Subchronic toxicity studies with the leukotriene D4 antagonist RG 12525.

Preclinical safety studies with the leukotriene D4 antagonist RG 12525 were conducted by the oral route in mice, rats, and monkeys. Oral administration of RG 12525 was repeated daily in studies up to 6 months in duration. RG 12525 was shown to have limited high-dose toxicity after repeated oral administration. The effects of RG 12525 were strongly dependent upon the species considered. High doses of RG 12525 caused significant increases in liver weight in mice, rats, and monkeys that were associated with diffuse hepatocellular hypertrophy in mice and rats but not in monkeys. No related clinical chemistry changes were observed in any of the species and hepatic activities of peroxisomal enzymes or cytochrome P450 were increased only slightly. Proliferation of brown adipose tissue (BAT) was observed in rats and mice but not in monkeys. The BAT reaction was more pronounced in the interscapular area but it was also observed in other subcutaneous locations as well as in mediastinal and bone marrow fat. In all locations, the RG 12525-induced BAT had some morphological similarities with cold-adapted BAT. Repeated administration of RG 12525 at high doses to female rats resulted in a lack of progression to the luteal phase of the estrous cycle that was reversible after discontinuation of treatment. Finally, RG 12525 was nephrotoxic in mice with males being more sensitive than females.

Proliferative pituitary lesions in rats treated with salmon or porcine calcitonin.

Calcitonin, the serum calcium-lowering hormone, has been used in the treatment of hypercalcemia of malignancy and postmenopausal osteoporosis in humans for several years without any adverse effects. Recent studies in rats have indicated that calcitonin may be associated with morphologic effects on the pituitary. A large study was performed on 2 strains of rats, Sprague-Dawley (SD) and Fischer-344 (F-344), with 2 types of calcitonin, salmon-derived (sCT) and porcine-derived (pCT) calcitonin to evaluate possible effects on the pituitary. Sixteen groups of 42 male and 42 female SD or F-344 rats were given 0 (vehicle control), 1.25, 5.0, or 80.0 IU/kg/day of sCT or pCT, once daily, subcutaneously, for 1 yr. An increased incidence of adenomas of the adenohypophysis was observed in male SD rats at all dose levels of sCT, female SD rats given 80 IU/kg/day of sCT, male SD rats at the high dose level of pCT, and male F-344 rats at the high dose level of sCT. Also, an increased incidence of total proliferative lesions, due mostly to an increased incidence of focal hyperplasia of the pars distalis, occurred in female F-344 rats given the high dose of sCT. These pituitary proliferations were histologically similar to those that occur spontaneously, and the incidences observed were comparable to those that could occur in rats on 2-yr or lifetime studies, indicating that the injection of calcitonin had decreased the latency period.

Glycoprotein hormone alpha-subunit-producing pituitary adenomas in rats treated for one year with calcitonin.

Calcitonin, a calcium-lowering hormone, has been associated with an increased incidence of nonfunctioning pituitary tumors in rats. In this study, rats were treated with calcitonin (80 IU/kg/d) for 52 weeks. After treatment with calcitonin, immunohistochemistry and in situ hybridization analyses demonstrated that most pituitary tumors expressed the glycoprotein hormone alpha-subunit. Expression of the alpha-subunit was identified rarely in hyperplastic lesions of control animals. Serum levels of GH, PRL, ACTH, LH, and FSH were unchanged in calcitonin-treated rats relative to controls. However, TSH levels were increased 2.1 fold after chronic treatment with calcitonin in both male and female rats (P less than 0.001). The level of glycoprotein hormone alpha-subunit was markedly increased (20-fold) in male rats with smaller elevations in female rats. Time course studies demonstrated that increases in serum alpha-subunit levels could be detected by 24 weeks of treatment and that elevations in alpha-subunit were present in the majority of animals by 40 weeks of treatment with calcitonin. The authors conclude that high doses of calcitonin, administered to rats for 6 months or longer, increases the incidence of alpha-subunit-producing pituitary tumors.

Spindle cell carcinoma of the conjunctiva. An immunohistochemical and ultrastructural study of six cases.

