Site Of Subcutaneous Inoculation Research Articles

ABCG2 Localizes to the Nucleus and Modulates CDH1 Expression in Lung Cancer Cells

Breast cancer resistance protein [BCRP/ATP-binding cassette subfamily G member 2 (ABCG2)] is a member of the ATP-binding cassette transporter family. The presence of ABCG2 on the plasma membrane in many kinds of human cancer cells contributes to multidrug resistance during chemotherapy, and it has been used as the side population marker for identifying cancer stem cells in lung cancers. We report here that, in addition to the membranous form, ABCG2 proteins are also found inside the nucleus, where they bind to the E-box of CDH1 (E-cadherin) promoter and regulate transcription of this gene. Increased expression of ABCG2 causes an increase of E-cadherin and attenuates cell migration, whereas knockdown of ABCG2 downregulates E-cadherin and enhances cell motility. In mice, xenografted A549 cells that have less ABCG2 are more likely to metastasize from the subcutaneous inoculation site to the internal organs. However, for the cancer cells that have already entered the blood circulation, an increased level of ABCG2, and correspondingly increased E-cadherin, may facilitate circulating cancer cells to colonize at a distant site and form a metastatic tumor. We propose a novel role for nuclear ABCG2 that functions as a transcription regulator and participates in modulation of cancer metastasis.

Establishment of a cell line from a Japanese patient useful for generating an in vivo model of malignant pleural mesothelioma

Malignant pleural mesothelioma is a refractory tumor with increasing incidence. In the present study, we established six mesothelioma cell lines possessing two allele deletions of the p16(INK4A) gene and one allele deletion of the neurofibromatosis type 2 gene, MM16, MM21, MM26, MM35, MM46 and MM56, from pleural effusion fluids or surgically resected tumors of Japanese patients. MM21, MM26 and MM46 cells failed to develop tumors in BALB/c-nude mice following subcutaneous inoculation. MM16 and MM35 cells slowly generated tumors at the site of subcutaneous inoculation in BALB/c-nude mice, but lost the expression of mesothelioma-related markers such as calretinin, D2-40 and Wilms' tumor 1 in the subcutaneous tumors. On the other hand, MM56 cells rapidly generated tumors with the expression of calretinin and D2-40 in BALB/c-nude mice following subcutaneous inoculation. In addition, orthotopic implantation of MM56 cells into BALB/c-nude mice developed diffusely growing thoracic tumors by 3 weeks after implantation. Pleural effusions were observed in these mice 4 weeks after implantation. Thoracic tumors invaded aggressively into the chest wall 5 weeks after implantation and often metastasized into the lung, rib, peritoneum and pericardial cavity. On the pleural surface, MM56 cells were growing as single or multiple cell layers with the reactive mesothelium of recipient mice. These results indicate that MM56 cells can behave in a manner characteristic of human malignant pleural mesothelioma in the thoracic cavity of BALB/c-nude mice. The in vivo model using MM56 cells may be useful for studying the biological behavior of malignant pleural mesothelioma and developing its diagnostic and therapeutic strategies.

Vaccine-Induced Control of Viral Shedding following Rhesus Cytomegalovirus Challenge in Rhesus Macaques

The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.

Distinct bacterial dissemination and disease outcome in mice subcutaneously infected with Borrelia burgdorferi in the midline of the back and the footpad

Subcutaneous inoculation of mice with Borrelia burgdorferi, the causative agent of Lyme disease, results in established infection and the development of acute arthritis and carditis, hallmarks of human disease. Because conflicting results may originate from the site of subcutaneous inoculation, we addressed the dissemination capacity of spirochetes injected in the shoulder region versus the footpad. Spirochetes inoculated in the footpad disseminated to a lesser extent to distant organs, such as the ear and the heart. This resulted in distinct degrees of joint and cardiac inflammation at the peak of the disease. The differences eventually leveled out. These results suggest that caution must be exercised in the interpretation of results obtained with routes of inoculation that do not closely represent the natural site of infection.

Transmission factors: Yersinia pestis genes required to infect the flea vector of plague.

Y. pestis Ymt and Hms proteins have complementary but distinct roles. Ymt is required for survival in the midgut compartment, but not the proventriculus compartment, of the flea digestive tract. Conversely, the hms genes are not required to infect the midgut, but are required to infect the proventriculus. In addition to ymt and hms, the outer surface plasminogen activator of Y. pestis is considered to be important for fleaborne transmission, although this role does not occur in the flea (Hinnebusch et al., 1998). Sodeinde et al., (2002) showed that Pla is important for dissemination from a subcutaneous inoculation site in mice. This finding suggested that pla is pertinent to the fleaborne life-cycle of Y. pestis, which demands that the bacteria be able to disseminate from the dermis where they are deposited by a flea. Notably, ymt and pla are located on plasmids that are unique to Y. pestis. Thus, acquisition of these two plasmids by horizontal transfer helps to account for the rapid evolutionary transition of Y. pestis to fleaborne transmission within the last 20,000 years.

