Reticulum cell neoplasms induced in C57BL/6 mice by cultured virus grown in stromal hematopoietic cell lines.

Thirty-one adherent cell lines have been established from the spleens, lymph nodes, and bone marrow of C57BL/6 mice carrying radiation leukemia virus (Duplan isolate)-induced reticulum cell neoplasms (RCN). The cell lines had a stable epithelial or fibroblastoid morphology, Supernatant virus from these lines induced splenic and lymph node RCN in 100% of inoculated C57BL/6 mice within 30 days. The disease was generalized and involved many organs. The monolayer cells themselves were not tumor cells and induced RCN through infection of the host with RCN virus. Simultaneous inoculation of in vitro-grown RCN-inducing virus any thymic lymphosarcoma virus induced each disease independently with unaltered incidence, latency period, and organ involvement; no mutual enhancement or inhibition was found, thus two separate mechanisms of action were indicated. Reextraction of the viruses from spleen, lymph nodes, and thymus gland indicated the specific organotropism of each agent. All the adherent cell lines that were derived from hematopoietic tissues produced ample, potent RCN-inducing virus. This high success rate suggests that in the hematopoietic organs the stromal, fibroblastoid cells are a natural habitat for the RCN-inducing virus. The RCN-inducing virus species may well be synthesized in these hematopoietic stromal cells. RCN-inducing virus from culture supernatants contained high-titer infectious ecotropic and xenotropic virus that was titrated. The cultures are being used to clone the RCN-inducing virus and to establish the virologic and molecular properties that endow it with specific RCN-inducing capacity.

Comparative long-term study of the toxicities of free and liposome-associated doxorubicin in mice after intravenous administration.

The toxicities of free doxorubicin (F-DOX) and liposome-associated doxorubicin (L-DOX) were investigated in inbred BALB/c and outbred Sabra mice treated iv with 5, 7.5, and 10 mg doxorubicin (DOX)/kg body weight every 2 weeks up to 8 injections and observed for 6 months. Sonicated liposomes containing phosphatidylcholine, phosphatidylglycerol, and cholesterol were used. The lethal effect was reduced in mice treated with L-DOX as compared to mice treated with F-DOX. At a dose of 7.5 mg DOX/kg, 100% of mice receiving the L-DOX survived a cumulative dose of 60 mg/kg administered over 98 days, while 92% of mice receiving the F-DOX died. Two distinct patterns of death were observed: an acute phase type occurring early after injection of high doses of DOX and apparently related to gastrointestinal toxicity and a delayed phase type requiring a long latency after initial drug exposure and characterized by a complex pattern of abnormalities. Delivery of DOX by liposomes effectively protected against both types of lethal effects. Reduced toxicity of L-DOX resulted in reduced body and organ weight losses, reduced severity of pathologic changes, and fewer blood biochemical alterations. The pathological damage to the heart muscle found in mice treated with L-DOX was less severe than with F-DOX, and in some cases it was reversible. Nephrotoxicity was extremely frequent and severe among F-DOX-treated mice, while it was totally insignificant among L-DOX-treated mice. Hyperlipidemia, hypoglycemia, and glycogen-depleted hepatocytes were characteristic findings in mice treated with F-DOX. Altogether, the data obtained in this study indicate that liposomes significantly diminish the toxicity of DOX with the use of an intermittent schedule of chemotherapy. In addition to changes in tissue distribution as a mechanism of reduced toxicity, it is proposed that DOX associated with liposomal lipids interacts less efficiently than the free drug with target intracellular phospholipids.

Studies on leukemia development in the SJL/J strain of mice.