Six cases of conjunctival spindle cell carcinoma, a rare variant of squamous cell carcinoma, were studied. The median age of the three men and three women was 63.5 years. The tumors appeared as a single nodule in some patients or diffusely involved the conjunctiva in others. Two of the four individuals with intraocular extension presented with phthisis bulbi. Polyclonal antikeratin antibody was helpful and gave the most consistent results when compared with monoclonal antikeratin antibodies, AE1/3 and PKK1. The electron microscopic study of four lesions also established the epithelial nature of the tumor cells. Intracytoplasmic tonofilaments and a few desmosomes were present. Histopathologically, this variant of squamous cell carcinoma is difficult to distinguish from other spindle cell tumors, and this study demonstrates the value of immunohistochemistry and electron microscopy in supporting the correct diagnosis.

Establishment of cell lines from somite stage mouse embryos and expression of major histocompatibility class I genes in these cells.

To study the regulation of MHC class I gene expression during embryonic development, we have characterized a number of clonal cell lines derived from somite stage mouse embryos that were established with or without infection by several transforming retroviruses in combination with murine leukemia viruses. Unlike embryonal carcinoma (EC) cells that have been used as a model for early embryos, the cell lines derived from somite stage embryos are negative for stage specific embryonic Ag-1 and do not appear to differentiate after retinoic acid treatment. Morphology varies from clone to clone and is distinct from that of F9 and other EC cells. In agreement with previous findings in in vivo embryos, expression of surface MHC class I antigen in 57 new clones is either undetectable or low (with variability). All of the clones respond to the addition of interferons and express MHC class I antigens at high levels, but the kinetics of mRNA accumulation vary considerably. To examine the basis of the generally low or absent MHC class I gene expression in these cells, we tested promoter activity of a MHC class I gene by CAT assay after transient DNA transfection. Regardless of the basal levels of mRNA or surface Ag, CAT activity directed by various portions of the 5' flanking region of the MHC class I gene was uniformly low. The cells showed neither the negative nor the positive regulation of MHC class I genes that had been noted respectively for EC cells and for cells expressing the Ag constitutively. The pattern seen in the new cell lines suggests that there is an intermediate stage in the developmental regulation of MHC class I gene expression that may operate during the middle to late stage of fetal development.

Immunohistochemical localization of hepatitis B surface antigen and hepatitis B core antigen in tissue sections. A source of false positive staining.

The occurrence of apparent false positive immunoperoxidase staining for hepatitis B surface antigen (HBsAg) led to the evaluation of several commercial antisera for usefulness in the diagnosis of hepatitis B by immunohistochemistry. One commercial antibody to hepatitis B core antigen (HBcAg) was tested and found to give sensitive and specific staining, with only a few false negatives and no false positives. Of three antibodies to HBsAg, one gave good staining results that were consistent with serologic data; one had many false positive stains due to contaminating antibodies to plasma proteins; and one (a monoclonal antibody) had many false negatives, probably due to its restricted antigenic specificity. Diagnosticians should be aware of the problems with false positive and false negative immunohistochemical stains. False positives in particular can be a significant problem, causing frequent misdiagnosis of hepatitis B.

Spindle-cell carcinoma of the aerodigestive tract. An immunohistochemical analysis of 21 cases.

Immunohistochemical analysis of 21 prototypic mucosal spindle-cell carcinomas of the aerodigestive tract was performed at the Armed Forces Institute of Pathology (AFIP) to establish the usefulness of selected immunohistochemical markers in distinguishing spindle-cell carcinoma from other mucosal spindle-cell neoplasms. Immunoreactive keratin could be demonstrated in only 13/21 (62%) of cases. Coexpression of keratin and vimentin was demonstrated in 10/17 (59%) of the tumors evaluated for both of these intermediate filaments. All spindle-cell carcinomas lacked S100 protein, which is an immunoreactivity we would expect to find in spindle-cell malignant melanoma, one of the principal considerations in a differential diagnosis. Both alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT) were demonstrated in the tumor cells in all cases. However, albumin had a similar distribution in the tumors, which suggested that passive uptake was a serious confusing factor. The results of this study indicate that AAT and ACT are unreliable markers for distinguishing spindle-cell carcinomas from malignant fibrous histiocytomas.

