma remains one disease for which there is no curative therapy. Clearly there is a need to explore new and innovative approaches for their treatment. In this report, we review our preclinical trial of a new adoptive immunotherapy protocol using cytotoxic T lymphocytes (CTL) which had been sensitized to glioma in vivo and then activated and their number expanded ex vivo using compounds which enhance signal transduction. These glioma-sensitized lymphocytes, when introduced systemically into rats with either an intracerebral or intradermal glioma, eradicated or slowed the progression of their tumor. These results indicate for the first time that a reproducible and sustained eradication of a malignant glioma could be achieved by the adoptive transfer of tumor-sensitized, ex vivo expanded CTL. A Phase I clinical trial is now underway to test the safety and potential efficacy of this immunotherapy in patients with recurrent malignant glioma.

Ex vivo expansion of tumor-draining lymph node cells using compounds which activate intracellular signal transduction. I. Characterization and in vivo anti-tumor activity of glioma-sensitized lymphocytes.

It has been shown that adoptive immunotherapy can be curative for established malignant tumors. The key to this treatment lies in obtaining sufficient numbers of lymphocytes which are sensitized to recognize tumor antigens and carry out immunological reactions to destroy tumor cells. Reported here are the results of experiments to: 1) sensitize lymphocytes to the antigens of rat glioma cells and expand them ex vivo for use in adoptive immunotherapy, 2) characterize the cells of the expanded population, and 3) evaluate antitumor activity in a cohort of rats with well-established intracranial gliomas. Viable RT-2 glioma cells were injected into the hind foot pads of syngeneic Fischer 344 rats. After 10 days, the tumor draining lymph nodes (DLN) were harvested from the injected limbs and mechanically dissociated. The cells of the DLN were then suspended in culture medium supplemented with low dose interleukin-2 (IL-2) and incubated for 18 hours with Bryostatin-1 and ionomycin (Bryo/Io) to stimulate expansion. The cells were next washed to remove the Bryo/Io and resuspended in culture medium and IL-2. Population expansions of 40- to 100-fold were seen after 8 days. Flow cytometric analysis showed these cells to be a nearly pure population of T lymphocytes of the CD3+CD8+ phenotype. Intravenous injection of the ex vivo expanded DLN cells did not significantly improve survival of rats with a seven-day intracerebral RT-2 glioma, although, compared to untreated controls, the tumors of the treated animals were smaller, showed no necrosis, and appeared to be less infiltrative. Furthermore, the treated animals had a pronounced lymphocytic infiltration of their tumors with greater associated degrees of hemorrhagic change and peritumoral edema. When the ex vivo expanded DLN cells were intravenously injected into three-day intracerebral RT-2 glioma models, tumors were almost always eliminated and the animals survived their tumor challenge. We conclude that successful expansion of glioma-sensitized DLN lymphocytes is possible and that adoptive immunotherapy using these cells is capable of effectively limiting the progression of large gliomas, while totally eradicating small ones.

Ex vivo expansion of tumor-draining lymph node cells using compounds which activate intracellular signal transduction. II. Cytokine production and in vivo efficacy of glioma-sensitized lymphocytes.

We have investigated the anti-tumor activity of ex vivo activated and expanded T cells which had been sensitized in vivo to one of two different syngeneic rat glioma cell lines; D74 or RT-2. Rats were sensitized by inoculation of irradiated tumor cells into each hind foot pad. After 10 days, the tumor-draining lymph node (DLN) from each popliteal region was excised and prepared as a single cell suspension. Tumor-DLN lymphocytes were next activated overnight in RPMI-1640 medium containing 10% fetal bovine serum (FBS), Bryostatin-1 (5 nM), ionomycin (1 microM), and 20 U human recombinant interleukin-2 (IL-2) per ml. Culture for seven days in RPMI-1640 supplemented with FBS and IL-2 resulted in approximately 100-fold expansion of the lymphocyte population. Both D74- and RT-2-sensitized T cells constitutively secreted tumor necrosis factor-alpha, and both lymphocyte populations produced comparable amounts of the cytokine when co-cultured with either glioma cell line. Neither D74- and RT-2-sensitized effectors constitutively secreted gamma-interferon (gamma-IFN), but both populations produced gamma-IFN when exposed to either glioma cell line in vitro. D74-sensitized T cells released significantly more gamma-IFN than the RT-2 DLN lymphocytes. In vitro Chromium-release assays indicated that RT-2-sensitized T cells were more cytotoxic for RT-2 targets than for the D74 line and that D74-sensitized effectors were also more cytotoxic for RT-2 targets. To assess in vivo therapeutic efficacy, rats who had been inoculated intradermally with RT-2 cells three days earlier received an intravenous injection of RT-2- or D74-sensitized DLN cells (10(6) cells/gram body weight) expanded after activation with Bryostatin-1 and ionomycin or an equal number of lymphokine-activated killer (LAK) cells. Tumor diameters were measured daily and revealed that injection of glioma-sensitized lymphocytes led to the elimination of tumor while treatment with LAK cells had no therapeutic benefit. These results indicate, that at least for these two glioma lines, gamma-IFN release, rather than in vitro cytotoxicity, was a better predictor for in vivo immunotherapeutic efficacy of the glioma-sensitized, expanded T cells.

