Pathogenesis of wasting disease after cyclophosphamide treatment of neonatal mice.

Cyclophosphamide is a potent immunosuppressive agent and is being widely used in organ transplantation. The effects of this anti-rejection drug on lymphoid organs are poorly understood. Newborn Swiss mice injected with various doses of cyclophosphamide suffered from wasting disease at 4 weeks post treatment. The incidence of wasting disease was dose dependent. Haematological picture of the wasting animals revealed leukocytosis of variable degree. Lymphocyte/granulocyte ratio was not inhibited. The cyclophosphamide treatment caused shrinkage of lymphoid organs. Bone marrow showed degeneration of haematopoietic cells. The failure to sustain lymphopoiesis by the potential lymphoid sites following cyclophosphamide treatment and the associated immunological insufficiency resulted in the fatal wasting disease.     

Nippostrongylus brasiliensis: Effect of cyclophosphamide treatment on worm expulsion in rats

The precise immunological mechanisms associated with expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis remain controversial. In order to investigate the effects of drug-induced immunosuppression on parasite burdens and expulsion, various regimens of cyclophosphamide were administered to parasitized Wistar rats. It was observed that both the number of worms established from an infective dose of 3000 larvae and the time of expulsion were markedly increased with higher doses of cyclophosphamide. Thus, at the highest sublethal level of treatment (100 mg/kg), 82% of the infective dose was recovered at Day 9 postinfection compared with 51% in nontreated controls. Furthermore, in such treated rats expulsion was delayed in 6 days beyond that of nontreated animals. As cyclophosphamide, at the levels used in the present study, is known to primarily effect B-cell function, the results support the view that antibody-mediated responses play an essential role in worm expulsion.     

Effect of bone marrow suppression on granulocyte opsonin levels.

Levels of serum opsonin for neutrophilic granulocytes were measured in dogs made neutropenic by cyclophosphamide administration. Heat-labile opsonin became elevated within 24 hr following cyclophosphamide (P less than 0.005) and remained elevated over the 4-5 day period of observation (P less than 0.005). In contrast, heat stable opsonin was not significantly effected. Bone marrow suppression by X-ray and busulfan also caused serum opsonin levels to increase. Changes in the levels of IgG, C3, and total hemolytic complement during the course of bone marrow suppression did not correlate with the granulocyte opsonin levels. These findings suggest that serum granulocyte opsonin levels respond to bone marrow suppression and may provide an improved environment for the function of transfused granulocytes.     

Reversibility of the effects of chronic paternal exposure to cyclophosphamide on pregnancy outcome in rats

Low-dose chronic treatment of the male rat with the antitumor drug cyclophosphamide causes a time- and dose-dependent increase in pre- and post-implantation loss in the untreated females to which he is mated. The objective of the present study was to determine whether such effects are reversed, and if so at what time after cessation of drug treatment. Adult male Sprague-Dawley rats were gavage fed daily, 6 times per week for 9 weeks, with saline (control) or with 1 of 3 doses of cyclophosphamide, 1.4, 3.4 or 5.1 mg/kg/day. After the 9 weeks of treatment and at 2-week intervals thereafter, each male was mated with 2 females in proestrus. The females were caesarian sectioned 20 days later and pregnancy outcome assessed. After 9 weeks of drug treatment, pre-implantation loss increased more than 3-fold from 6% in the control group to 21% in the 5.1 mg/kg/day cyclophosphamide treatment group. Post-implantation loss increased in a dose dependent fashion from 5% in the control group to 74% in the 5.1 mg/kg/day cyclosphosphamide treatment group. Pre-implantation loss rapidly decreased upon cessation of treatment with cyclophosphamide: within 2 weeks it had returned to within the control range. Within just 2 weeks after termination of drug treatment in the 5.1 mg/kg/day cyclophosphamide treatment group, post-implantation loss decreased by half to 44%; it had decreased to 11% by 4 weeks and then was maintained at 4-6% thereafter. In the 3.4 mg/kg/day cyclophosphamide treatment group, post-implantation loss returned to the control range by 4 weeks. Thus, the effects of paternally administered cyclophosphamide on progeny outcome are reversible. The timing of reversal suggests that the effects on pre-implantation loss are due to a drug effect on spermatozoa either in the epididymis or near the time of spermiation while those on post-implantation loss are due to an additional effect on spermatids in the seminiferous tubules.     