Lymphoid interstitial pneumonia: clinicopathological and immunopathological findings in 18 cases.

Eighteen patients with lymphoid interstitial pneumonia (LIP) were studied. The diagnosis was established by the microscopic finding of interstitial infiltrates of lymphocytes and plasma cells. Forty-seven% of patients also had germinal centres, while 72% showed interstitial giant cells. Cases studied by the immunoperoxidase technique showed the interstitial plasma cells to be polytypic. The median age of patients was 56 years; most had cough, dyspnea, or chest pain. Chest X-rays showed either patchy interstitial infiltrates (usually bilateral) or poorly defined nodules. Ten patients had hypergammaglobulinemia; one had hypogammaglobulinemia. Two patients had Sjögren's syndrome, two had biopsy-proven chronic active hepatitis, and two had a clinical diagnosis of primary biliary cirrhosis. Follow-up examination of 14 patients showed clearing of symptoms, X-ray infiltrates or stable infiltrates in 4 cases each. Five patients died (mean survival, 41 months), one of whom succumbed to disseminated lymphoma and a second to respiratory failure. Our results support the hypothesis that LIP is a non-neoplastic cellular proliferation in which lymphoma may supervene. The high incidence (22%) of chronic liver disease has not previously been noted.

Kupffer cells in hepatocellular adenomas.

Hepatocellular adenomas are usually visualized as defects on technetium-99m-sulfur colloid liver scans, a fact which has been attributed to the absence of phagocytic Kupffer cells in the tumors. To determine whether this is true, seven hepatocellular adenomas were subjected to immunoperoxidase staining for lysozyme, a marker of mononuclear phagocytes. The Kupffer cells were counted in the tumors and surrounding non-neoplastic liver. All hepatocellular adenomas studied were found to contain Kupffer cells. Three tumors had fewer Kupffer cells than the surrounding liver. Three had about the same number as the surrounding liver, and one had more Kupffer cells than the non-neoplastic liver. Thus, the lack of phagocytosis of colloid in liver scans is probably due to something other than a deficiency of Kupffer cells in the hepatocellular adenomas.

Effect of thymosin immunostimulation with and without corticosteroid immunosuppression on chimpanzee hepatitis B carriers.

Carriers of hepatitis B surface antigen (HBsAg) are at a high personal risk of developing chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma, and they pose a potential health threat to others. Accordingly, erradication of the carrier state is an important therapeutic goal. Several categories of drugs have been evaluated for this purpose, with, at best, limited success. The immune stimulants constitute a drug group considered to have potential benefit, since altered cell-mediated immunity (CMI) appears to have a pathogenic role in the perpetuation of the carrier state. One such immune stimulant is the thymic hormone, thymosin, which is known to enhance suppressor T-cell activity. We therefore examined its possible therapeutic role by evaluating its effect on four chronic HBsAg- and hepatitis B e antigen (HBeAg)-positive chimpanzees. After baseline biochemical, serological, immunological, and histochemical studies were conducted, all four chimpanzees received parenteral thymosin for a period of 10-14 weeks; two of them were pretreated for 4 weeks with corticosteroids. All four were then reevaluated in the same manner at regular intervals during the 14-week period. Neither immunosuppression nor immunostimulation significantly affected biochemical, serological, or histological measures. Indices of CMI were altered, however: both T4 and T8 cells increased with thymosin treatment, although the T4/T8 ratio declined because of the relatively greater increase of the T8 than of the T4 cells. Thymosin did not affect the mitogen assays. Thus, while immunostimulation with thymosin did slightly alter CMI, it had no affect on the HBsAg carrier state or on measures of chronic hepatitis, even when preceded by corticosteroid immunosuppression.

Sarcomas and sarcomatoid neoplasms of the major salivary gland regions. A clinicopathologic and immunohistochemical study of 67 cases and review of the literature.

