Late appearance of resistance to tumor rechallenge following cryosurgery: A study in an experimental mammary tumor of the rat

Resistance to tumor challenge following surgical and cryosurgical eradication of the tumor was studied, using an experimental mammary tumor of the rat, MRMT-1. It was revealed that rejection rate of the challenged tumor increased gradually following cryosurgery and reached its peak at 10 weeks after cryosurgery. No such phenomenon was observed after surgical excision of the tumor. Decreased incidence of lymph node metastases and decreased tumor weights in “take” cases also suggested an increased immunological activity against the tumor at 10 weeks after cryosurgery.     

Antitumor immunologic reactivity in the relatively early period after cryosurgery: Experimental studies in the rat

An experimental investigation was performed on antitumor immunity in the relatively early postoperative period after cryosurgery, using a metastasizing rat's mammary tumor, MRMT-1. Two weeks after its inoculation, surgical excision of the tumor, cryosurgery, surgical excision plus inoculation with freezing-thawing produced vaccine, or surgical excision plus fasting for 72 hr was performed, and postoperative follow-up was done on incidences of metastases, those of metastatic death, etc. Specific immunologic reactivity was examined in the surgical excision (SE) and cryosurgery (CR) groups.The FTV and fasting groups showed more metastatic deaths as compared with the SE group. The CR and SE groups did not differ significantly from each other in incidences of lung and lymph node metastases.Specific footpad reactivity at 2 and 3 weeks after treatment was lower in the CR group than in the SE group.Winn's neutralization assay showed an inhibition of tumor growth at 1 and 3 week(s) after treatment both in the SE and in the CR groups, the inhibitory effect tending to be lower in the latter.Inactivated serum obtained at 1 week after treatment showed a facilitation of tumor growth in the SE group and a tendency of tumor suppression in the CR group, showing a significant difference between them.A mild reduction in antitumor immunity seen in the relatively early postoperative period following cryosurgery probably was not due to a blocking effect by superfluous antigens. Rather it was considered to be due to activation of suppressor cells, consequent on cryosurgical stress, and/or slow and steady absorption of antigens.     

Experimental study on mechanism of absorption of cryonecrotized tumor antigens

On Day 7 of subcutaneous MRMT-1 tumor inoculation in the thigh of Sprague-Dawley rats, the tumor was treated cryosurgically by contact method. Postoperative changes in local blood circulation were observed by colloidal carbon perfusion and hydrogen-clearance methods, and the pathway and time course of tumor antigen absorption were observed by measuring uptake in serum and lymph nodes of [3H]thymidine injected intratumorally. Results were: At 0.5 to 1 hr after cryosurgery, vascular stasis, dilatation, and tortuosity were observed. There was no inflow of carbon and almost no uptake of [3H]thymidine. At 6 hr, vascular stasis and sludging become more marked. There was no inflow of carbon, while uptake of [3H]thymidine markedly increased. At 24 hr, some carbon inflow in fin vessels and partial recovery of blood circulation in the peritumoral region were observed. Rate of [3H]thymidine uptake was further increased. At 72 hr, carbon inflow and blood circulation increased further, while the cryonecrotized tumor showed hyaline degeneration. At 168 hr, increase of newly formed vessels and recovery of blood circulation were remarkable, but uptake of [3H]thymidine was decreased in parallel with increased demarcation of the cryonecrotized tumor. From these results, it was suggested that the absorption of cryonecrotized tumor antigens started through lymphatic channels in the early period and then took place also through newly formed capillaries surrounding the tumor at 24 hr after cryosurgery and continued until 72 hr after cryosurgery.     

Effect of gravitational loads in different directions on the structure of the lymphatic bed of the pelvic extremities in the rabbit in vivo]

The method of lymphorentgenography was used in order to study alterations in the lymph vessels of pelvic extremities of rabbits at different terms (from 1 week up to 6 months) after exposure to gravitation stresses of different directions, value and duration. After exposure to stresses of cranio-caudal direction the amount of vessels increased. They had weel pronounced sinuosity and clearly protruding valves. The diameter of the vessels was more than normal and was equal to 0,3-0,8 mm. Popliteal lymph nodes were also increased up to 12,6-6,7 mm. Within 6 months after stresses these changes retained. Stresses of caudal-cranial direction during all periods of observation (from 1 week up to 6 months) caused dilatation of vessels (0,3-0,6 mm), their sinuosity and enlargement of popliteal lymph nodes up to 10,5-6,2 mm. All these changes returned to normality within 6 months. Thus, most pronounced and resistant morphological changes of the lymphatic bed were caused by greatest endurable stresses of cranio-caudal direction.     