Spontaneous reticulum cell neoplasms developed in 70-80% of intact male and female SJL/J mice at a mean age of 380 days. The neoplasms had a basic histological pattern of a multicellular type-B reticulum cell neoplasm, as designated by Dunn. The tumors were transplanted in isogenic mice. Serial passages of cell suspensions of these tumors were carried on through at least 4 tumor-inducing generations, and the growing transplanted tumors maintained the structure of a reticulum cell neoplasm similar to the original tumor. A histological survey of spontaneous lesions in SJL/J mice revealed different characteristics of reticulum cell neoplasms, some similar to Hodgkin's lesions. Five or ten feedings of 7,12-dimethylbenz-[alpha]anthracene (DMBA) in polyethylene glycol 400 (1 mg DMBA per feeding) to SJL/J mice induced lymphosarcomas in 74-83% of the mice at a mean age of 157-188 days. A single feeding of DMBA induced lymphosarcomas in only 27% of the mice at a mean age of 246 days. These DMBA-induced lymphatic leukemias do not appear to depend on the thymus for development--a 60% incidence of lymphosarcomas was obtained in adult thymectomized mice treated with DMBA, and 27% of the thymectomized DMBA-treated mice developed myeloid leukemia at a mean age of 180 days. Urethan was only slightly leukemogenic in SJL/J mice.

Enhancement and retardation of spontaneous reticulum cell neoplasm development in SJL/J mice.

The enhancement or delay in the appearance of spontaneous reticulum cell neoplasms (RCN-B) in SJL/J mice was tested. Immunosuppressive treatment with antithymocyte serum and prolonged antigenic stimulation by repeated injections of sheep erythrocytes, leukemogenic cell-free centrifugates, and multichain synthetic polypeptides had no effect on the incidence or latency of spontaneous tumor appearance. Treatment with mineral oil or incomplete Freund adjuvant markedly enhanced tumor appearance (though the abundance of plasma cells induced by these treatments did not evoke the development of plasmacytomas, nor did it affect the incidence of serum paraproteinemia among mice with spontaneous tumors). Treatment with cortisone acetate, however, markedly retarded spontaneous tumor development. Tumors occurring early due to mineral oil and adjuvant treatment all had the characteristics of "RCN-B plasma cell type," and tumors occurring late due to cortisone treatment were all of the "RCN-B reticulum cell type." The possible involvement of the stem cell pool size in the enhancement or delay of spontaneous tumor development was discussed.

Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy.

In mice, 4-nitrobenzylidene malononitrile (AG1714), which belongs to the tyrphostin family, reduced toxicity induced by doxorubicin and cisplatin without impairing their antitumor efficacy. AG1714 reduced mortality induced by doxorubicin and cisplatin. It prevented, in a dose-dependent manner, cisplatin-induced nephrotoxicity as assessed by measurement of serum creatinine and blood urea nitrogen levels. The protective effect of AG1714 was most pronounced on its administration 2 h before cisplatin. AG1714 also prevented doxorubicin-induced myelosuppression as assessed by the scoring of bone marrow nucleated cells and colony-forming units. Cisplatin-induced small intestinal injury was also protected by AG1714 as assessed by histopathological analysis. In vitro, AG1714 reduced cisplatin-induced apoptosis in a murine fibroblastic cell line (A9) and did not affect doxorubicin-induced apoptosis of B-16 melanoma cells. In contrast to its protective effect against mortality and injury of normal tissues induced by chemotherapy, AG1714 did not impair its antitumor activity and in some tumor models enhanced it. This was evident by using the murine tumors B-16 melanoma, Lewis lung carcinoma, and methylcholanthrene-induced fibrosarcoma and the human tumors SK-28 melanoma and human ovary carcinoma xenografts in nude mice. Experiments in which low and high doses of cisplatin and doxorubicin were administered to tumor-bearing mice demonstrated that AG1714 reduced mortality of high-dose chemotherapy and increased its therapeutic index. AG1714 could provide a novel, useful tool to improve chemotherapy by allowing dose intensification.

Enhancement of adriamycin delivery to liver metastatic cells with increased tumoricidal effect using liposomes as drug carriers.