Sixty-seven cases of sarcomas and sarcomatoid neoplasms of the major salivary gland regions were studied in order to determine the clinical and histomorphologic features and biologic behavior. Fifty-seven of these proved to be sarcomas and the two most common types were malignant schwannoma (11) and fibrosarcoma (9). Nine sarcomas could not be subclassified morphologically. Ten cases, originally believed to be sarcomas, proved by means of immunohistochemistry to be either carcinomas (five cases) or melanomas (five cases). Fifty-nine of the 67 cases occurred in the parotid gland regions, and the remaining eight occurred in the submandibular regions. Twenty of the 67 cases were thought to arise from within the gland, nine from paraglandular tissues, and insufficient data was present to anatomically categorize the other 38 cases. The mean age of occurrence was 42 years for men and 38 years for women. A swelling was the presenting symptom in 64 cases, with a mean duration of 4.3 months. Pain, tenderness, or paralysis were noted in 17 cases, but the swelling was painless in seven cases. Follow-up data of 42 sarcoma patients revealed that 17 experienced recurrences, 16 developed metastases, and 15 died of disease. These rates were lowest among patients with tumors arising from within the gland (Group I) and highest among those patients with tumors of paraglandular origin (Group III). Mean survival time for those dying of disease was 2.4 years, and a 5-year survival time appeared to be a significant indicator of cure. The most successful therapy was either parotidectomy (superficial or total) or a combination of surgery and radiation. The morphologic and the immunohistochemical evidence suggest that the majority of the tumors represent true sarcomas that may arise from undifferentiated pluripotential cells, but that the remainder (15%) represent epithelial malignancies.

Lymphomatoid granulomatosis: a clinicopathologic study of 42 patients.

We studied the histological and clinicopathological findings in 42 patients who had lymphomatoid granulomatosis (LYG). In addition to small round lymphocytes, small to intermediate lymphocytes with serpentine nuclei, large immature mononuclear lymphoid cells, abundant histiocytes, and vascular invasion by the cell infiltrate were observed in all cases. Fifty percent of lesions had occasional "atypical" cells with multi-lobed nuclei. Three of four follow-up autopsies showed large cell lymphoma, while one other autopsy and the single repeat biopsy showed increased numbers of large immature mononuclear lymphoid cells. Patients were most frequently men 40-60 yr old who had a history of pulmonary symptoms, such as cough or chest pain, and who showed multiple bilateral lung nodules without hilar adenopathy in the chest x-ray. Thirteen patients (38%) died of disease, 11 of them within 12 mth of initial diagnosis. The presence of neurological signs and symptoms, increased mitoses, or increased numbers of atypical multi-nucleated cells in the initial biopsy were not statistically significant predictors of survival.

Estrogen receptor protein in bone and soft tissue tumors.

Thirty-three histologically diverse bone and soft tissue tumors were analyzed biochemically for the presence of estrogen receptor protein (ERP) and progesterone receptor by means of a conventional, commercially available, steroid-binding assay (dextran-coated charcoal method) on fresh frozen tissue. These results were compared with analysis of ERP by using a specific monoclonal antibody both in an enzyme immunoassay and on frozen tissue sections by using immunohistochemical procedures. Frozen tissue sections were also examined for the presence of estrogen and progesterone receptors using fluorescein-labeled steroids. Six of the 33 tumors (18%) contained low levels of ERP ranging from 19 to 73 fmol/mg as determined by the dextran-coated charcoal method. The remaining 27 cases contained no (less than 10 fmol/mg) ERP. The ERP-positive group included a fibromatosis, leiomyosarcoma, liposarcoma (2 cases), neural sarcoma, and a synovial sarcoma. Four were high grades sarcomas, and two were low grade sarcomas. There was excellent agreement between the ERP levels determined by the dextran coated charcoal method and those determined by enzyme immunoassay. ERP could not be demonstrated immunohistochemically on frozen tissue sections of the tumors even though it could be demonstrated in breast carcinomas serving as positive controls. The failure of the immunohistochemical technique may be related to the low levels of ERP in these tumors and the difficulty of detecting antigen at threshold levels. Cytochemical localization of receptor protein employing fluoresceinated steroids did not correlate with cytosolic ERP as determined by enzyme immunoassay or the dextran coated charcoal method. Moreover, the high level of background fluorescence gave rise to a significant amount of intraobserver and interobserver variation. Although the clinical significance of ERP protein in mesenchymal tumors is still uncertain, the present findings, coupled with various clinical observations suggesting hormonal dependency of some mesenchymal tumors, indicate that investigation of a larger group of patients amenable to statistical analysis is warranted.

