Single Injection Of Cyclophosphamide Research Articles

Susceptibility to influenza A virus infection in mice immunosuppressed with cyclophosphamide.

It is known that immunocompromised hosts show an enhanced susceptibility to microbial infections. Among these, viral infections represent a particular problem because of the lack of really efficient antiviral drugs. In the present report we have studied the effect of pharmacological immunosuppression with cyclophosphamide (CY) in virus infection in vivo, using Balb/c mice infected with influenza A virus (PR8). At the dose of 0.1 hemagglutinating units of viral inoculum, intranasal administration of PR8 virus caused the death of 50 to 60% of the animals within a period of 3-10 days. A single injection of CY (200 mg/kg) significantly increased mouse mortality to 90%, when PR8 virus challenge was performed 4 days after chemotherapy pretreatment. When the PR8 virus infection was performed at different times after CY-treatment, a similar appreciable effect was not observed. Severe alterations of some immunological parameters such as NK activity and analysis of lymphoid spleen cell subsets were related to the increased susceptibility to virus infection.

Administration of IL-7 to mice with cyclophosphamide-induced lymphopenia accelerates lymphocyte repopulation.

Lymphopenia was induced in mice by a single injection of cyclophosphamide. IL-7 or a control protein were administered to the mice twice daily and the cellularity and composition of the spleen, lymph node, bone marrow, and thymus were determined at various time points thereafter. In comparison to the control cyclophosphamide-treated mice, animals receiving cyclophosphamide and IL-7 had an accelerated regeneration of splenic and lymph node cellularity. There was no significant difference in the rate of recovery of the bone marrow and thymus of the control and IL-7-treated mice. Assessment of the pre-B cell compartment revealed a dramatic increase in total pre-B cell numbers in the spleen and bone marrow of the IL-7-treated mice as measured by both flow microfluorimetry and a pre-B cell colony-forming assay. This was followed in a few days by a significant increase in surface IgM+B cell numbers to levels above normal values in both the spleen and lymph node. IL-7 administration to cyclophosphamide-treated mice also resulted in an accelerated recovery of peripheral CD4+ and CD8+ cell numbers in the spleen and lymph node. The numbers of CD8+ cells were increased by twofold over normal levels in cyclophosphamide-treated mice receiving IL-7. Myeloid recovery was determined in cyclophosphamide treated mice by assessing the numbers of CFU-granulocyte-macrophage and Mac 1+ cells. There was no significant difference in myeloid recovery between cyclophosphamide-treated mice receiving IL-7 or control protein. These results suggest that administration of IL-7 after chemical-induced lymphopenia may have therapeutic benefits in shortening the period required to achieve normal lymphoid cellularity.

Chemoimmunotherapy with cyclophosphamide and bestatin in experimental metastasis in mice.

Bestatin has significant therapeutic activity (even following oral administration) for the treatment of metastatic disease, an activity which is limited by tumor burden. Therefore, the therapeutic potential of bestatin was examined in combination with chemotherapy to determine if there is additive activity for heavy tumor burdens. Bestatin significantly increased therapeutic activity and decreased the myelotoxicity of cyclophosphamide following a single injection of cyclophosphamide or split daily doses. In immune function studies, in tumor-bearing animals, bestatin increased the number of colony-forming units (granulocyte-macrophage) (CFU) and alveolar macrophage tumoricidal activity. However, when bestatin was combined with cyclophosphamide, which depressed bone marrow and macrophage activity, it did not show apparent augmentation of macrophage and NK cell activity, but did significantly increase bone marrow CFU activity. Thus, in combined chemoimmunotherapy, bestatin appears to enhance therapeutic activity by accelerating the recovery of hematopoiesis. We suggest, therefore, that a combination chemotherapy protocol, with oral bestatin, may facilitate myelorestoration following aggressive chemotherapy. The majority of biological response modifiers require parental administration; thus, the identification of an orally active, synthetic immunoaugmenting agent with a defined receptor is of particular interest.

Immunotherapeutic studies on EN-L2C guinea pig leukemia

Effect of various immunomodulators in combination with chemotherapeutic agents were studied using EN-L2C cell line, an acute lymphoblastic leukemia (ALL) of inbred strain-2 guinea pig.Results obtained were followings.1) Typical leukemic cells appeared in peripheral blood 12 days after the inoculation of 105 cells and the hosts died within 5 days. When EN-L2C cells were inoculated together with BCG or N-CWS (Nocardia rubra cell wall skeletones), the host survived significantly longer than controls.2) Single injection of cyclophosphamide (CY ; 35 mg/kg, BW) 14 days after animals receiving inoculation of EN-L2C cells induced complete remission. However, it lasted 2 weeks. A repeated administration of BCG, N-CWS or irradiated EN-L2C cells plus BCG during the remission period did not prolong the period of remission.3) Immunization of animals during the remission period with Mitomycin C-treated ENL2C cells either conjugated or mixed with PPD did not delay the relapse.4) During hematological relapse, meningeal involvement was proven histologically. The animals received irradiation over cerebrospinal area with 60Co on the following day after they were given CY survived several days longer than the non-irradiated controls. An additional injection of N-CWS to the irradiation plus CY regimen did only slightly but significantly prolong the survival days. Passive transfer of specifically immunized lymphocytes to the animals after they received CY did only slightly but significantly prolong the survival days with or without the addition of craniospinal irradiation.

Modulation of c-myb transcription in autoimmune disease by cyclophosphamide.

This study explores the relationship between autoimmunity and the myb proto-oncogene, a gene important for T cell development. The lpr/lpr mice had very large amounts of myb RNA in the lymph node (LN) cells; but unexpectedly, they had abnormally low levels of myb RNA in the thymus, an organ normally rich in myb RNA. Mice with the gld/gld genotype had high myb RNA levels in peripheral LN, similar to lpr/lpr mice, but had normal thymic myb RNA levels. Both lpr/lpr and gld/gld mice and an AILD patient with lymphadenopathy and high myb RNA in peripheral blood cells were treated with cyclophosphamide (CY). In all cases, the CY eliminated the lymphadenopathy and corrected the abnormal myb expression. However, there were significant differences in the clinical and cellular responses to this drug. A single large dose of CY led to marked regression of the lymphadenopathy of gld/gld mice and long-term amelioration of their autoimmune syndrome. In contrast, similar treatment of lpr/lpr mice failed to alter either the lymphadenopathy or the disease process. Consistent with these clinical findings, LN myb was normalized in gld/gld mice by a single injection of CY, whereas there was no effect on myb expression in lpr/lpr mice. The AILD patient reacted much like the gld/gld mice in that myb RNA levels in the peripheral blood, and bone marrow returned to normal after only three doses of CY. The lymphadenopathy and high levels of LN myb mRNA of the lpr/lpr mice could be normalized; this occurred only after long-term treatment with CY. These events were accompanied by an increase in thymic myb mRNA from low levels. These studies have combined a molecular probe with CY therapy to provide insights into the cellular bases for lymphoproliferative autoimmune diseases.

Nature of the suppressor cells mediating prolonged graft survival after administration of extracted histocompatibility antigen and cyclosporine.

Antigen-specific suppressor T cells are induced by donor histocompatibility antigen extracted from spleen cells with 3M KCl combined with cyclosporine (Ag-CsA). A single i.v. injection of 5 mg 3M-KCl-extracted donor Buffalo (Buf, RT1b) antigen (Ag) combined with a three day course of CsA prolonged renal allograft survival in Wistar-Furth (WFu, RT1u) hosts to a greater extent (MST 26.5 days) than CsA alone (MST 11.8 days). Peripheral blood lymphocytes (PBL) or spleen cells harvested from Ag-CsA-treated recipients ten days after transplantation inhibited the mixed lymphocyte reaction (MLR) between normal responder WFu cells and irradiated Buf cells (55.6% and 64.4% suppression, respectively, P less than 0.025), but not third-party Brown-Norway (BN, RT1n) stimulator cells (13.6% and -18.3% suppression, respectively, NS). The suppressor effect was not mediated by cytolytic cells; there was neither primary nor secondary cytolytic activity against 51Cr-labeled Con-A blastoid Buf cells. The suppressor cells were neither adherent to plastic dishes nor to nylon-wool columns. PBL irradiated with 800 rads, but not 1500 rads, suppressed the MLR. A single injection of cyclophosphamide (CY, 25 mg/kg) seven days after transplantation abrogated the suppression induced by Ag-CsA treatment. Moreover, PBL from Ag-CsA recipients failed to suppress the MLR, if depleted either of all T cells by treatment with monoclonal antibody (Mab) W3/13 HLK (pan T cells; % suppression -15.8), or of cytotoxic/suppressor cells with Mab OX-8 (-19.3% suppression) together with rabbit antimouse immunoglobulin and complement. On the other hand, PBL treated with the Mab W3/25 (helper) showed suppressor cell activity (+56.4%, P less than 0.001) similar to untreated cells (62.4%, P less than 0.001). Moreover, adoptive transfer of suppressor T cells purified from pooled lymphocytes by rosetting using Mab significantly prolonged the survival of donor-specific, but not third-party, test grafts in naive secondary hosts. Thus, these studies demonstrated antigen-specific suppressor T cells mediate the long-term unresponsiveness induced by the Ag-CsA regimen.

Immunologic requirements for the adoptive transfer of ectromelia virus meningitis.

Intracerebral infection with the Hampstead egg strain of ectromelia virus in mice produced a meningitis with clinical signs and death. A single injection of cyclophosphamide three days after infection delayed the onset of pathology and clinical signs. Adoptive transfer of immune spleen cells into preinfected recipients could induce the meningitis earlier. These donor-immune cells were virus-specific in their action. The Ig- population, deficient in B lymphocytes, could transfer meningitis; but donor-immune cells depleted of T lymphocytes by anti-theta treatment or the Ig+ population, deficient in T-cells, were unsuccessful. Studies with labeled cells showed a preferential accumulation of labeled donor cells in cerebrospinal fluid in the first 24 hours post-transfer, whereas labeled recipient cells accumulated between 24 and 48 hours. The results demonstrated that the adoptive transfer of ectromelia virus meningitis was a cell-mediated inflammatory process.

The effects of cyclophosphamide on antibody dependent cell-mediated cytotoxicity in mice.

The effect of cyclophosphamide (CY) on antibody dependent cell-mediated cytotoxicity (ADCC) was evaluated. The results indicated that a single injection of CY markedly increased ADCC activity of murine splenic cells. This effect was dose dependent and was maximal on day 7 after the drug injection. The number of lymphoid cells in treated mice decreased to approximately 50%; however the percentages of EA rosettes were similar to those of control animals suggesting that the augmented cytotoxic activity was not due to a relative increase in Fc receptor bearing cells. CY treatment also enhanced delayed type hypersensitivity, but suppressed antibody production to sheep red blood cells. The possibility that CY eliminates a suppressor cell subpopulation that normally regulates ADCC levels is discussed.

Host treatment with cyclophosphamide elicits transient changes in graft-versus-host reactivity of donor cells.

We have investigated the effects of a single injection of cyclophosphamide (CY) in F1 host mice. One day after CY treatment, F1 host mice show increased susceptibility to graft-versus-host (GVH)-reactive parental strain spleen cells when measured by the popliteal lymph node (PLN) enlargement assay. F1 host mice challenged with parental strain cells 7 days after CY treatment show decreased PLN enlargement as compared with CY-untreated F1 hosts receiving parental strain cells. The PLN reactivity of normal F1 spleen cells injected into F1 hosts pretreated with CY was also measured. F1 spleen cells elicited marked PLN enlargement when injected into F1 hosts that were treated with CY 1 day previously. By day 7 after CY treatment of F1 hosts, F1 cells no longer produced significant PLN enlargement as compared to untreated F1 hosts receiving F1 cells. We discuss these results in terms of the possibility that CY initiates changes in host antigenicity that can lead both to an increase in responsiveness of GVH-reactive cells and to susceptibility to attack by syngeneic cells.

Modulation of granulomatous hypersensitivity. II. Participation of Ly 1+ and Ly 2+ T lymphocytes in the suppression of granuloma formation and lymphokine production in Schistosoma mansoni-infected mice.

The regulatory mechanism(s) active in the spontaneous suppression of parasite egg-induced granulomatous response was analyzed in Schistosoma mansoni-infected mice. A single injection of cyclophosphamide (CY) given to chronically infected mice with diminished granulomas and impaired ability to produce MIF restored the enhanced granulomatous response and lymphokine production by spleen cells. Drug-treated animals also showed a rise in the level of circulating anti-egg antigen antibody. After CY treatment, spleen cells were still capable of adoptively suppressing the vigorous granulomatous response and MIF production in acutely infected recipients. Adoptive suppression, however, was abrogated when spleen cells of chronically infected mice were pretreated with anti-Lyt 1.1 alloantiserum and C before transfer. Based on the present and previous observations, it is proposed that the modulated granulomatous inflammatory response in mice with chronic infection is maintained by a dynamic equilibrium between effector and regulator lymphocytes. Although the inflammation is maintained by Ly 1+ Ia- TDH cells, the intensity of the response is regulated by Ly 1+ Ia+ TH, Ly 2+ Ia+ Ts and possibly a putative precursor population of lymphocytes.

Motor function recovery after axotomy: Enhancement by cyclophosphamide and spermine in rat

The rate of motor function recovery after sciatic nerve freezing was measured in rats treated with (i) a single injection of cyclophosphamide (75 mg/kg) or (ii) a daily injection of spermine (10 mg/kg). The axotomy was made at one of three levels and the recovery of motor function was indicated by the return of the toe spreading reflex. A linear regression analysis of the recovery time on the length of degenerating peroneal nerve was used to calculate the recovery rate of the motor function. The recovery rate increase in both treated groups was highly significant compared with a control group. According to the known cellular activity of cyclophosphamide and spermine, these results support the hypothesis that an accelerated metabolic activity of the severed motoneurons could enhance the motor function recovery.

Mechanisms of antigen-induced blockade of immune response and cyclophosphamide-promoted tolerance to Salmonella typhi Vi antigen.

Blockade of the immune response, caused by a high dose of Salmonella typhi Vi antigen (200 microgram i.v.) and cyclophosphamide (CY)-induced tolerance to Vi antigen, were analyzed. The results of the study show that blockade of the immune response cannot be attributed to masking of the response resulting from neutralization of antibodies by the excess of non-cell-bound antigen. A high dose of Vi-antigen induced triggering and proliferation of specific B precursors but reversibly suppressed synthesis or secretion of antibody by plaque-forming cells. A single injection of CY (200 mg/kg i.p.) 2 days after a high dose of Vi antigen markedly prolonged the antigen-induced state of unresponsiveness. CY-induced tolerance to Vi antigen is due to elimination or long-term inactivation of specific B precursors. Dissimilarities in the characteristics of immune response blockade and CY-induced tolerance are discussed as well as their possible implications for the mode of action of CY.

Immunity Against MOPC 104E Plasmacytoma: Effects of Tumor Size and Time Post Therapy on In Vivo Tumor Immunity 2

BALB/c mice with the plasmacytoma MOPC 104E were cured of palpable tumors (6-15x10(7) cells) with a single injection of cyclophosphamide (10 mg/kg). Animals cured of tumor showed a considerable increase in their ability to reject secondary challenge with graded numbers of viable tumor cells. Mice with palpable subcutaneous tumors were cured therapeutically and rechallenged 22, 44, or 120 days post therapy. The ability of such animals to reject secondary tumor cell challenge was similar in all groups, which implied that in vivo tumor immunity remained relatively constant for at least 4 months post therapy. A second group of animals was treated therapeutically (10 mg cyclophosphamide/kg) 4, 11, or 20 days post tumor cell injection. These therapeutically treated animals were then rechallenged with various numbers of viable tumor cells 30 days post therapy. Mice given cyclophosphamide 4, 11, or 20 days post tumor injection rejected 6, 60, or 400 times as many tumor cells, respectively, as did control animals. These results implied that, over the range of tumor sizes investigated, exposure to greater amounts of tumor antigen resulted in increasing amounts of residual tumor immunity following cure.

Effects of cyclophosphamide treatment before implantation on the development of rat embryos after implantation

After treatment of pregnant rats 24 h before implantation with a single injection of cyclophosphamide (20--80 mg/kg), a dose-dependent increase in resorption was observed at term but no malformed fetuses could be found. The lowest cyclophosphamide dose that caused 100% resoprtion was 60 mg/kg. Somite number and wet weight indicated retardation of about 24 h during organogenesis. Determination of the time of implantation revealed that the developmental retardation in treated embryos was not due to delayed implantation. At implantation, 24 h after cyclophosphamide treatment, a significant and dose-dependent decrease of the cell number of blastocysts was found. Embryo transplantation experiments showed that early cyclophosphamide treatment interfered with the subsequent development of both the embryo and the mother. The decidual reaction seemed to be more affected by the treatment than the embryos. Most teratologists hold that mouse embryos after treatment in the preimplantation period either die before implantation or survive to term without being malformed. The present study, however, proves that the reaction of drugs at this early stage of pregnancy is more complex than is generally assumed.

Effect of cyclophosphamide treatment on the response of rat peripheral blood lymphocytes to phytohemagglutinin

The lymphoproliferative responses of rat peripheral blood lymphocytes to phytohemagglutinin (PHA) were studied following treatment with single or multiple doses of cyclophosphamide. A dose-dependent lymphocytopenia was observed with both regimes. The remaining lymphocytes had decreased responses to PHA. Serum collected 24 hr after a single injection of cyclophosphamide and used at a concentration of 5% enhanced the response of cells from normal or cyclophosphamide-treated rats. Serum collected after a course of treatment did not have this effect, but it lacked the marked suppressive activity, at a concentration of 20%, which was shown by normal rat serum. The enhancing activity was not dialysable. Doses of cyclophosphamide adequate to abolish primary antibody production to sheep erythrocytes did not totally abrogate responsiveness to PHA. Thus, the pattern of immunological defects in cyclophosphamide-treated rats consisted of decreased primary antibody production, lymphocytopenia with a decreased response of the remaining lymphocytes to PHA, and diminution of serum suppressive activity.

Effects of variation in time and dose of cyclophosphamide injection on delayed hypersensitivity and antibody formation

A single injection of Cyclophosphamide (CY) in a dose of 300 mg of CY/kg of mice resulted in enhanced delayed hypersensitivity (DH) when administered between at least 7 days prior to and 15 days after intracutaneous (ic) immunization of sheep red blood cells in Freund's complete adjuvant. The maximal enhancement occurred when CY was applied 8 hr before the antigen. Using the latter interval, the effect of varying the dose of CY before ic or intraperitoneal (ip) injections of antigen was studied. Combined with ic immunization, increasing doses of CY resulted in increasing DH. The ip route of immunization needed CY in amounts of at least 100 mg/kg to augment DH to a detectable level. The enhancing effect of lower doses of CY was more pronounced when the interval between immunizing and eliciting injections was reduced. Administration of 300 or 200 mg of CY/kg before ip immunization suppressed the antibody formation, when measured S and 7 days later. A dose of 100 mg of CY/kg caused a suppression of antibody formation on Day 5, but an enhancement on Day 7. With this dose, a maximal enhancement of DH was found on both days. The results suggest that CY interferes with more than one regulatory mechanism of the immune response.

Interference of cyclophosphamide with the development of T helper activity in rabbits.

In rats antibody formation against Brucella was suppressed by a single injection of cyclophosphamide given between 1 day before and 4 days after antigenic stimulation (1). A selective suppression of 7S antibody synthesis was described in rats immunized with Sheep Red Blood Cells (SRBC) after treatment with a course of 5 daily injections of cyclophosphamide, started 2 days after antigen injection (2). In mice a cyclophosphamide induced immunological tolerance to SRBC was found when the drug was given simultaneously with or 2 days after antigen injection (3.4). In most of these systems, coöperation between T and B cells is required for antibody production. In rats (5), guinea pigs (6) and rabbits (7) the histological changes caused by a single injection of cyclophosphamide were described. In all species cells homing in the non-thymus-dependent area’s of spleen and lymph nodes proved to be sensitive to the drug.KeywordsMemory CellAntigenic StimulationAntigen InjectionAccelerate ResponseStimulate Lymph NodeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Appearance of foci of hematopoiesis and of hematopoietic stem cells in the mouse liver after a single injection of cyclophosphamide

On the 6th day after a single intraperitoneal injection of cyclophosphamide in a dose of 200 mg/kg body weight into mice of lines DBA/2, C57BL/6j, and F1(C57BL/6j×CBA) foci of hematopoiesis appeared in the liver and lymph glands. This process was accompanied by the appearance of hematopoietic stem cells in the liver. Their number reached a maximum on the 6th and 9th days. On the 12th day their number fell, but it was still higher than in animals receiving an injection of a suspension of normal liver cells. After exhaustion of the pool of bone marrow and spleen stem cells by myleran, a subsequent injection of cyclophosphamide did not induce the appearance of foci of hematopoiesis and stem cells in the liver.

Drug-induced tolerance: selective induction with immunosuppressive drugs and their synergistic interaction.

Drug-induced tolerance to polyinosinic-polycytidylic acid was studied in NZB/W mice. A single injection of cyclophosphamide (CY) or methotrexate (MTX) induced tolerance when given 24 h after antigen, whereas azathioprine (AZ) and methylprednisolone (MP) did not. However, MP and MTX each synergized with a nontolerogenic dose of CY. In addition, MP and AZ further reduced the tolerance induced by low but tolerogenic doses of CY. Drug combinations in the induction of tolerance may have wide application in the fields of clinical immunology and transplantation.

Studies on the effect of cyclophosphamide on T and B lymphocytes in the blood, lymph nodes, and thymus of normal guinea pigs.

Cyclophosphamide administered three times at 48-hour intervals to normal guinea pigs caused a fall in the percentage of B lymphocytes and a rise in that of T lymphocytes in the blood. A single injection of cyclophosphamide caused little change in the percentage of T and B lymphocytes. There was a marked reduction in total lymphocyte count with both dose regimes with resultant falls in T and B cell levels when these were expressed as reacting cells/mm<sup>3</sup> of blood. Cyclophosphamide, when injected three times at 48-hour intervals, caused the percentage of B lymphocytes to fall and that of T lymphocytes to rise in lymph nodes. There was no alteration in T or B lymphocyte levels in thymus following cyclophosphamide treatment.