A D-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.     

The intraperitoneal delivery of radiolabeled monoclonal antibodies: studies on the regional delivery advantage

Summary The i.p. delivery of murine monoclonal antibody was compared with i.v. delivery in normal mice and rats, in normal nude mice and in those with i.p. human ovarian carcinoma xenografts. In normal rats, all classes of antibodies and antibody fragments evaluated were cleared from the peritoneal cavity at comparable rates. The regional delivery (Rd¹) advantage to the peritoneal cavity following i.p. delivery was thus most dependent on the rate of clearance of the antibody or fragment from the blood stream. Determining the exact i.p. delivery advantage was problematic due to the difficulty in reliably obtaining peritoneal fluid later than 9–10 h after i.p. injection in normal animals. During the first 9 h following i.p. injection, the Rd(0–9/0–9) was, for a murine IgG2ak Fab>F(ab)₂>IgG (at 13.6>10>7.9). Two murine IgMs evaluated differed in Rd(0–9) at 27.1 and 9.2 respectively. When blood levels were extrapolated to infinity, these Rd (0–9/) values were considerably lower with the Fab having the highest Rd at 4.67. The i.p. Rd advantage was almost solely due to the i.p. antibody levels seen in the first 24 h after injection, as after that time, blood levels become comparable to those seen following i.v. injection. Normal tissues obtained at sacrifice 5–7 days after i.p. injection. Normal tissues obtained at sacrifice 5–7 days after i.p. or i.v. injection in rats showed comparable levels of radioantibody activity, whether the injection was i.p. or i.v. (except for higher diaphragmatic levels following i.p. delivery). In nude mice with i.p. human-derived ovarian tumors, intact IgG clearance from the peritoneal cavity to the blood was considerably slower than in normal animals, and early i.p. tumor uptake of specific antibody was significantly higher than that following i.v. antibody delivery. With higher early tumor uptake and lower systemic exposure, early tumor/nontumor ratios were significantly greater than those for i.v. delivery, though not beyond 48 h after i.p. injection. This study demonstrates the pharmacokinetic rationale for i.p. monoclonal antibody delivery, especially for agents cleared rapidly from the blood, such as antibody fragments. In addition, definite i.p. delivery benefit for antibody specific to i.p. tumors in the i.p. ovarian cancer system was shown soon after injection. These data regarding i.p. antibody delivery should be useful in rationally planning diagnostic and therapeutic studies involving the i.p. delivery of unmodified and immunoconjugated monoclonal antibodies.Rd is area under the curve (AUC) for peritoneal fluid activity/AUC for blood radioactivity. Rd (0–9/0–9) is the Rd measured from 0 to 9 h for both peritoneal fluid and blood. Rd (0–9/) is the conservative estimate of Rd with i.p. fluid AUC measured to 9 h, with blood levels extrapolated to infinity. Rd₂ is Rd/(AUC i.p. fluid (0–9)/AUC blood (0–9)) after i.v. injection.     

Functional activity in vivo of effector T cell populations. II. Anti-tumor activity exhibited by syngeneic anti-MoMULV-specific cytolytic T cell clones

The functional activity in vivo of murine tumor-specific cytolytic T lymphocyte populations and clones was studied. Tumor cell destruction induced after the i.v. injection of cytolytic effector cells was quantitated by monitoring the elimination of 131IUdR-labeled tumor cells in the peritoneal cavity by using whole-body counting techniques. Mixed leukocyte-tumor cell cultures were established by using spleen cells from C57BL/6 regressor mice that had rejected an intramuscular tumor induced by the injection of MSV-MoMuLV virus. This effector cell population was observed to eliminate syngeneic MoMuLV-induced tumor cells in a dose-dependent manner. Treatment of the effector cell population with monoclonal anti-Lyt-2 antibodies plus complement totally abrogated their ability to induce tumor cell destruction in the peritoneal cavity. MSV-MoMuLV-specific Lyt-2+ cytolytic T cell clones derived by micro-manipulation of T lymphocyte-tumor cell conjugates were also tested for functional activity in vivo. Several clones induced a rapid, specific elimination of 131I-labeled MBL-2 tumor cells from the peritoneal cavity after i.v. injection, whereas others were inactive. Both active and inactive clones were highly cytolytic and secreted MAF/IFN-gamma lymphokines. In contrast to previous results obtained in a tumor allograft model, the MSV-MoMuLV-specific cytolytic T cell clones that were active in vivo did not proliferate in vitro in response to stimulation with irradiated tumor cells plus filler spleen cells in the absence of an added source of interleukin 2.     