Lung neoplasms in rodents after chronic administration of dimethyl hydrogen phosphite.

Dimethyl hydrogen phosphite (DMHP), an intermediate in the production of insecticides or herbicides, was administered by p.o. gavage for 2 yr to male Fischer 344/N rats and male and female B6C3F1 mice at doses of 0, 100, or 200 mg/kg and to female Fischer 344/N rats at doses of 0, 50 or 100 mg/kg. Dose related toxicity was seen in the lungs of treated male and female rats. The lung lesions were most prevalent in the high dose male rat group which received a dose twice that given to the high dose female rats. Lung lesions included alveolar epithelial hyperplasia, chemically related pneumonia, alveolar-bronchiolar adenoma, alveolar-bronchiolar carcinoma, and squamous cell carcinoma. DMHP also caused neoplastic and nonneoplastic lesions of the forestomach in male rats; a similar but less pronounced effect was observed in female rats. Nonneoplastic lesions associated with administration of DMHP included mineralization of the cerebellum in male rat and focal calcification of the testis in male mice. Under the conditions of this study, there was clear evidence for carcinogenicity for male rats, equivocal evidence for carcinogenicity in female rats, and no evidence for carcinogenicity in either male or female mice. DMHP caused the highest incidence of lung tumors in the male rat of all chemicals studied to date in the National Cancer Institute-National Toxicology Program Carcinogenesis Testing Program.

Coexpression of keratin and desmin in a carcinosarcoma involving the maxillary alveolar ridge.

Immunohistochemical determination of the class of intermediate filaments (keratins, vimentin, desmin, neurofilaments, and glial fibrillary acidic protein) expressed by tumors has become an important diagnostic procedure for the histogenetic classification of neoplasms. A case of a poorly differentiated malignant neoplasm of the maxillary alveolar ridge which coexpressed keratin and desmin types of intermediate filaments is reported. These intermediate filaments have been associated with epithelial and muscle cell differentiation, respectively. The morphologic features of the tumor plus the expression of both these intermediate filaments indicate that this neoplasm represents a true carcinosarcoma.

Pigmented dermatofibrosarcoma protuberans (Bednar tumor). A pathologic, ultrastructural, and immunohistochemical study.

Described by Bednar as a "storiform neurofibroma," pigmented dermatofibrosarcoma protuberans is a rare neoplasm accounting for approximately 1-5% of all cases of dermatofibrosarcoma protuberans (DFSP). The lesion commonly presents as an exophytic, multinodular neoplasm of the dermis or subcutaneous tissue. It occurs predominantly in blacks. The majority are located on the trunk, and the remainder are more or less equally distributed in the upper and the lower extremities and the head and neck. Microscopically the lesion is characterized by spindled cells arranged in a tight storiform pattern and admixed with a small population of melanin-containing dendritic cells. The dendritic cells are the primary feature distinguishing this lesion from conventional DFSP. Three cell populations are identifiable by electron microscopy. The majority of cells resemble fibroblasts. A second population of cells exhibits long slender cell processes partially or completely invested by basal lamina. The third population of cells, also invested by basal lamina, contains both melanosomes and premelanosomes. The histogenesis of this neoplasm remains controversial. Although Bednar considered these lesions as variants of neurofibroma, S-100 protein could not be identified, and this finding contrasts significantly from the description of conventional neurofibroma, which almost always contains this antigen. Follow-up information available in nine cases indicates that this lesion may recur locally. Although distant metastases were not observed in our material, complete excision in conjunction with close follow-up care is indicated for this neoplasm of probable intermediate malignant potential.

Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study.