Innate immune function as a bioindicator of pollution stress in fish.

Immunotoxicological studies, based on processing of samples in the field and laboratory, were conducted on fish collected from a stream receiving point-source contaminants near its headwaters. Previous studies in this stream have revealed that cytochrome P4501A activity, liver somatic indices, macrophage aggregates, and parasitic liver lesions are significantly elevated in sunfish with the degree of impact decreasing with distance from the contaminant source. Fish collected from each sampling site were equally divided, One group was sacrificed in the field and the spleen and anterior kidney tissues were removed and placed in buffer on ice. The other group was kept in MS-222 for 2 hr and transported to the laboratory for processing. The spleen and anterior kidney from each fish were then prepared as a single cell suspension and shipped overnight to Mississippi State University. Cells were then evaluated for PMA-stimulated phagocyte oxidative burst and non-specific cytotoxic cell (NCC) activity against K562 tumor targets. Oxidative burst responses were dramatically suppressed in both groups at sampling sites near the headwaters but returned to reference levels further downstream. There were no differences between processing strategies at each station. NCC activities did not follow gradient-response patterns observed with phagocyte oxidative burst data and there were inconsistent differences between processing strategies at each site. These data indicate that simple immune function assays, such as phagocyte oxidative burst responses, can be used as a ancillary bioindicator in fish health monitoring and that immune function in these fish can be reliably assessed even if samples are not immediately processed.

Delayed tarsal eversion following periorbital trauma.

Delayed onset of upper lid edema with exuberant chemosis developed in a 3-year-old girl following blunt periorbital trauma. Examination under anesthesia demonstrated a tightly everted upper tarsus that focally compressed the underlying conjunctiva at the superior tarsal border. Injection of subconjunctival hyaluronidase followed by local compression and temporary tarsorrhaphy resulted in rapid resolution of the chemosis and restoration of the normal lid position.

The effects of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on glioma-specific cytotoxic T-cell activity in Fischer 344 rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent environmental immunomodulating agents identified so far. Historically, mice have been used to model mammalian immunobiology and most of the data gathered on the immunotoxicity of TCDD has been obtained from studies with mice. However, rats have been used more extensively in toxicological research to establish human risk assessment criteria. A need exists, therefore, to develop a database using the rat model in immunotoxicology so that complete animal toxicity studies can be conducted. We have treated female Fischer 344 rats with a single i.p. dose of 0.3, 3.0, or 30.0 micrograms/kg TCDD or corn oil vehicle and examined cytotoxic T-cell (CTL) activities 24 days following treatment. Syngeneic in vivo tumor-specific CTLs were generated that model cell-mediated immune reactions against neoplastically transformed self antigens. RT2, a virally-induced Fischer 344 rat glioma, and D74, a ethylnitrosurea-induced Fischer 344 rat glioma were used as targets. This immunological parameter was compared to body, thymic, and liver weights as well as liver ethoxyresorufin deethylase (EROD) activity on day 24 post-TCDD treatment. The results indicate that Fischer 344 rats are very sensitive to TCDD as indicated by severe thymic atrophy and EROD induction at all three doses. In contrast, CTL activity was only marginally affected by these same doses of TCDD with only a modest suppression noted at the highest dose. These results indicate that the CTL response in rats may not be useful in characterizing the effects of this xenobiotic on immunocompetence in the rat.

Intraoral vesicles occurring after alginate impressions.

In 47 of 227 dental students intraoral vesicles developed after multiple alginate impressions. The lesions were generally solitary and clear, and appeared within 24 to 48 hours after the impression. They were most frequently located inside the vermilion border of the lips and resolved spontaneously in 2 to 5 days. The purpose of this study was to determine the cause of these reactions. Histopathologically one lesion was suggestive of a contact allergy. Cutaneous patch tests, which proved negative, were performed on 14 students to determine whether an allergy to the alginate flavoring existed. The surface of three lesions were cultured and the organisms identified. Contamination studies were carried out on seven unopened containers of the alginate powder and resulted in the isolation of some organisms similar to the mucosal cultures; however, no relationship can be proved. These findings indicate that the cause of the vesicles remains unknown, and further studies are necessary to establish the cause.

Systemic treatment with murine recombinant interleukin-1 beta inhibits the growth and progression of malignant glioma in the rat.