We have investigated the tissue distribution of liposome-entrapped Adriamycin (ADM) in mice with metastatic spread to the liver and spleen after inoculation of J-6456 lymphoma cells. Sonicated phosphatidylserine:phosphatidylcholine:cholesterol liposomes were used as carriers of ADM, based on previous studies on the drug entrapment, stability, and tissue distribution of ADM-containing liposomes of various compositions (A. Gabizon, A. Dagan, D. Goren, Y. Barenholz, and Z. Fuks. Cancer Res., 42: 4734-4739, 1982). Increased hepatic and splenic levels of ADM were found in tumor-bearing mice when the drug was injected in the liposome-entrapped form. Concomitantly, decreased cardiac uptake of ADM was observed in tumor-bearing mice treated with liposome-entrapped ADM. In order to measure the concentration of ADM directly in metastatic cells, J-6456 lymphoma cells were isolated from the liver by Percoll density gradients. It was found that the ADM levels were significantly augmented in tumor cells from mice given injections of liposome-entrapped ADM as compared to those given injections of free ADM at all time intervals checked after drug injection. In addition, the in vitro and in vivo growth ability of these isolated metastatic cells was significantly more impaired when they were obtained from mice receiving liposome-entrapped ADM as compared to mice which received free ADM. The histopathological damage to the normal liver parenchyma of mice treated with liposome-entrapped ADM was mild and confined to discrete foci and was not significantly different from that observed in mice treated with free ADM. These results indicate that liposome delivery may provide an efficient means of improving the therapeutic efficiency of ADM in certain forms of metastatic liver disease, while diminishing the potential hazard of cardiotoxicity.

Thermal sensitivity and thermotolerance in normal porcine tissues.

We have previously presented a histopathological grading scheme for thermal damage in normal porcine adipose and skeletal muscle tissues. Here we have used this scheme to assess the heat sensitivity of these tissues, and evaluate the protective benefit of thermotolerance as induced by a prior thermal exposure. Tissues were exposed to temperatures ranging from 40-50 degrees for 30 min. Half of all sites also received a thermal exposure of 41.0-43.0 degrees 4 hr earlier. Biopsies for histological evaluation were obtained at 18 to 24 hr ("acute") and at 28 to 31 days ("chronic") following treatment. Only mild acute injury was seen in the early samples, following either single or double heat exposures, at all temperature levels. Minimal chronic damage was also seen in the late samples following single exposures of 45 degrees or less. Higher single exposures caused important chronic lesions, the severity of which was dose dependent. Regions that had received the earlier conditioning thermal exposure showed a significant protection against the subsequent thermal exposure. In such regions, mean (chronic) pathology scores were reduced by 76 to 86% over the temperature range 45-48 degrees. The degree of acute damage failed to predict the degree of chronic damage. Overall, induction of thermotolerance provided an advantage of 2 degrees or more in normal tissue protection.

The effect of exposure to diazepam through the placenta or through the mother's milk. Histological findings in slices of rat brain.

Exposure to diazepam during the prenatal or early postnatal developmental period has been reported to result in later behavioural deficits. In the present study morphological changes in the brains of rats that were exposed to diazepam (DZP) prenatally or through the mother's milk postnatally were investigated. The results showed that prolonged prenatal exposure (16 days) to diazepam (10 mg/kg) resulted in characteristic and extensive pathological changes, i.e. gliosis and perivascular cuffing in the brains of the rats. These changes could be observed under the light microscope a long time after exposure to the drug had been terminated. Limiting the prenatal exposure to a single trimester of 7 days reduced somewhat the number of lesions but did not prevent their occurrence. Rats exposed to diazepam postnatally through the mothers' milk showed very few lesions.

T-cells specific for soluble recombinant oligodendrocyte-specific protein induce severe clinical experimental autoimmune encephalomyelitis in H-2(b) and H-2(s) mice.