A critical role for sphingosine kinase in anaphylatoxin-induced neutropenia, peritonitis, and cytokine production in vivo

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-alpha and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-alpha and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.     

Spontaneous and induced leukemias of myeloid origin in recombinant inbred BXH mice

BXH-2 recombinant inbred (RI) mice produce high titers of B-ecotropic murine leukemia virus beginning early in life and have a high incidence of non-T-cell leukemias that occur before 1 year of age. The leukemias that develop are in some cases associated with hind limb paralysis. In addition, a dualtropic mink cell focus-forming virus has been isolated from leukemic cells of BXH-2 mice. Immunological and cytochemical characterization of the BXH-2 leukemias showed that they are of the myeloid lineage. To assess the oncogenicity of the BXH-2 viruses, newborn mice of several BXH RI strains were inoculated at birth with biologically cloned B-ecotropic or mink cell focus-forming murine leukemia virus. These studies demonstrated that the B-ecotropic virus can induce myeloid leukemias in other BXH RI strains, whereas the dualtropic mink cell focus-forming isolates were nononcogenic in the strains tested. DNA-DNA reassociation analysis indicated that the organotropism of the B-ecotropic murine leukemia virus is confined to lymphoid tissues. Southern analysis of tumor DNAs showed that there was amplification of ecotropic virus-specific sequences in BXH-2 myeloid tumors and in all leukemias induced in other BXH RI strains by inoculation of the BXH-2 B-ecotropic virus. Although B-ecotropic virus is expressed in central nervous tissues of paralyzed BXH-2 mice, we were unable to induce the disorder in several BXH RI strains inoculated intracranially at birth with either the B-ecotropic or dualtropic virus. These results suggest that the paralysis that occurs in BXH-2 mice is due to the infiltration of leukemic cells into the central nervous system.     

Differences in initial rate of intracellular killing of Salmonella typhimurium by granulocytes of Salmonella-susceptible C57BL/10 mice and Salmonella-resistant CBA mice

The contribution of granulocytes to differences in the innate susceptibility of mouse strains to infection by Salmonella typhimurium was assessed on the basis of the size and composition of the inflammatory exudate after i.p. injection of bacteria and the intracellular killing of the bacteria by exudate peritoneal cells and blood granulocytes of resistant CBA and susceptible C57BL/10 mice. The increase in the numbers of both peritoneal granulocytes and macrophages 24 hr after i.p. injection of various numbers of live S. typhimurium was two to four times higher in C57BL/10 mice (p less than 0.05) than in CBA mice. However, despite the larger number of phagocytes in the inflammatory exudate, the numbers of viable S. typhimurium in the peritoneal cavity 24 hr after injection was higher (p less than 0.01) in C57BL/10 mice than in CBA mice. Because the proportion of noningested bacteria was similar in the two mouse strains (less than 30%), these findings indicate a difference in the rate of intracellular killing of the bacteria by exudate peritoneal cells (greater than 75% granulocytes) of the two mouse strains. Subsequent determination of the initial rate of intracellular killing (Kk) of S. typhimurium revealed that after phagocytosis of the bacteria in vivo, exudate peritoneal granulocytes (harvested 24 hr after i.p. injection of 10(3) live S. typhimurium) of CBA mice killed S. typhimurium twice as efficiently (Kk = 0.014 min-1; p less than 0.01) as exudate granulocytes of C57BL/10 mice (Kk = 0.008 min-1) did. Similarly, the initial rate of intracellular killing of the ingested S. typhimurium by blood granulocytes of CBA mice (Kk = 0.017 min-1) was two times higher (p less than 0.01) than that of C57BL/10 mice (Kk = 0.007 min-1). These findings may be specific for S. typhimurium, because L. monocytogenes were killed with equal efficiency by exudate granulocytes and blood granulocytes of these mouse strains (p greater than 0.20). The results of the present study are relevant with respect to the innate resistance of mice to S. typhimurium, particularly during the initial phase of infection when the inflammatory exudate contains predominantly granulocytes.     