Cyclophosphamide induced generation of giant hypersegmented granulocytes in rat bone marrow: cell cycle distribution and silver nucleolar staining

Daily injection of cyclophosphamide (20 mg/kg body weight) for 7 days resulted in accumulation of 50% of rat bone marrow granulocytes in G2. Tetraploid neutrophils were hypersegmented (7.25 +/- 0.33) in comparison with diploid ones (3.92 +/- 0.33). After 14 days of cyclophosphamide treatment tetraploid hypersegmented neutrophils could be found in peripheral blood. Diploid neutrophils in these animals were also hypersegmented (4.78 +/- 0.14 versus 3.15 +/- 0.02 in control, p less than 0.001). Nucleolar ribosomal gene activities, evaluated by morphometry of silver nucleolar grains, decreased on bone marrow granulocytes in the course of differentiation in control rats. After cyclophosphamide treatment mature granulocytes contained more silver grains than in controls which may be explained by conservation of silver binding sites of nucleoli from the stages of promyelocytes and myelocytes. These results suggest two mechanisms of hypersegmented neutrophil, generation in cyclophosphamide treated rats: the first, via maturation of myelocytes arrested in G2, and second, a direct one, without tetraploid granulocyte involvement.     

The effect of plasma on the reaction of cyclophosphamide with 4(-p-nitrobenzyl)-pyridine.

Cyclophosphamide is not structurally modified by blood plasma and may be recovered quantitatively from it. The apparent loss of alkylating ability of cyclophosphamide, as measured by its ability to react with 4-(p-nitrobenzyl)-pyridine (NBP), following treatement with plasma is not, as has been reported, due to metabolism of the drug but rather to an inhibition of the colorimetric reaction by a constituent of the plasma. This inhibition is strongly pH dependent, reaching 100% when the pH of the solution of cyclophosphamide in plasma is above 8, and falling to less than 20% when the pH is below 6, but extraction with chloroform at pH 7 separates cyclophosphamide from the inhibitor. Although the nature of the inhibitor has not been elucidated, its presence in plasma is of great importance in the quantitative determination of cyclophosphamide, and may also be of significance in the biological effects of cyclophosphamide and other alkylating agents in vivo.     

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.     

Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny

Cyclophosphamide is an anticancer and immunosuppressive agent commonly used in men of reproductive age. The relationship between the effects of paternal cyclophosphamide treatment on the male reproductive system and the pregnancy outcome is unknown. To study this relationship, adult male Sprague-Dawley rats were administered saline or cyclophosphamide (1.4, 3.4, and 5.1 mg/kg) daily for 11 wk by gavage. Each male was mated weekly with two females in proestrous; 20 days later, the females were caesarean-sectioned and the number of corpora lutea, resorptions, and normal and abnormal fetuses were noted. After 11 wk of treatment, none of the drug-treated males showed any significant difference compared to controls with respect to male reproductive organ weights, serum testosterone, luteinizing hormone or follicle-stimulating hormone, epididymal sperm counts or fertility. Despite the apparent minimal effects of the treatment regimen on the male reproductive system, there were a number of effects on pregnancy outcome. There was a dose-dependent increase in preimplantation loss at 5-6 wk that was not evident at other times, a progressive dose-dependent increase in postimplantation loss starting at 2 wk, and an increase in malformed and growth-retarded fetuses at 3-4 and 7-9 wk. These results indicate that low dose chronic cyclophosphamide treatment of the male rat can affect the outcome of his progeny; such effects are seen in the absence of any apparent alteration of a number of measures of male reproductive function.     

Elevation of a potassium current in differentiating human leukemic (HL-60) cells

Human promyelocytic leukemia (HL-60) cells display a novel voltage-dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL-60 cells after exposure to phorbol-12-myristate-13-acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl-. From a holding potential of -80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1-2 X 10(-6) M). These properties appear to differ significantly from the Ca2+-activated K+ channel and the delayed rectifier. The increase of this voltage-activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.     

Increased resistance to Listeria monocytogenes following subchronic cyclophosphamide exposure: Relationship to altered bone marrow function

Mice administered multiple doses of cyclophosphamide demonstrated a marked resistance to infection with the bacterium, Listeria monocytogenes. In contrast, acute exposure rendered mice more susceptible to infection than untreated controls. Resistance to infection with Listeria, a facultative intracellular organism, is thought to be dependent upon normal antimicrobial activity early after infection and subsequently through generation of primed T cells. Examination of various macrophage and immune functions, however, failed to demonstrate a significant difference between the two cyclophosphamide-treated groups although both groups were immunosuppressed when compared to untreated controls. Adoptive transfer studies into X-irradiated recipients revealed that repopulation with bone marrow cells from subchronic but not acute cyclophosphamide-treated mice, restored resistance. Furthermore, the numbers of granulocyte/macrophage progenitor cells in the bone marrow were elevated in subchronically treated mice but not acute or unexposed controls. These data suggest the selection of a granulocyte/macrophage progenitor cell possessing a high degree of antilisterial activity following subchronic cyclophosphamide treatment. The effects induced by this exposure regimen are probably related to the enrichment of this cell population resulting from the cell cycle stage specific activity of the drug.     

Leucokinetic study. Morphology of the bone marrow and blood after experimental induction of marrow hypoplasia by cyclophosphamide in laboratory rats

Experimental hypoplasia of femoral bone marrow in rats was induced by cyclophosphamide, injected i.p. at various clock hours (08.00, 12.00, 16.00, 20.00, 24.00, and 04.00). Cyclophosphamide-induced neutropenia persisted for six days and was followed by transitory granulocytosis with subsequent decrease in the cont of circulating mature granulocytes. The absolute counts of circulating segmented neutrophils changed in parallel with the absolute counts of segmented neutrophils in the bone marrow. The count of circulating neutrophils was not essentially influenced by the clock hour of cyclophosphamide injection. The toxic action of cyclophosphamide upon the bone marrow exhibited a circadian rhythmicity: the greatest decrease in the count of nucleated marrow cells was found in the morning, and the least, in the evening. The minimal compartment transit times of the development stages of bone-marrow neutrophils, and the daily granulocyte production in the rat were calculated.     

Suppression of cellular immune responses following transfer factor: Report of a case

A girl with chronic mucocutaneous candidiasis and recurrent staphylococcal infections had abnormal cellular immunity and defective granulocyte chemotaxis. Administration of dialyzable transfer factor caused conversion of the candida skin test and MIF production by candida-stimulated lymphocytes but had no effect on granulocyte chemotaxis and produced no clinical benefit.Nine months later, while her cellular immunity was still intact, she received four additional doses of dialyzable transfer factor. Following these injections, the delayed skin tests and antigen-induced lymphocyte transformation and lymphokine production temporarily became negative. There were no effects on granulocyte chemotaxis.These observations suggest that transfer factor may have both suppressive and activating immunologic activities.     

Bronchial hyperreactivity occurs in steroid-treated guinea pigs depleted of leukocytes by cyclophosphamide

The effects of cyclophosphamide and cortisone acetate treatment on O3-induced changes in airway mucosal morphology and bronchial reactivity were assessed in guinea pigs. Animals in groups of four were studied at 2 or 6 h after O3 (3.0 ppm, 2 h) and in one control group. Reactivity was determined by measuring specific airway resistance during intravenous acetylcholine infusion in intact, unanesthetized, spontaneously breathing animals. After testing, tracheal tissue was obtained from all animals for light microscopic examination. Another group of 10 drug-treated and 10 normal animals were tested at 2 h, 6 h, 1 day, and 4 days after O3. Drug treatment resulted in substantial decreases in both circulating and airway mucosal granulocytes. Two hours after O3, a marked decrease in airway mucosal goblet cells as well as ciliated cell damage occurred in both normal and treated animals. However, only in normal animals did neutrophilic infiltration develop thereafter. Nonetheless, hyperreactivity postozone occurred and progressed similarly in both groups. Our results indicate that acute O3-induced bronchial hyperreactivity at 2 h is associated with signs of airway mucosal injury but appears independent of granulocyte changes. Airway neutrophilic infiltration and eosinophil depletion seem to be consequences of mucosal injury from O3 and not causes of the bronchial hyperreactivity that results.     

Cyclophosphamide pretreatment in tumor cryotherapy: A murine model

Following the protocol of Turk and Lagrange for selective potentiation of cell-mediated immunity, a single high dose of cyclophosphamide was given to inbred mice bearing subcutaneous tumors 3 days before cryosurgery. Cyclophosphamide pretreatment caused a rise in the rate of total tumor ablation and local recurrence was delayed. The dose response to tumor size, cyclophosphamide, and freeze parameters indicated that these effects were the result of synergistic rather than additive processes. Instances of metastatic tumor growth and immunologically mediated complications of cryosurgery were also lower when the cryosurgery was accompanied by cyclophosphamide pretreatment.     

Characterization of a novel SOD-1(G93A) transgenic mouse line with very decelerated disease development

Amyotrophic Lateral Sclerosis (ALS) is a fatal motoneuron disease, characterized by progressive weakness, muscle wasting and death ensuing 3-5 years after diagnosis. The etiology of ALS is complex and therapeutic approaches rely mostly on transgenic animal models with SOD-1 mutations. Most frequently employed is a mouse line transgenic for SOD-1 (SOD-1 Tg) that contains a point mutation at amino acid position 93 (G->A), present in patients suffering from a familial form of amyotrophic lateral sclerosis. Here we report on a SOD-1 (G93A) Tg mouse line with abnormally delayed onset of disease and prolonged survival. This phenotype arose spontaneously in our colony of the classic SOD-1 (G93A) line. We found that the copy number of the SOD-1 transgene was drastically decreased. We established a new breeding colony, the SOD-1 (G93A)(PS) line (PS for prolonged survival) where the phenotype is stably inherited for 4 generations now. The mice develop symptoms at an age of approximately 12 months and die at 15 months of age. The delayed development of disease may more closely mimic human pathophysiology, and studying drug effects in this model may yield added confidence for potential efficacy of ALS drug candidates.     

Interference of anti-tumor and immunosuppressive effects of cyclophosphamide in tumor-bearing rats

Summary Adult rats were given 10⁵ or 10⁶ Yoshida ascites sarcoma (YAS) cells IP and were treated with cyclophosphamide (CY) given IP in single doses of 20 mg/kg or 100 mg/kg, 2 or 5 days after YAS inoculation. Both the curative effect of CY and subsequent resistance to tumor challenge in rats that survived depended on the dose of injected tumor cells and on the dose and time of administration of CY. These three factors determined whether the host's immune response to tumor antigens would develop and contribute to the overall anti-tumor effects of the chemotherapy. The curative effects of CY were significantly less pronounced in T-cell-deficient than in normal rats. Anti-tumor and immunosuppressive activities of CY exerted opposite influences on the ultimate result of the chemotherapy. Adverse immunosuppressive effects prevailed when the drug was administered early (2 days) after YAS inoculation. In this case the chemotherapy was less efficient and the surviving rats were susceptible to a subsequent tumor challenge. Further analysis showed that the injection of CY 2 days after inoculation of YAS antigens induced strong and specific immunologic tolerance to the tumor. In contrast, when a sufficient amount of tumor antigens (higher dose of tumor cells injected and CY injection delayed) elicited an anti-YAS immune response that was not suppressed by early injection of CY (CY administered 5 days after the tumor) effective eradication of tumor cells and anti-YAS resistance in cured animals were observed.Abbreviations YAS Yoshida ascites sarcoma - CY cyclophosphamide - MRBC mouse red blood cells     

Role of circulating platelets and granulocytes in PAF-induced pulmonary dysfunction in awake sheep

The effects of a single intravascular bolus injection of platelet-activating factor (PAF) on pulmonary hemodynamics, lung mechanics, and lung fluid and solute exchange were studied in 13 chronically instrumented unanesthetized sheep. Since PAF has profound effects on both platelets and granulocytes, we investigated the effects of platelet and granulocyte depletion on the sheep's response to exogenous PAF. Sheep received PAF when granulocyte and platelets counts were normal and after platelet depletion with rabbit antisheep platelet antibodies (n = 5) or granulocyte depletion with hydroxyurea (n = 5). Sheep served as their own controls, and the order of experimentation was varied. Bolus injections of PAF had reproducible effects on pulmonary hemodynamics (pulmonary arterial pressure increased acutely to 85 +/- 3.7 cmH2O) and lung mechanics (dynamic compliance of the lungs decreased to 24.5 +/- 3.8% of base line and resistance to airflow across the lungs increased greater than 10-fold) and caused marked increases in lung lymph concentrations of thromboxane B2 and 6-ketoprostaglandin F1 alpha. The single bolus injection of PAF also caused marked prolonged elevations in lung lymph flow and increases in the lymph-to-plasma protein concentration ratio for 3 h after PAF. PAF had profound effects despite platelet and granulocyte depletion. Platelet depletion slightly attenuated the pulmonary hypertension observed after PAF injection. Platelet depletion also attenuated the increases in thromboxane B2 concentrations in lung lymph, and lung mechanics normalized more rapidly in platelet-depleted sheep. There were no statistically significant effects of granulocyte depletion to less than 200 granulocytes/mm3 on any of the measured variables.(ABSTRACT TRUNCATED AT 250 WORDS)     

A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems

We have found previously that daily treatment of male rats for 11 wk with low doses of the anticancer drug cyclophosphamide had no apparent effect on male reproductive organ weights, epididymal sperm counts, or serum hormones at the end of the treatment period; yet, upon breeding to untreated females, these males produced a high rate of post-implantation loss and fetal anomalies. The present study was designed to investigate the time course and dose response of the effects of chronic cyclophosphamide treatment on the male reproductive and hematologic systems. Male Sprague-Dawley rats were gavage-fed for 1, 3, 6 and 9 wk with saline (control), or 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide. After each of the treatment periods, males were mated to determine the effect on pregnancy outcome, then killed, and the effects on the male reproductive and hematologic systems were assessed. After 6 wk of treatment, a sharp increase in mortality was found between the 5.1 and 6.8 mg/kg/day doses of cyclophosphamide. The high dose of cyclophosphamide induced higher levels of pre- and post-implantation loss but fewer fetal anomalies than did the low dose. The low dose of cyclophosphamide did not affect reproductive organ weights; in contrast, the high dose caused decreases in epididymal, ventral prostate, and seminal vesicle weights after 3, 6, and 9 wk. Testicular and epididymal sperm counts were decreased in a dose-dependent manner after 3 wk; in addition, the high dose led to a decrease in epididymal sperm counts after 6 wk of treatment. Another rapidly proliferative tissue, the bone marrow, was dramatically affected by both doses of cyclophosphamide at all time points, with leukocyte counts decreasing to 40% of control by 1 wk. After 9 wk of treatment, effects on the male reproductive system were less marked, compared to earlier time points, whereas those on the hematologic system and pregnancy outcome persisted. Thus chronic low-dose treatment of male rats with cyclophosphamide not only had early and striking effects on the bone marrow and the pregnancy outcome but also affected the male reproductive system in a clear time- and dose-dependent manner.     

Inhibition of normal granulopoiesis by cytostatic agents.

Rabbits were treated with cyclophosphamide and 5-fluorouracil, myelosuppressive cytostatic agents, applied with a single dose of 1/3 LD50 or daily doses of 1/30 LD50 given for 14 days. Functional tests for evaluation of granulopoiesis were regularly performed at standard intervals and were following: leukocytosis, bone marrow picture with mitotic index, 3H-thymidine incorporation in vitro followed by autoradiography of labeled promyelocytes and myelocytes, serum lysozyme activity, mobilization of granulocyte reserve pool by staphylococcal alpha-toxin, cytochemistry of granulocytes, phagocytosis ability and Nitro-BT reduction. It has been found that 6-10 days after application of cytostatics, a marked depression of proliferation of young granulocyte forms and lowered reserve pool, are regularly observed. This was followed by spontaneous regeneration of granulopoiesis. No changes were noted in functional tests of mature granulocytes in peripheral blood. It is suggested that for investigation of the impairment of granulopoiesis after application of cytostatic agents, most suitable is evaluation of mobilization of the bone marrow reserve pool, lysozyme activity in blood serum and labelling of promyelocytes and myelocytes with 3H-thymidine in vitro.     

Potentiation by blastolysin of the effect of cyclophosphamide in preventing recurrence in mouse T-cell leukemia

Injection of cyclophosphamide in a dose of 100 mg/kg two or four times at 18- or 7-day intervals, respectively, prolonged the remission in EL-4-leukemia-bearing C57Bl/6j mice induced by cytosar under deoxycytidine protection. Injection of blastolysin in a dose of 25 mg/kg once a week at the same period had no effect. However, blastolysin potentiated the antirelapse effect of cyclophosphamide. It is suggested that the potentiation of the cyclophosphamide effect with blastolysin is mediated by activation of antitumor immunological reactivity.