To investigate the immunogenicity and encephalitogenicity of oligodendrocyte-specific protein (OSP), recombinant soluble mouse OSP (smOSP) was produced from a synthetic gene engineered to lack the sequences coding for the hydrophobic transmembrane domains of the native molecule. SmOSP was immunogenic and encephalitogenic for SJL/J, C3H.SW and C57BL/6J mice, but not PL/J or BALB/c mice. SmOSP-specific T-cells from SJL/J, C3H.SW and C57BL/6J mice induced severe chronic clinical experimental autoimmune encephalomyelitis upon transfer. These findings indicate that autoimmune T-cell responses to OSP should be investigated in the context of multiple sclerosis.

A rapid method for obtaining murine bone marrow cells in high yield.

A rapid and efficient method for obtaining murine bone marrow cells is described, which yields up to twice the amount of cells obtained by the conventional method of flushing through the bones. The femoral and tibial bones are partially broken by an Omni-Mixer in the presence of phosphate-buffered saline to allow their bone marrow content to extrude into the liquid suspension. Murine bone marrow cells obtained by this method were found to be more than 95% viable, and their differential counts were comparable to those of bone marrow suspensions obtained by the flushing method. Moreover, no contamination by cells from the bone or other surrounding tissues has been observed. Transplantation of bone marrow, obtained by the new extrusion method and depleted of T cells, resulted in long-term stable chimeras in which the hematopoietic reconstitution was comparable to that found in mice transplanted with bone marrow obtained by the flushing method. This new method for obtaining murine bone marrow cells may serve as a time- and mice-sparing alternative to the conventional flushing method, and may also prove useful in other animal models.

The effects of hyperthermia on normal mesenchymal tissues. Application of a histologic grading system.

The alterations produced by radiofrequency-induced hyperthermia of 42 to 48 degrees C for 30 minutes were studied in the subcutaneous adipose tissue and skeletal muscle of swine. Acute lesions (18 to 24 hours) included edema, hemorrhage, necrosis (predominantly of myocytes) and granulocytic exudate in fat or muscle. The most important chronic lesion (28 to 31 days) was fibrosis replacing either tissue. There was a histiolymphocytic exudate with foreign-body giant cells around large lipid vacuoles. Muscle necrosis persisted and there was variable myocyte regeneration. Several specimens showed deep necrosis and abscesses. A grading system was developed to quantitate independently acute and chronic damage in each tissue. Acute lesions were usually less severe and extensive than chronic ones, without obvious dose response. Chronic lesions showed clearly a dose response, which began at 43 degrees C and increased with temperature. The latter appear to be reliable indicators of hyperthermic damage in deep soft tissues.

The riddle of the uvula.

Since ancient times, the uvula has been a subject of interesting and contradictory observations. On the one hand, it was regarded as having a functional role in speech and in immunology, but on the other hand it was regarded as a potentially hazardous organ, possibly responsible for sudden infant death syndrome. None of these hypotheses, however, has been proved. In a previous study on patients undergoing uvulopalatopharyngoplasty, we suggested that the most important function of the uvula is connected with the muscularis uvula. Its function could be related to drinking while bending over. This previous assumption was that the uvula is a phylogenetic remnant from mammals that drink while bending their neck downward. In the present study, the soft palate of eight different mammals was macroscopically and microscopically studied and compared. Of all animals in the study, a small underdeveloped uvula was found only in two baboons. We found that the human uvula consists of an intermix of serous and seromucous glandular masses, muscular tissue, and large excretory canals. The serous and seromucous glands are absent in the other mammals. Thus, the uvula is a highly sophisticated structure, capable of producing a large quantity of fluid saliva that can be excreted in a short time. Both uvula and speech serve to differentiate human beings from animals. Our conclusion is that the uvula is possibly an accessory organ of speech, and may be another marker of human evolution that differentiates man from other mammals.

Effects of probenecid on blood levels and tissue distribution of ampicillin in fowls and turkeys.