Depletion of macrophages and disappearance of postcapillary high endothelial venules in lymph nodes deprived of afferent lymphatic vessels

Summary The afferent lymphatic vessels of rat popliteal lymph nodes were interrupted, and the histological alterations in the lymph nodes occurring 1 to 14 weeks after operation were studied. One week after operation the number of macrophages was considerably reduced and continued to decrease during the subsequent time periods studied. A 6 weeks most macrophages had disappeared. Simultaneously the immunological activity diminished and had completely disappeared 8 weeks after operation. Three weeks after operation the endothelial cells of the postcapillary high endothelial venules had flattened, and the number of immigrating lymphocytes was greatly reduced. Subsequently the lymph nodes became depleted of both macrophages and lymphocytes, leaving only the reticuloendothelial framework.     

The immune response of male DSN hamsters to a primary infection with Ancylostoma ceylanicum

The immune response of hamsters to a chronic hookworm infection has been investigated. Ancylostoma ceylanicum caused long term infections in hamsters which were associated with prominent changes in secondary lymphoid organs. The mesenteric lymph nodes and spleens increased rapidly in size stabilizing at approximately 3-4 times the weight in control animals by weeks 3-7. Cells from both the mesenteric lymph node and spleen, after an initial period of increased blast cell activity, became less reactive in the latter stages of infection. Serum antibody responses were marked, commencing in weeks 3-4 and increasing in intensity throughout the 10 week period of measurement. The results are discussed in relation to their contribution to the understanding of human hookworm infection.     

Inhibition of mitogen-induced lymphocyte proliferation correlated to anticomplementary activity in sera from melanoma patients

Summary Sera from 98 melanoma patients and 90 normal donors were analyzed for antibody (Ab) to melanoma extracts, melanoma-associated antigen (Ag) and anticomplementary (Ac) activities by the microcomplement consumption technique. Sera were also tested for their ability to inhibit mitogen(phytohemagglutinin: PHA)-ininduced blastogenesis of normal lymphocytes. The results of the complement consumption assays were correlated with the results of inhibition of PHA-induced blastogenesis. Of 98 melanoma sera, 22% were Ab-positive, 30% were Ag-positive, and 44% were Ac-positive, in contrast to only 6% Ab-positive, no Ag-positive, and 7% Ac-positive in 90 normal sera. Fifty-nine percent of melanoma sera were inhibitory to PHA-induced blastogenesis, as against 12% of normal donors' sera. Ac-positive melanoma sera were significantly more inhibitory than Acnegative melanoma sera. The inhibitory activity of Acpositive sera was potentiated by the simultaneous presence of detectable Ag activity and was diminished by detectable Ab activity. Presence of Ab or Ag activity alone did not correlate with the inhibitory activity of the melanoma sera. Increasing incidence of Ac activity and ability to inhibit PHA-induced blastogenesis was observed with increasing tumor burden or advancing clinical stage. Sera from patients who displayed a delayed cutaneous hypersensitivity reaction to 2,4-dinitrochlorobenzene (DNCB) were less inhibitory to PHA-induced blastogenesis than the sera from patients who did not respond to this contact allergen. Thus, Ac activity may be one of the circulating factors responsible for the immunosuppressive effect of cancer patients' sera.     

恒河猴艾滋病模型中枢神经系统及淋巴结病变的电镜观察

目的观察恒河猴艾滋病模型不同感染阶段中枢神经系统及淋巴结的超微结构改变,探讨艾滋病的发病机制和病理改变的发展过程。方法 15只恒河猴,1只为正常对照,其余14只静脉注射感染SIVmac239猴艾滋病病毒,分别于感染后1周,2周,1个月,2个月,12个月,18个月取腹股沟淋巴结及下丘脑组织进行透射电镜检查。结果感染后1周即可出现淋巴结内淋巴细胞病变,表现为线粒体肿胀,嵴溶解,出现自噬体等。感染后2周及1个月时淋巴细胞病变主要表现为线粒体肿胀,嵴溶解;感染2个月时淋巴细胞细胞核形态出现明显改变。感染后12个月淋巴结内多数淋巴细胞出现病变,细胞肿胀,细胞器正常形态大部分消失;部分细胞出现溶解性坏死的特征。至感染后期（18个月时）,淋巴结内局部淋巴细胞稀疏,细胞核肿胀或形态不规则。中枢神经系统的病变在感染后1周出现,表现为神经纤维肿胀,感染后2周出现神经元的病变,表现为神经元线粒体肿胀,粗面内质网脱颗粒,尼氏体消失;神经纤维内出现空泡。感染后12个月及18个月时,神经元和神经纤维的病变加重。结论在SIV感染早期,淋巴结内淋巴细胞及中枢神经系统即可出现病变,维持一段相对稳定的时间后,至疾病后期,病变加重。中枢神经系统与淋巴结的超微结构病变发展规律有一定的相似性。     

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by bronchoalveolar lavage fluid.