Successful immunotherapy with intraperitoneal Corynebacterium parvum in a murine ovarian cancer model is associated with the recruitment of tumor-lytic neutrophils into the peritoneal cavity

The rejection of a murine ovarian teratocarcinoma (MOT) after i.p. injection of Corynebacterium parvum was investigated. Treatment with C. parvum (1400 micrograms) 24 hr after i.p. inoculation of a lethal number of tumor cells (10(5] induced an antitumor effect that cured 75 to 95% of the mice. Morphologic analysis and an in vivo cytotoxicity assay that measured the rate of disappearance of radioactivity from the peritoneal cavity after injection of 125IUdR-labeled tumor cells indicated that the antitumor effect was initiated during the first 24 hr after C. parvum injection. During this period of time, host effector cells retrieved from the peritoneal cavity prevented tumor growth in a Winn assay and lysed radiolabeled MOT targets in a 4-hr Cr-release assay. After separation of peritoneal inflammatory cells on a Percoll gradient, neutrophil-enriched fractions demonstrated significant in vitro tumor lysis, but neutrophil-depleted populations were ineffective. Microscopic analysis of lysis at the single cell level confirmed that neutrophils were binding to and lysing MOT targets. Further characterization of these tumor cytolytic neutrophils revealed that they are nylon wool-adherent, not generated in indomethacin-pretreated mice (but effectively generated in whole body-irradiated mice), and achieve lysis within 30 min after binding to MOT targets. These results indicate that neutrophils must be considered potential antitumor effectors that can be recruited by treatment with biologic response modifiers.     

Impaired calcium mobilization in CD4+ and CD8+ T cells in a retrovirus-induced immunodeficiency syndrome, murine AIDS.

After infection with LP-BM5 murine leukemia viruses, susceptible strains of mice develop a severe and progressive immunodeficiency disease, termed murine AIDS (MAIDS), features of which include markedly impaired T cell response to mitogens or specific Ag stimulation and decreased production of IL-2. Since an elevation of intracellular calcium concentration resulting from binding of Ag to the TCR is associated with IL-2 production, T cells from mice either uninfected or infected with LP-BM5 murine leukemia viruses were examined by a calcium mobilization assay. Both CD4+ and CD8+ T cells from infected mice manifested impaired calcium mobilization responses upon in vitro stimulation with anti-CD3 mAb or Con A. The abnormalities appeared early after virus inoculation and showed no difference in time course between subsets of T cells. Frequencies of prestimulation calcium-positive cells among both CD4+ and CD8+ cells in mice with MAIDS were significantly higher than those for uninfected mice. These abnormalities were associated with presence of the MAIDS-inducing defective virus genome, but were not induced by infection of mice genetically resistant to development of MAIDS or with nonpathogenic helper murine leukemia virus, a virus component that induces high spontaneous proliferation of T cells, even in MAIDS-resistant mice.     

Macrophage activation by Lactobacillus casei in mice

Effects of Lactobacillus casei YIT 9018 (LC 9018), which has antitumor activities against allogeneic and syngeneic murine tumors, on macrophage functions were examined. By intraperitoneal (i.p.) injection of LC 9018, acid phosphatase activity and phagocytic activity of peritoneal macrophages were enhanced significantly compared with those of normal peritoneal macrophages. The phagocytic activities showed peaks 2-3 days after the LC 9018-injection. LC 9018 accelerated the phagocytic function of the reticuloendotherial system in ICR mice tested by the carbon clearance test. The cytostatic activity of peritoneal exudate cells (PEC) induced by i.p. injection of LC 9018 into C57BL/6 mice against EL4 cells was also enhanced. On the other hand, PEC induced by L. fermentum YIT 0159, which has no antitumor activity, did not have cytostatic activity. These observations showed that LC 9018 was able to activate macrophages in mice.     