Combined hepatocellular-cholangiocarcinoma is a rare form of primary liver cancer showing features of both hepatocellular and biliary epithelial differentiation. In a review of 24 cases of this tumor, three histologic types were encountered. Four cases were Type I or "collision tumors," apparently a coincidental occurrence of both hepatocellular carcinoma and cholangiocarcinoma in the same patient. Twelve cases were Type II or "transitional tumors," in which there were areas of intermediate differentiation and an identifiable transition between hepatocellular carcinoma and cholangiocarcinoma. Eight cases were Type III or "fibrolamellar tumors" which resembled the fibrolamellar variant of hepatocellular carcinoma but which also contained mucin-producing pseudoglands. Type III tumors differ from other combined tumors, occurring at a younger age, in the absence of cirrhosis, and having a slightly longer survival. Immunohistochemical (immunoperoxidase) staining for intracellular antigens showed that alpha-fetoprotein is a fairly specific, although insensitive, marker of hepatocellular differentiation in primary liver cancers, being present in 50% of typical hepatocellular carcinomas and in hepatocellular areas in 29% of combined tumors, but in no cholangiocarcinomas or cholangiocellular areas of combined tumors. Keratin is a good marker of biliary epithelial differentiation, being found in 90% of cholangiocarcinomas and in 52% of combined hepatocellular cholangiocarcinomas, but in no hepatocellular carcinomas. Alpha-1-antitrypsin, fibrinogen, IgG, and carcinoembryonic antigen may be found in both hepatocellular carcinoma, cholangiocarcinoma, and in combined tumors; these antigens are therefore of limited use in differential diagnosis.

A search for cytomegalovirus and herpes viral antigen in brains of schizophrenic patients.

The peroxidase-antiperoxidase method was employed to search for evidence of cytomegalovirus (CMV) or herpes simplex virus (HSV) antigen in the brains of 25 patients with a diagnosis of schizophrenia, 25 nonschizophrenic neuropsychiatric patients, and 16 nonpsychiatric control subjects. Brain specimens from patients with acute CMV and herpes encephalitis served as positive controls. Although early results with low-titer CMV antisera suggested immunoreactivity in specific brain regions of a small number of schizophrenic and control cases, the present studies with high-titer anti-CMV IgG did not give a positive immunoperoxidase reaction in sections from the basal forebrain, hypothalamus, or midbrain. Scattered neurons in the lateral vestibular nucleus and hippocampus showed questionable staining with CMV IgG in one schizophrenic patient and none in control subjects. No schizophrenic or control cases demonstrated an immune reaction to HSV antisera.

Keratin in epithelioid sarcoma. An immunohistochemical study.

Intermediate-sized filaments have been noted in epithelioid sarcoma by previous investigators, two of whom have reported that the filaments represent vimentin. We utilized polyclonal antibodies directed against keratin and immunoperoxidase techniques (PAP) to stain 32 of the more than 300 cases accumulated at the AFIP . All of our material was formalin-fixed, paraffin-embedded. Seventy-five percent of our cases (24/32) showed positive immunoreactivity, a feature that may be of diagnostic help in distinguishing epithelioid sarcoma from modular fasciitis, benign and malignant fibrous histiocytoma, malignant melanoma, and necrotizing granuloma. In these cases, the reaction was enhanced using predigestion with trypsin. The immunoreactivity varied from tumor to tumor, perhaps due to formalin fixation. Since synovial sarcoma and mesothelioma may also be cytokeratin-positive, our findings indicate that keratin immunoreactivity is not confined to epithelial tumors and may also occur in neoplasms traditionally regarded as mesenchymal.

Differentiation of vascular prostheses in dogs with serial tests of in vivo platelet reactivity.

Three different vascular prostheses (standard weight knitted Dacron, double velour knitted Dacron, and expanded polytetrafluoroethylene) were implanted in the aortas of dogs, and serial determinations of platelet survival and platelet serotonin were monitored at 12-week intervals for 1 year. Prostheses were then removed and luminal coverage with endothelialized neointima and production of prostacyclin were measured. Changes in platelet survival were correlated with changes in platelet serotonin, and both measurements reflected in vivo platelet reactivity with the vascular prostheses. These changes were unique for each type of prosthesis and were dependent upon physical characteristics and the rate and degree of coverage of the prosthetic surface with endothelialized neointima that produced prostacyclin. Prostheses that reduced platelet survival and platelet serotonin the least as shown by serial evaluation were found at harvest to be the most completely paved with nonthrombogenic neointima. In dogs, these techniques allow differentiation of vascular prostheses and provide a useful animal model for their evaluation.