The effect of probenecid (a benzoic acid derivative which competitively inhibits active secretion of weak organic acids by the renal tubules) on serum ampicillin concentrations and the distribution of ampicillin in body organs was examined in fowls and turkeys. An aqueous solution of probenecid coadministered intramuscularly, at 200 mg/kg, with sodium ampicillin solution, at 25 mg/kg, resulted in peak serum antibiotic concentration of 16.5 microgram/ml. A similar dose of ampicillin administered alone produced a peak level of 4.6 microgram/ml. Subcutaneous injections of sodium ampicillin at 25 mg/kg with aqueous probenecid at 200 mg/kg resulted in a peak serum ampicillin concentration (12.8 microgram/ml) three times as high as the peak produced by the subcutaneous injection of ampicillin alone at 50 mg/kg (4.2 microgram/ml). The elimination half-life (t 1/2) of the drug (30 min) was increased to 1.5 hr by coadministration of probenecid parenterally, and serum antibiotic levels greater than or equal to 5.0 microgram/ml were maintained during 3 hours. Ampicillin seemed to be poorly absorbed from the gastrointestinal tract of fowls. A single oral bolus administration of ampicillin trihydrate aqueous suspension produced a peak of 0.6 microgram/ml, and coadministrations of aqueous probenecid suspension at 20, 50, and 100 mg/kg respectively produced peaks of 0.9, 1.25, and 1.5 microgram/ml. During 4 and 5 days, when ampicillin was added to the drinking water at rates of 200 and 50 mg/liter, serum ampicillin levels were rather low (peaks of 0.20 and 0.12 microgram/ml, respectively), and although these levels were increased by 50% with the coadministration of probenecid they were considered to be of limited clinical value for treating systemic bacterial infections. Probenecid did not change the distribution of ampicillin in the organs.

Epidermoid cysts in the central nervous system of mice.

Two epidermoid cysts are described in mice, one intracranial, in the fourth ventricle and the other in the thoracic spinal canal. They were lined by compressed squamous epithelium and contained keratinaceous squamae. These incidental findings suggest that such cysts might be detected more often if more extensive examinations of the CNS were carried out in group studies.

Histological findings in rabbits which died with symptoms of mucoid enteritis.

Histological and aetiological similarities between rabbit mucoid enteritis and human ulcerative colitis are briefly discussed. Escherichia coli seems to be associated with the rabbit disease, and treatment aimed at this organism has been followed by a period of 4 years free from mucoid enteritis.

Temperature measurements and histology of the canine prostate during transurethral hyperthermia.

Intraprostatic temperature measurements during transurethral hyperthermia at 44.5C were obtained in 5 dogs. Temperatures were also recorded in the bladder neck wall and the rectal wall. After completion of the temperature measurements, hyperthermia was continued for 3 hours. The prostates were then removed and taken for histologic examination immediately after hyperthermia and 1 week and 1 month later. The mean temperatures obtained in the 5 canine prostates were 44.5 +/- 0.4C at the heating electrode; 43.8 +/- 0.4C at a distance of 3 mm. from the electrode; 42.6 +/- 0.5C, 40.8 +/- 0.4C and 39.4 +/- 0.5C, 6, 9 and 12 mm. from the heating electrode, respectively, in the right, prostatic lobe. Similar temperatures were measured in the left lobe. The thermal gradient in the prostatic tissue was therefore about 4C per 1 cm. On histology, hemorrhagic necrosis of the prostatic tissue adjacent to the urethra was found. These histologic changes were found as much as 5 mm. from the heating antenna (where the temperatures measured were above 43C). The findings of our study may have major clinical importance. We found that thermal energy above 43C provides enough penetration to cause tissue damage and be clinically effective in most patients, while the thermal gradient around the heating electrode of 4C per 1 cm. is steep enough to confine histologic damage within the prostate.