Accumulation and chemotaxis of natural killer/large granular lymphocytes at sites of virus replication

A model for monitoring the accumulation of natural killer cell/large granular lymphocytes (NK/LGL) at a site of virus replication was studied by using mice infected i.p. with either lymphocytic choriomeningitis virus (LCMV), murine cytomegalovirus (MCMV), mouse hepatitis virus (MHV), Pichinde virus, or vaccinia virus. An i.p. but not i.v. infection resulted in a localized increase in NK/LGL cell number (a fourfold to greater than 20-fold increase) and augmentation (a 10- to 20-fold increase) of NK cell activity associated with virus-induced peritoneal exudate cell (PEC) populations. An increase in NK/LGL cell number was detected as early as 12 hr postinfection (p.i.) and peaked at 3 days p.i. with MHV. The initial LGL recruited into the peritoneal cavity at 1 to 3 days p.i. were nonadherent to plastic and were demonstrated to have an NK cell phenotype: asialo GM1+, Thy-1.2 +/-, Lyt-2.2-, and J11d-. The peak number of LGL appeared at 7 days after infection with the NK cell-resistant virus, LCMV. This LGL population had been previously demonstrated to contain cytotoxic T lymphocyte/LGL (CTL/LGL) as well as NK/LGL. During an MHV infection the number of LGL decreased between days 3 and 7 p.i., suggesting that the second wave of CTL/LGL was absent. These findings may explain the absence of a good MHV-CTL model. Virus-induced, activated NK/LGL responded to chemotactic signals by migrating in a unidirectional manner across two 5-microns pore size polycarbonate filters during 7 hr in vitro chemotaxis assays. Wash-out fluid obtained from the peritoneal cavity contained chemotactic activity for NK/LGL as well as for other cell types. We conclude that production and/or release of chemotactic factors at sites of virus replication are at least partially responsible for the accumulation of NK/LGL at these sites.     

Moloney murine leukemia virus-induced myeloid tumors in adult BALB/c mice: requirement of c-myb activation but lack of v-abl involvement.

BALB/c mice treated with pristane and Abelson virus have been used as an animal model system for the rapid induction of plasmacytomas. Myelomonocytic tumors with helper Moloney murine leukemia virus clonally inserted into the c-myb locus were observed in about 10% of pristane-primed BALB/c mice infected with Abelson virus. However, v-abl was absent in almost all of those tumors. Since Moloney virus is thought to induce mostly T-cell lymphomas, we have carried out studies to investigate this alteration of disease specificity and to determine whether v-abl played an obligatory role in the development of these tumors. We found that, whereas lymphomas developed late (greater than 3 months) in both pristane-primed and unprinted control mice, the myelomonocytic tumors arose at a high frequency, within 3 months, but only in pristane-treated mice. Clonal Moloney virus insertion was again found in each of the seven myelomonocytic tumors examined. Northern blot analyses and S1 mapping studies revealed the presence of virally promoted chimeric mRNAs that lack the three 5'-most myb coding exons. Hence it appears that the requirement for the v-abl gene product in tumor induction is not obligatory. Our results also indicate that tumor-specific alteration at the 5' end of the myb gene plays an important role in the development of these tumors.     

Novel integration sites at the distal 3' end of the c-myb locus in retrovirus-induced promonocytic leukemias.

In BALB/c nu/nu and sublethally irradiated DBA/2 mice, promonocytic leukemia was induced by intravenous inoculation of Friend murine leukemia virus (F-MuLV) strain C57 in conjunction with intraperitoneal injection of pristane. These tumors appear to be identical morphologically to previously reported ones induced by other MuLVs, such as Moloney, amphotropic 4070A, and F-MuLV FB29, which most commonly have provirus integrations in the 5' end of the c-myb locus. Interestingly, 2 of the 16 F-MuLV-induced tumors had viruses integrated in the distal 3' end of c-myb. To determine the precise locations of these integrations, it was necessary to clone sequences encoding the 3' c-myb exons and to prepare a physical map of this region. Exons 10 to 15 were positioned on the map, and it was found that the proviruses in the aforementioned tumors were located within narrow region in the beginning of the large (greater than 11 kb) intron 14. The predicted protein product encoded by the affected alleles is truncated by 38 amino acids. This represents a novel virus integration site which is most likely associated with oncogenic activation of the c-myb gene during leukemogenesis.     