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2+, Lyt-1+, L3T4-, Lyt-2- phenotype and asialo-Gm1+ cells in BALF significantly increased and L3T4+ cells slightly increased in number. By week 3, the numbers of Lyt-2+ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy-1.2+ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2+ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2+ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.     

AUTORADIOGRAPHIC STUDY OF TYPE II-CELL HYPERPLASIA IN LUNGS OF MICE CHRONICALL EXPOSED TO URETHANE

Hyperplasia of pulmonary alveolar epithelium was induced by exposure of adult mice to 0–1% urethane in drinking water, and their lungs were analysed using combined autoradiographic and morphometric methods. The response of pulmonary epithelium showed three phases: an initial one of 3 weeks in which the number of type II cells was consistently low and, after a transient decrease in ³H-thymidine labeling index, the latter steadily increased. Degenerating or dead type II cells were found, consequently this period was considered one in which cell death was disproportionately increased over cell production. Between the 3rd and 6th weeks the number of type II cells doubled, and thereafter increased at a slower rate. The ³H-thymidine labeling index reached its peak at 6 weeks. The third phase was marked by a decline in labeling index which returned to near-normal levels by the 16th week. The number of type II cells declined slowly after the 10th week and was still elevated at the end of the observation period. Tumors consisting of type II cells continued to arise even during the period of declining labeling index. The dissociation between the proliferative response which was reversible, and the neoplastic response which was not, supports the assumption that the major part of the observed population growth or hyperplasia induced by urethane was a form of regeneration repair secondary to cell death and may be unrelated to tumor growth.     

Dynamics of lymph node function in disordered innervation

A mechanotron 6MX1C was used to study lymphodynamic indices of the dog popliteal lymph nodes after cutting the ischiatic and femoral nerves in the left limb at 1 week, 3 and 5 months. The lymph nodes of the contralateral limb and the lymph nodes from intact dogs were used as controls. It has been demonstrated that denervation causes acceleration of the periods of the filling and emptying of the lymph nodes both in the denervated and contralateral limbs as compared with the initial period. The amplitude of fluctuations under maximal filling of the lymph nodes decreases significantly at the long-term postoperative periods. The lymphodynamic changes seem to be determined by pathomorphological alterations in lymph node tissues.     

Characterization of early pathogenic effects after experimental infection of calves with bovine immunodeficiency-like virus.

The early pathogenic effects of bovine immunodeficiency-like virus (BIV) were studied in calves experimentally inoculated with BIV. All animals inoculated with BIV R29-infected cells seroconverted by 6 weeks postinoculation, and BIV was recoverable from each animal at 2 weeks postinoculation. However, levels of BIV replication in vivo appeared to be low. In situ hybridization studies indicated that during peak periods of viral replication in vivo, less than 0.03% of peripheral blood mononuclear cells were expressing detectable levels of viral RNA. Moreover, the levels of viral RNA in these cells in vivo were less than 1/10 the levels observed in persistently infected cells in vitro. BIV-inoculated calves had significantly higher numbers of circulating lymphocytes, and follicular hyperplasia was observed in lymph nodes, hemal nodes, and spleen. The histopathological changes observed in BIV-infected calves were similar to changes found early after infection with the immunosuppressive lentiviruses, including human immunodeficiency virus type 1.     

Investigation of the mechanism and the effect of cryoimmunology in the Copenhagen rat

This study examined the potential for "cryoimmunology" to increase the destruction of the Dunning AT-1 prostate tumor after cryosurgery. Two possible mechanisms explaining the cryoimmunologic response were studied. The first was that an antitumor antibody is produced after cryosurgery. The second was that freezing induces an immunostimulatory signal that creates a T-cell response to the tumor. Six groups of animals (three experimental groups and three control groups) were treated once per week for 4 weeks with different therapies designed to investigate these mechanisms. Three types of immune response were measured: (1) the anti-AT-1 tumor immune titer (Ab response) by serum ELISA, (2) the effect on secondary tumor growth after challenge with live AT-1 cells (size and weight of the secondary tumor over time), and (3) the nature of the immunologic infiltrate into the secondary tumors by immunoperoxidase stain. ELISA showed that immune titers were present in the experimental groups after therapy, but the presence of an immune titer did not have a significant effect on tumor propagation. Histology showed the immunologic infiltrate was similar in all groups. These results showed that an immune response to AT-1 tumor was measurable by serum antibody, but it did not significantly limit secondary tumor growth or affect tumor histology. This suggests that the growth of AT-1 tumors is not inhibited by a cryoimmunological response. Thus, the effect of in vivo cryosurgery in the AT-1 tumor system would likely be limited to cellular and vascular changes.     

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by broncho alveolar lavage fluid

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2⁺, Lyt-1⁺, L3T4⁻, Lyt-2⁻ phenotype and asialo-Gm1⁺ cells in BALF significantly increased and L3T4⁺ cells slightly increased in number. By week 3, the numbers of Lyt-2⁺ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy1.2⁺ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2⁺ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2⁺ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.     

Kinetics of lymphoid cells in tumor-bearing mice

The distribution patterns of ⁵¹Cr-labeled lymphoid cells from normal C3H and tumor-bearing 6C3HED mice were studied by the method of Bainbridge and Gowland (1). An increased localization of normal recirculating lymphocytes was observed in draining lymph nodes of tumor bearing animals which reached a maximum of 8.4% by the 8th–10th day following transplantation. The proportion of recirculating cells from draining nodes of tumor-bearing animals decreased with the progression of the tumor although their actual numbers increased. This decrease is thought to be related to the invasion of draining nodes by tumor cells.     

Citrobacter rodentium infection causes iNOS-independent intestinal epithelial dysfunction in mice

Attaching-effacing bacteria are major causes of infectious diarrhea in humans worldwide. Citrobacter rodentium is an attaching-effacing enteric pathogen that causes transmissible murine colonic mucosal hyperplasia. We characterized colonic inflammation and ion transport at 3, 7, 10, 30, and 60 d after infection of C57Bl/6 mice with C. rodentium. Macroscopic damage score was significantly increased 7 and 10 d after infection. Colonic wall thickness was increased at 7, 10, 30, and 60 d. Myeloperoxidase (MPO) activity was significantly increased at 3, 7, and 10 d and returned to control levels by days 30 and 60. The expressions of inducible nitric oxide synthase and cyclooxygenase-2 were increased by C. rodentium infection. Significant reductions in the epithelial secretory response to carbachol, but not to electrical field stimulation or forskolin, were observed at 3 and 10 d of infection. Translocation of enteric bacteria into the mesenteric lymph nodes was observed 10 d following infection. There was no difference in response to infection between animals deficient in inducible nitric oxide synthase and wild-type controls. The COX-2 inhibitor rofecoxib caused decreased wall thickness and MPO activity at day 10. However, COX-2 inhibition did not alter infection-induced changes in ion transport. Citrobacter rodentium infection causes colonic inflammation, mucosal hyperplasia, and nitric-oxide-independent epithelial dysfunction in association with increased permeability to luminal bacteria.     

A critical role for macrophages in promotion of urethane-induced lung carcinogenesis

Macrophages have established roles in tumor growth and metastasis, but information about their role in lung tumor promotion is limited. To assess the role of macrophages in lung tumorigenesis, we developed a method of minimally invasive, long-term macrophage depletion by repetitive intratracheal instillation of liposomal clodronate. Compared with controls treated with repetitive doses of PBS-containing liposomes, long-term macrophage depletion resulted in a marked reduction in tumor number and size at 4 mo after a single i.p. injection of the carcinogen urethane. After urethane treatment, lung macrophages developed increased M1 macrophage marker expression during the first 2-3 wk, followed by increased M2 marker expression by week 6. Using a strategy to reduce alveolar macrophages during tumor initiation and early promotion stages (weeks 1-2) or during late promotion and progression stages (weeks 4-16), we found significantly fewer and smaller lung tumors in both groups compared with controls. Late-stage macrophage depletion reduced VEGF expression and impaired vascular growth in tumors. In contrast, early-stage depletion of alveolar macrophages impaired urethane-induced NF-κB activation in the lungs and reduced the development of premalignant atypical adenomatous hyperplasia lesions at 6 wk after urethane injection. Together, these studies elucidate an important role for macrophages in lung tumor promotion and indicate that these cells have distinct roles during different stages of lung carcinogenesis.     

Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.     

Dynamics of the functional activity of the lymph nodes under conditions of sympathectomy

By means of mechanotron 6MYIC and perfusion with physiological solution using the "running vesicle", dynamics of functional activity of the popliteal lymph nodes have been studied in 17 non-inbred dogs under conditions of unilateral sympathectomy for 1 week up to 5 months of the experiment. Dependence of the lymph node functional activity on the adrenomediator concentration level in their tissues has been determined by means of the microfluorometry method in the preparations treated after Falck-Hillarp method in E. M. Krokhina modification. Lymph nodes of intact dogs serve as a control. Unilateral sympathectomy of the lymph node is stated to produce an enhancement of the period for its filling and emptying with lymph, resulting from an increased level of the mediator in the fibrillar stroma at the expense of compensatory enhancement of functional activity of the nervous fibers of the contralateral origin. During the remote postoperative period the amplitude of the lymph node capsule fluctuations is stipulated by certain pathomorphological changes, produced by deficiency in concentration of catecholamines in the organ's parenchyma. As to the periodicity of the capsule fluctuations in the intact lymph nodes, it is limited with the cycle of 3-6 min.