We investigated the effects of daily subcutaneous (SC) injections of 100, 200, or 400 micrograms/kg murine recombinant interleukin-1 beta (rIL-1 beta) or its excipient on normal Fischer 344 rats and ones harboring a malignant RT-2 glioma. The tumor model has a predictable course with animals dying on days 14-17 following an intracerebral inoculation of 10(4) RT-2 glioma cells. Treatments with rIL-1 beta or excipient began on day seven post-tumor inoculation and continued for 7 days. We observed no significant effect on core body temperatures although there was a significant (p less than 0.05) decrease in body weight in all rIL-1 beta treated animals. When tumor-bearing animals became moribund, they received an intraperitoneal injection of bromodeoxyuridine (BUdr) and were sacrificed two hours later. Blood samples were obtained prior to their sacrifice by transcardiac perfusion with a buffered aldehyde solution. Recombinant IL-1 beta affected blood differentials; causing neutrophilia, lymphopenia, and slight thrombocythemia. The BUdr labeling index of glioma cells did not significantly differ between treatment groups, although tumors differed histologically at the time of necropsy. Tumors of rIL-1 beta treated animals had more extensive necrosis and a greater degree of leukocyte infiltration. Survival studies were conducted in which rats were given continuous daily SC injections of rIL-1 beta until day of death. Overall survival between the two groups differed significantly in studies using 100 micrograms/kg/d (p less than 0.05); rIL-1 beta treated rats had a mean survival time of 22 (+/- 3.0) days while excipient controls had a mean survival time of 17 (+/- 0.5) days. Similarly, at a dose of 200 micrograms rIL-1 beta/kg/d, mean survival was significantly (p less than 0.05) increased as compared to excipient controls (18.75 +/- 1.5 vs. 15.25 +/- 1.7 days, respectively). Daily injections of 400 micrograms/kg did not significantly increase the survival of glioma bearing animals, possibly as a consequence of rIL-1 beta toxicity at this dose.

Orbital intramuscular schwannoma.

In an 8-year-old girl with asymptomatic proptosis, computed tomographic scans showed a large medial orbital mass that contoured the globe anteriorly, bowed the optic nerve laterally, and extended posteriorly to the orbital apex. T1-weighted coronal magnetic resonance images showed the mass to be a diffusely enlarged medial rectus muscle. Histopathologic examination of a medial rectus muscle biopsy specimen disclosed a multinodular, intramuscular schwannoma, separating and infiltrating normal skeletal muscle fibers. The intramuscular location and multinodular configuration of this tumor, together with its occurrence in a child, distinguish it from previous orbital schwannomas.

Aerobic bacterial contamination in dental materials.

Current concern about disease transmission points out the need for better infection control in dentistry. The purpose of this study was to test samples of dental materials in factory-sealed containers for aerobic bacterial contamination. Multiple unopened containers of 12 different dental materials were obtained from the dental school dispensary. Samples were removed from each container and incubated at 38 degrees C in standard broth medium for 1 week. Those that exhibited visual signs of possible bacterial growth were subjected to a Gram stain for verification. The results of that test indicated that 20% to 30% of the samples of alginate, glass ionomer cement and base powders, and retraction cord contained bacterial contamination. The remaining eight dental materials exhibited no apparent bacterial growth. Thus viable aerobic organisms were found in samples from 4 of 12 dental material products.

Primary orbital melanoma associated with orbital melanocytosis.

We report a case of primary orbital melanoma in a 17-year-old girl. The patient presented with painless proptosis during the first trimester of pregnancy. Computed tomography demonstrated a well-circumscribed mass located infra-temporally in the right orbit. The tumor was bluish-black, grossly encapsulated, and associated with orbital blue nevi. Histologic examination of the mass revealed a pigmented spindle-cell neoplasm. On electron microscopy, the presence of premelanosomes and the absence of basal lamina supported the diagnosis of melanoma. Malignant transformation of a preexisting nevus is postulated since perineural foci of benign dendritic melanocytes were seen within the melanoma. There has been no recurrence or metastasis in a 2-year follow-up. Of 30 primary orbital melanomas reviewed, 12 (40%) were associated with periorbital pigmentary disorders, such as oculodermal melanocytosis, blue nevus, and ocular melanocytosis. Our case is unique since the pigmentary lesions were limited to the orbital tissues.

Ocular and systemic findings in the Aarskog (facial-digital-genital) syndrome.

The Aarskog (facial-digital-genital) syndrome is an X-linked disorder in which short stature is accompanied by hypertelorism, digital anomalies, and shawl scrotum. Except for hypertelorism and blepharoptosis, ophthalmic abnormalities have been rarely noted in this condition. We examined four patients who had Aarskog syndrome and unilaterally or bilaterally decreased vision on initial examination. Three family members had V-pattern esotropia, latent nystagmus, inferior oblique overaction, and amblyopia. A fourth patient had bilateral blepharoptosis and severe astigmatism. Other ocular features included hyperopia, anisometropia, deficient ocular elevation, blue sclerae, and posterior embryotoxon. These findings underscore the need for ophthalmic examination in asymptomatic patients with Aarskog syndrome to rule out treatable causes of visual loss.