Mutation of all Runx (AML1/core) sites in the enhancer of T-lymphomagenic SL3-3 murine leukemia virus unmasks a significant potential for myeloid leukemia induction and favors enhancer evolution toward induction of other disease patterns

SL3-3 murine leukemia virus is a potent inducer of T-lymphomas in mice. Using inbred NMRI mice, it was previously reported that a mutant of SL3-3 with all enhancer Runx (AML1/core) sites disrupted by 3-bp mutations (SL3-3dm) induces predominantly non-T-cell tumors with severely extended latency (S. Ethelberg, J. Lovmand, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:7273-7280, 1997). By use of three-color flow cytometry and molecular and histopathological analyses, we have now performed a detailed phenotypic characterization of SL3-3- and SL3-3dm-induced tumors in this mouse strain. All wild-type induced tumors had clonal T-cell receptor beta rearrangements, and the vast majority were CD3(+) CD4(+) CD8(-) T-lymphomas. Such a consistent phenotypic pattern is unusual for murine leukemia virus-induced T-lymphomas. The mutant virus induced malignancies of four distinct hematopoietic lineages: myeloid, T lymphoid, B lymphoid, and erythroid. The most common disease was myeloid leukemia with maturation. Thus, mutation of all Runx motifs in the enhancer of SL3-3 severely impedes viral T-lymphomagenicity and thereby discloses a considerable and formerly unappreciated potential of this virus for myeloid leukemia induction. Proviral enhancers with complex structural alterations (deletions, insertions, and/or duplications) were found in most SL3-3dm-induced T-lymphoid tumors and immature myeloid leukemias but not in any cases of myeloid leukemia with maturation, mature B-lymphoma, or erythroleukemia. Altogether, our results indicate that the SL3-3dm enhancer in itself promotes induction of myeloid leukemia with maturation but that structural changes may arise in vivo and redirect viral disease specificity to induction of T-lymphoid or immature myeloid leukemias, which typically develop with moderately shorter latencies.     

Antitumor effect of the streptococcal preparation OK-432 in a murine model of ovarian cancer

Summary The antitumor effects of the streptococcal preparation OK-432 were analyzed in a murine ovarian teratocarcinoma (MOT) model. Administration of OK-432 i.p. prevented tumor outgrowth in 75% of mice challenged with 10³ MOT cells i.p. 24 h previously. Treatment was less successful in mice challenged with 10⁴ or 10⁵ cells, preventing tumor growth in 25% of the former and only 5% of the latter group. Tumor-challenged mice cured by injections of OK-432 were not rendered resistant to a subsequent challenge with 10³ MOT cells 75 days after initial treatment. Only the i.p. route of administration was effective as i.v. OK-432 did not prolong survival of tumor-challenged mice. An antitumor response was detected as early as 24 h after i.p. treatment. This correlated temporally with an influx of neutrophils into the peritoneal cavity. Peritoneal cells obtained between 6 and 24 h after treatment were capable of lysing MOT targets in vitro. A single cell cytotoxicity assay demonstrated that peritoneal neutrophils, elicited by i.p. injection of OK-432, could bind to and lyse MOT targets. These data indicate that OK-432 is effective against small tumor cell inocula in this murine model of ovarian cancer and, furthermore, that the neutrophilic response into the peritoneal cavity plays a role in tumor rejection.     

Leukemogenicity and cell transformation mechanisms in vitro by Gross murine leukemia virus: analysis of virus subpopulations.