Early detection of Leishmania (Leishmania) chagasi in draining lymph node after subcutaneous inoculation in hamster

To study the route of migration of Leishmania (Leishmania) chagasi to the viscera from the subcutaneous inoculation site, ultrastructural studies were carried out. These studies on popliteal draining lymph nodes of hamsters inoculated with 10 7 promastigotes of Leishmania (Leishmania) chagasi in the hind footpad were carried out. Parasites were detected in lymph nodes just 2 h after inoculation. Parasites were either preserved or degenerated in macrophages. In 24 h signs of binary division of the parasites were found in macrophages. This fact suggests that the parasites migrate from skin to the lymph node early in the infection within phagocytes and proliferate in lymph nodes before dissemination to the viscera.

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation.

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.

Neurovirulence of the UC-2 and UC-8 Strains of Bluetongue Virus Serotype 11 in Newborn Mice

In vivo and in vitro experiments were done to investigate whether the difference in neurovirulence between the two strains of bluetongue virus 11, UC-2 and UC-8, is based on a different capability to gain access to the brain from the subcutaneous inoculation site or on a different tropism for neural cells. In newborn Balb/c mice subcutaneous inoculation of UC-8 at doses between 10(-0.2) plaque forming units (PFU) and 10(4.8) PFU caused a severe necrotizing encephalitis whereas UC-2 at doses of up to 10(4.4) PFU did not affect newborn Balb/c mice. However, intracranial inoculation of 10(2.4) PFU of either virus strain produced severe necrotizing encephalitis. In vitro both virus strains infected dissociated brain cell cultures similarly. Double labelling immunofluorescent staining with markers specific for neural cells did not reveal differences in the target cells for the two viruses. The difference in neurovirulence between UC-2 and UC-8, therefore, appears to be determined by the ability of UC-8 to infect the brain from a subcutaneous inoculation site.

Effect of androgen on the expression of the sex difference in susceptibility to infection with Strongyloides ratti in C57BL/6 mice.

The possible site of expression of the sex difference in susceptibility to infection with Strongyloides ratti was studied in C57BL/6 mice. The intensity of infection, measured by daily larval output in faeces or by intestinal worm burden, was significantly higher in males than in females. This sex difference was already expressed during the migration of larvae from the site of subcutaneous inoculation to the cranial cavity or lung as early as 24 h after infection. Conversely, there was no significant difference in the larval output in faeces or in the worm burden of male or female recipients after implantation of adult worms using a stomach tube. When the susceptibility to infection with S. ratti of gonadectomized animals was examined, the daily larval output in faeces of orchidectomized males was significantly reduced to the level of intact females, while ovariectomy had no effect, suggesting that androgen is one of the important regulatory factors for the expression of sex difference. In fact, testosterone treatment of orchidectomized males or normal females could increase their susceptibility to or above the level of intact males. These results strongly suggest that sex difference in the susceptibility to infection with S. ratti in C57BL/6 mice is mediated by 'natural defence mechanisms' which are under the regulation of testosterone.

Establishment of Theileria parva-infected bovine tissue culture in Swiss and athymic (nude) mice

Theileria parva-infected bovine lymphoid cells, grown in culture, were inoculated by different routes into neonatal and adult Swiss mice immunosuppressed by irradiation, thymectomy or inoculation of anti-lymphocyte serum. Tumour-like masses, composed of parasitized bovine lymphoid cells, formed at the site of subcutaneous inoculation in immunosuppressed neonatal and adult mice, but consistent establishment of cells following intra-peritoneal inoculation occurred only in neonatal mice. In all cases the degree of cellular establishment was proportional to the degree of immunosuppression. The best “take” was in irradiated neonatally thymectomized mice. Cells underwent short-term multiplication in mice but, as immune competence returned, the cells were rejected. There was no evidence that cells, on passage, became more adapted to grow in mice, nor that mouse cells became parasitized. Culture-derived cells were also inoculated subcutaneously into irradiated and non-irradiated nu/nu, nu/+ and Swiss mice. Tumour-like masses, composed of parasitized bovine lymphoid cells, developed at the site of inoculation in all irradiated mice. In nu/+ and Swiss mice these masses regressed after 2–3 weeks, but in the athymic nu/nu mice there was generally no rejection or cellular degeneration and parasitized cells became widely disseminated in the host's tissues and organs, in some cases causing death. T. parva-infected cells could not be established in non-irradiated nu/nu mice, nor when irradiated nu/nu mice were inoculated by the intra-peritoneal route. “Take” in irradiated neonatal nu/nu mice was also poor. Cells were passaged three times in irradiated nu/nu mice inoculated subcutaneously and it seems probable that indefinite passage of T. parva in mice can now be achieved.

Observation of M. leraemurium in Subcutaneous Tissue of Mice by Spread Tissue Preparations

Mice of 6 inbred strains (C3H, CF#1, BALB/C, KK, DDD and C57BL/6) were inoculated subcutaneously in the back with 0.25 ml of a 1:1000 leproma suspension (Hawaiian strain). Spread tissue preparations were made from the inoculation site at about weekly intervals until 10 weeks to observe growth patterns of murine leprosy bacilli in subcutaneous tissues.There were no remarkable differences among these 6 strains during the first 3 weeks after inoculation. In 1 week, an acute inflammatory reaction with polymorphonuclears disappeared and elongation of the bacilli without increase in number was observed in mononuclears. The bacilli showed longer and thinner forms with a length of about 2 to 3 times the initial size. At 3 weeks, enlarged mononuclears, being crowded with the long bacilli, could easily be demonstrable by low magnification.Four weeks after inoculation, however, significant differences were observed in growth patterns of murine leprosy bacilli among these mouse strains. In C3H and CF#1 mice, inflammatory cells were consisted mainly of mononuclears, most of which were heavily loaded with the long bacilli. On the contrary, in the other 4 strains, many lymphocytes and polymorphonuclears were found in the subcutaneous inoculation site, surrounding a smaller number of the bacillus-containing mononuclears. Such differences between mouse strains became more remarkable at 5 weeks, because of more pronounced cellular reactions in these 4 strains.The significant differences between C3H and CF#1 mice were manifested in 10 weeeks. In CF#1 mice lymphocyte infiltration, which surrounded the lesions of mononuclears containing the bacilli, was evident, whereas no host cellular reactions were seen in C3H mice.From the results of these observations, this experimental method can be recommended for early evaluation of development of mouse leprosy, with special reference to the relation of the host cells to the organism, and these mouse strain differences are presumably due to the cell-mediated immunity developed in the hosts.

Response of whole-body irradiated mice to inoculation of Theileria parva infected bovine lymphoid cells

Theileria parva-infected bovine lymphoid cells produced tumour-like masses at the site of subcutaneous inoculation in whole-body irradiated Swiss mice. The optimal dose range for irradiation was 800 to 900 rad. Intact parasitized cells were detected in these masses up to about d. 17 after inoculation and were then gradually rejected as the host's defence mechanisms recovered. The masses appeared to be produced by the continued growth of tissue culture in the immuno-suppressed host. There was no evidence that mouse cells became parasitized.

Resistance to reinfection in experimental brucellosis.

Summary and Conclusions1. The purpose of the present work was to determine if a relationship exists between resistance to superimposed infection and the presence of the organisms of primary inoculation in the body of guinea pigs infected with Br. abortus. The organisms of reinoculation never persisted in the tissues in the presence of the brucellae of primary infection, except in one instance. The exception was a guinea pig reinoculated 11 days after infection. Some animals, from which the organisms of the original infection were not recovered from any of the tissues, were resistant to reinfection. Resistance waned gradually after the organisms of primary inoculation could no longer be recovered from the tissues. 2. A local reaction with abscess formation was produced at the site of subcutaneous inoculation in infected guinea pigs; brucellae were recovered in large numbers from these lesions. The organisms of reinfection penetrated into the deep tissues but disappeared promptly. This suggests that the loc...

Persistence of Antigen at the Site of Inoculation of Vaccine Emulsified in Oil

Summary A technic was developed for the in vivo quantitative estimation of Shigella paradysenteriae type III (Z) antigen in mice, by utilization of the minimum quantity found necessary to induce an agglutinin response (0.002 μg). This method was used to determine the fate of this antigen in a mineral oil emulsion at the site of subcutaneous inoculation in mice. Active Shigella antigen was recovered from the site of inoculation in mice at periods up to 18 and 24 weeks, depending on the original dose of antigen. There was a gradual diminution with time in the amount recovered. The deterioration of antigen at the site of inoculation was paralleled by a fall in the agglutinin titers of the mice receiving the oil vaccine.

Effect of Ferric Chloride Injections on Spread of Tuberculosis from Site of Subcutaneous Inoculation.

Previous studies have demonstrated that repeated intravenous injections of a 0.25% solution of ferric chloride crystals in tuberculous rabbits are followed by an accumulation of iron in the caseous areas of the lungs.1, 2 Subsequent studies revealed that concomitant with this accumulation of iron in tuberculous areas there was a definite increase in the survival time of infected rabbits.3, 4 When control and experimental animals were sacrificed at given intervals of time after inoculation with tubercle bacilli, in the majority of instances the extent of the lesion in experimental animals was distinctly less than that found in the controls.4 The conclusion drawn was that with the accumulation of iron in tuberculous areas, the course of development of the disease is protracted in the rabbit.Further experiments were set up to determine whether the repeated administration intravenously of ferric chloride solution would retard the dissemination of tubercle bacilli and hence the progress of the disease when mic...