The leukemogenic activity of Gross murine leukemia virus adapted to rats was tested in W/Fu rats and NIH/Swiss mice. All animals infected with this virus developed thymic and nonthymic T-cell leukemia with a short latency period. It was observed that cell-free extracts from thymic lymphoma tissue of mice and rats, induced by either Gross murine leukemia virus or Gross murine leukemia virus adapted to rats, consisted of both small-plaque-forming and large-plaque-forming viruses, as determined by the XC plaque test. MCF-type virus was found in these virus complexes. Transformed cell foci were induced in SC-1 cell layers by double infection of the cloned MCF-type virus and an ecotropic virus. SC-1 cells containing transformed cell foci were shown to be tumorigenic upon inoculation into nude mice. The formation of transformed cell foci in mink lung cells was also observed after double infection with the cloned MCF-type virus and a xenotropic virus. The possible mechanism of leukemogenesis by endogenous viruses is discussed.     

Protection by polyI:polyC against infection with herpes simplex virus in mice pretreated with Corynebacterium parvum

The effect of Corynebacterium parvum on the protection by polyinosinic-polycytidylic acid (polyI:polyC) against lethal infection with Herpes simplex virus type 1 (HSV) was studied in mice. Pretreatment with C. parvum resulted in prolonged survival times in all experiments. One third of the mice survived an infection with 100 LD50, whereas all mice died when treated with polyI:polyC alone. Increased protection was observed up to 6 weeks after pretreatment and only seen when both C. parvum and polyI:polyC were given at the same site of injection (intraperitoneally). Protection against HSV correlated with increased interferon (IFN) activities induced by polyI:polyC in the peritoneal cavity of C. parvum-pretreated mice. In these mice, natural killer cell activity of peritoneal exudate cells (PEC) was also augmented in response to polyI:polyC. Protection was markedly decreased by intraperitoneal injection of silica or of an antiserum against murine IFN. It appears that increased local levels of IFN presumably produced by macrophages in response to polyI:polyC in C. parvum-pretreated mice play the major role in the antiviral defence in our model and that activation of NK cells may be a secondary effect of IFN.     

Induction of histiocytic sarcoma in mouse skeletal muscle

Myeloid sarcomas are extramedullary accumulations of immature myeloid cells that may present with or without evidence of pathologic involvement of the bone marrow or peripheral blood, and often coincide with or precede a diagnosis of acute myeloid leukemia (AML). A dearth of experimental models has hampered the study of myeloid sarcomas and led us to establish a new system in which tumor induction can be evaluated in an easily accessible non-hematopoietic tissue compartment. Using ex-vivo transduction of oncogenic Kras(G12V) into p16/p19(-/-) bone marrow cells, we generated transplantable leukemia-initiating cells that rapidly induced tumor formation in the skeletal muscle of immunocompromised NOD.SCID mice. In this model, murine histiocytic sarcomas, equivalent to human myeloid sarcomas, emerged at the injection site 30-50 days after cell implantation and consisted of tightly packed monotypic cells that were CD48+, CD47+ and Mac1+, with low or absent expression of other hematopoietic lineage markers. Tumor cells also infiltrated the bone marrow, spleen and other non-hematopoietic organs of tumor-bearing animals, leading to systemic illness (leukemia) within two weeks of tumor detection. P16/p19(-/-); Kras(G12V) myeloid sarcomas were multi-clonal, with dominant clones selected during secondary transplantation. The systemic leukemic phenotypes exhibited by histiocytic sarcoma-bearing mice were nearly identical to those of animals in which leukemia was introduced by intravenous transplantation of the same donor cells. Moreover, murine histiocytic sarcoma could be similarly induced by intramuscular injection of MLL-AF9 leukemia cells. This study establishes a novel, transplantable model of murine histiocytic/myeloid sarcoma that recapitulates the natural progression of these malignancies to systemic disease and indicates a cell autonomous leukemogenic mechanism.     

Delayed hypersensitivity and immune protection against herpes simplex virus: suppressor T cells that regulate the induction of delayed hypersensitivity effector T cells also regulate the induction of protective T cells

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.     

Minor effect of depletion of resident macrophages from peritoneal cavity on resistance of common carp Cyprinus carpio to blood flagellates

Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection.