RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivoGalleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutansIMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

Increased urinary excretion of cyclic guanosine monophosphate in rats bearing Morris hepatoma 3924A.

Urinary excretion of cyclic guanosine monophosphate (GMP) increased in rats bearing Morris hepatoma 3924A, and a correlation coefficient of .842 was observed comparing nucleotide excretion and tumor size. Irradiation of tumor or 5-fluorouracil administration delayed the increases in urinary cyclic GMP and tumor size. Surgical removal of tumors resulted in a rapid decline in cyclic GMP excretion to baseline levels. Cyclic adenosine monophosphate excretion was not altered by implantation, irradiation, or excision of tumor.

Changes in serum putrescine and spermidine levels following local radiation to hepatoma 3924A of the rat.

Local irradiation of tumors of rats bearing 3924A hepatomas resulted in more than a doubling of the putrescine levvel and nearly a doubling of spermidine concentration in the serum within 12 hr. Within 24 hr, the putrescine concentration had increased four-fold in the serum, along with a continued increase in the spermidine concentration. The decrease in the spermidine concentration of the tumor paralleled increased levels of spermidine in the serum, whereas the concentrations of polyamines in the liver were unchanged. These changes are similar to previously reported changes in the spermidine concentration in sera and in tumors following the administration of a single dose of 5-fluorouracil to rats with 3924A hepatomas. Since local irradiation was confined to the tumor, we conclude that the increases in putrescine and spermidine detected in the serum are derived from the tumor tissue with no involvement of the host tissues.

A hematopoietic stromal lesion associated with fractionated radiotherapy (FxRT): time- and dose-effects.

Earlier, we reported that the local exposure of femoral bone marrow to a clinically-relevant course of fractionated radiotherapy [FxRT; 2.0 Gy (q24h x 5) --> 74 Gy] resulted in the delayed appearance of a stromal lesion which temporally appeared after exposures to a threshold dose of 20 Gy FxRT. To further define this threshold dose, the temporal recovery of blood-forming elements ("cobblestone area" forming colonies; CAFC(7-28d)) and the microenvironmental stroma (long-term bone marrow cultures; LTBMC) of the marrow was examined over a 17-week period following 10 and 30 Gy FxRT. After a subthreshold dose of 10 Gy, each of the 4 CAFC subpopulations were significantly dampened, with recovery initiated within a 6-week interval of 10 Gy of FxRT. Above the threshold dose (30 Gy FxRT), the CAFC subpopulations were again reduced to a level similar to that observed with 10 Gy FxRT. However, after exposures to 30 Gy FxRT, CAFC recovery was significantly well beyond the 6-week interval observed with a 10 Gy Dose of FxRT. Similarly, cell production in LTBMC prepared from marrow exposed to either 10 or 30 Gy FxRT was significantly dampened for at least 3 weeks following the FxRT. Moreover, while cell production in LTBMC derived from marrow exposed to 10 Gy was eventually restored to normal, the dampened cell production observed in LTBMC prepared after 30 Gy FxRT persisted for a period in excess of 17 weeks. Collectively, these observations provide additional support to our earlier observation suggesting that FxRT generates two forms of dose-dependent damage in the marrow: the first an early lesion arising in the blood-forming CAFC subpopulations; the second form, a delayed lesion that involves the persistent expression of a dysfunctional microenvironmental phenotype, appearing to disrupt the normal regulation of hematopoietic stem cell (HSC) proliferation and differentiation of the HSC during the recovery process.

Absence of interleukin 1 alpha radioprotection in tumor-bearing animals: elevated plasma levels of prostaglandin E versus a preexisting primed marrow.

Recombinant human interleukin 1 (IL-1) administered as a "priming" agent 24 h prior to hematopoietically lethal doses of total body irradiation (TBI) confers radioprotection to normal C57B1/6 (B6) mice, but not to B6 tumor-bearing animals (TBAs) known to have altered hematopoietic steady states. Using the Lewis lung tumor (LLca) in the B6 mouse, studies were carried out to determine whether the failure of IL-1 to radioprotect the LLca TBA was related to a preexisting "primed" hematopoietic state in the TBA or resulted from inhibition of myelopoietic activity associated with the production of prostaglandin E (PGE) by, or in response to, the tumor. Both normal B6 and LLca B6 TBAs were injected (every 24 h x 1-5) with 100 micrograms of indomethacin (IND) prior to the administration of IL-1. A single treatment with IND was sufficient to reduce the elevated levels of PGE found in the plasma of the TBAs. After five treatments, IND reduced the PGE level to below that of controls. Neither the acute nor the protracted IND treatment, however, affected the expansion of the stem and progenitor cell compartments of the marrow in the LLca TBA. Furthermore, no evidence of restoration of the radioprotective properties of IL-1 was observed in TBAs pretreated with IND. Collectively, these data suggest that the failure of IL-1 to provide radioprotection to the LLca TBA is not a direct result of the elevated plasma PGE levels associated with growth of the LLca tumor. In addition, these studies provide insight into the importance of examining in vivo effects of biological molecules in altered, as well as normal, physiological states.

Effect of prolonged expansion of marrow progenitor compartments following doxorubicin (ADR) on subsequent radiation tolerance.

The long-term effects of a maximum tolerated dose of doxorubicin (ADR) (10 mg/kg, LD10/60) on the recovery of the hematopoietic compartments of the femoral marrow from radiation (450 rad) were investigated over a 32-week interval using a mouse model. Comparative radiation response curves, estimating hematopoietic proliferative potential, were used to establish response deficits (RD) for individual compartments of ADR-treated marrow. The RD data suggest that two potentially discrete lesions result from ADR treatment: one lesion associated with acute toxicity and a second developing 8-16 weeks after drug treatment. A depletion of the older hematopoietic stem cell (CFU-S8d) compartment was observed to accompany the development of the second lesion. Data are presented suggesting that both initial stem cell kill, as well as an accelerated aging of the CFU-S8d by prolonged expansion of progenitors, eventually lead to a depletion of stem cell reserve manifested by a progressive loss of radiotolerance with time.

Development of latent residual drug damage to the hematopoietic marrow during the subsequent growth of tumors.

Growth of the Lewis lung (LLca) tumor in BDF1 mice was found to be accompanied by a marked expansion of the multipotential stem (CFUs-8) and committed erythroid (BFUe) and myeloid (CFU-gm) progenitor cells of the marrow with a concomitant depression of more differentiated compartments. The long-term effects of adriamycin (AdR), busulfan (BU), cis-diaminedichloroplatinum II (DDP), and 5-fluorouracil (5-FU) on the LLca-induced expansion of the CFUs-8 and CFU-gm were investigated at eight weeks after drug treatment. Of the four drugs studied, only BU demonstrated a reduction of CFUs-8 at eight weeks after treatment and prior to tumor inoculation. However, all of the drugs were found to prevent the expansion of the CFUs-8 compartment after 16 days of tumor growth. BU also resulted in a depressed CFU-gm compartment at the time of tumor inoculation, while CFU-gm in ADR-, DDP-, and 5-FU-treated animals was either at control levels (AdR), or unexpectedly elevated (DDP and 5-FU). Similar to the observations made for CFUs-8, all drugs prevented the expansion of the CFU-gm associated with tumor growth. The data suggest that qualitative differences observed between the long-term effects of the drugs on the marrow compartments may be more accurately related to the temporal "fixation" of residual drug damage brought about by enhanced differentiation of a drug-limited pluripotential CFUs, than to the actual magnitude of hematopoietic damage.

Synergy between recombinant human IL-1 alpha (rHuIL-1) and M-CSF (rHuM-CSF) during the recovery of murine hematopoietic activity in myelosuppressed animals: abbreviated versus chronic administration of rHuM-CSF.

The ability of highly purified, recombinant human macrophage colony-stimulating factor (M-CSF) and recombinant human interleukin 1 alpha (IL-1) to rescue hematopoietic activity from the myelosuppressive effects of 5-fluorouracil (5-FU) was investigated in the C57Bl/6 mouse. IL-1 (q24 h x 4) stimulated granulopoietic recovery in the 5-FU-treated animals and reduced the period of severe neutropenia associated with this drug by 7 days. Chronic M-CSF administration (q24 h x 14), on the other hand, resulted in a modest retardation of granulocyte recovery, and, when combined with IL-1, the chronic administration of M-CSF significantly dampened the accelerated recovery of granulopoietic activity observed with IL-1 alone. Consistent with their effects on neutrophil recovery, IL-1 alone markedly enhanced the recovery of the granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), macrophage colony-forming units (CFU-M), and erythroid burst-forming units (BFUe) in the marrow, whereas M-CSF failed to demonstrate a significant influence on the restoration of these hematopoietic progenitors (with the exception of delaying the recovery of the BFUe). Unexpectedly, the combination of IL-1 plus M-CSF (q24 h, days 1-4) followed by M-CSF (q24 h, days 5-14) resulted in a more than additive stimulation of progenitor recovery in both the marrow and the spleen that was observed as early as day 3 following 5-FU treatment. Furthermore, in the absence of protracted M-CSF administration on days 5-14, the 4-day rescue with a combination of IL-1 plus M-CSF also resulted in a more than additive effect on the recovery from 5-FU-induced neutropenia. Collectively, these observations demonstrated that IL-1 and M-CSF can interact synergistically to stimulate granulopoietic recovery in the 5-FU-treated animal. However, the data also suggest that the continued administration of M-CSF following the 4-day IL-1 plus M-CSF rescue may interfere with the restoration of neutrophils in the myelosuppressed animal.

Temporal recovery of short-term repopulating HSC subpopulations in marrow following schedule-dependent administrations of IL-1 alpha and M-CSF.

Studies were carried out to establish the temporal effects of abbreviated administrations of IL-1 and IL-1 plus M-CSF as rescue agents on multipotential and short-term repopulating hematopoietic stem cell (HSC) subpopulations in murine marrow treated with a myelosuppressive dose of 150 mg/kg 5-FU. The recovery kinetics for high-proliferative-potential colony-forming cells (HPP-CFC), CFU-S8 and -S12, and both CFU-M and CFU-G compartments were monitored over a 14-day interval in 5-FU-treated bone marrow (FUBM) following daily cytokine injections over a 4-day interval. Both IL-1 and the coadministration of IL-1 and M-CSF rapidly enhanced the recovery of the HPP-CFC in FUBM to supranormal levels and maintained these levels for extended intervals. Moreover, since M-CSF was unable to influence the recovery of the HSC subpopulations in FUBM by itself, the results of the two cytokines amounted to a synergistic effect on the recovery of the HPP-CFC in FUBM and a reduction of severe neutropenia in the myelosuppressed animal. Scheduling studies demonstrated that these synergistic effects were restricted to those schedules in which M-CSF was coadministered with IL-1 during the first 2 days of cytokine rescue. Finally, the recovery curves generated for the HSC and CFU-M subpopulations in response to IL-1 (with or without M-CSF) also suggest that these cytokines may conceivably alter the normal balance between proliferation and differentiation within CFU-S8 and -S12 during the accelerated recovery of hematopoiesis in FUBM.

Enhanced platelet recovery in myelosuppressed mice treated with interleukin-1 and macrophage colony-stimulating factor: potential interactions with cytokines having megakaryocyte colony-stimulating activity.

Studies were carried out to determine whether the combination of IL-1 + M-CSF, similar to the effect of these cytokines on neutropenia, was able to reduce the duration of thrombocytopenia in the 5-fluorouracil (5-FU)-myelosuppressed mouse. In addition, comparisons were made between the in vivo effects of IL-1 + M-CSF and other "thrombopoietic" cytokines (e.g., IL-3, IL-6, and GM-CSF) that demonstrate some form of megakaryocytopoietic activity in vitro. Of the five cytokines studied, only IL-1 and IL-6, by themselves, were able to effect thrombopoietic recovery in the myelosuppressed mouse. IL-1, either when acting alone or interacting synergistically with M-CSF, was able to reduce significantly the period of thrombocytopenia, but the effects of IL-6 were restricted to enhancing platelet production during the period of rebound thrombocytopenia without altering the kinetics of thrombopoietic recovery. Moreover, none of the cytokine combinations studied were found to interact to reduce further the duration of thrombocytopenia beyond that observed with IL-1 + M-CSF. Nonetheless, IL-3, IL-6, and, to a lesser extent, GM-CSF were each able to interact with IL-1 + M-CSF to extend further the period of enhanced platelet production in the animal. However, scheduling studies suggested that these thrombopoietic cytokines interacted in sequence, rather than in concert, with IL-1 + M-CSF to enhance platelet production during thrombopoietic recovery. Furthermore, the data presented are consistent with the hypothesis that IL-1 + M-CSF initially acts on a multilineage, 5-FU-resistant target cell and that IL-6 (and possibly IL-3 and GM-CSF) serves as a secondary cytokine further to enhance platelet production during rebound thrombopoiesis in the 5-FU-treated mouse.

Secondary cytokines interact in sequence with interleukin-1alpha (IL-1alpha) with or without macrophage colony-stimulating factor (M-CSF) to further accelerate granulopoietic recovery in myelosuppressed animals.

Interleukin-1alpha (IL-1), by itself, accelerates both granulopoietic and thrombopoietic recovery in the 5-fluorouracil (5-FU) myelosuppressed mouse (FUM). As a primary cytokine, IL-1 also interacts in concert with macrophage colony-stimulating factor (M-CSF) to synergistically enhance hematopoietic recovery in the FUM. As part of our continuing interest in cytokine sequencing, studies were carried out to determine whether the addition of several secondary cytokines (GM-CSF, IL-3, and IL-6) to IL-1 (+/-M-CSF) would further enhance the stimulatory effects of the primary cytokine(s) on hematopoietic recovery in FUM. Throughout these studies, IL-1 (+/-M-CSF) was administered for 2 days to the FUM, and the secondary cytokines were given either in concert (days 1 and 2) or in sequence (days 3-6) or both with the primary cytokine(s). Based on the magnitude of 7-day post-5-FU granulocyte recovery, the results demonstrated that the synergistic effects of IL-1 + M-CSF treatment on granulopoietic recovery in FUM could not be duplicated by substituting either IL-3, IL-6, or GM-CSF for M-CSF. Nonetheless, the secondary cytokines were observed to enhance the stimulatory effects of IL-1 under the following administration schedules: (1) 2 days of IL-1, followed by a sequential treatment (days 3-6) with either IL-3 or IL-6, (2) 2 days of IL-1 + GM-CSF followed by an additional 4 days of GM-CSF alone, and (3) 2 days of IL-1 + GM-CSF followed by 3-4 days of a combination of GM-CSF and either IL-3 or IL-6. Although these cytokine treatment schedules led to an enhanced granulocyte recovery (vs. IL-1 alone) in FUM, the day 7 granulocyte numbers never exceeded those observed after 2 days of IL-1 + M-CSF. Similarly, granulocyte recovery in FUM receiving 2 days of IL-1 + M-CSF followed by either GM-CSF or IL-3 also was significantly greater than that observed with IL-1 + M-CSF alone. In contrast, however, the sequential administration of IL-6 with IL-1 + M-CSF, unlike IL-1, failed to further enhance granulopoietic recovery, suggesting that there may be an antagonism between IL-6 and M-CSF in the FUM. In summary, therefore, the secondary cytokines were found to interact more effectively when they were administered in sequence, rather than in concert, with both IL-1 and IL-1 + M-CSF.

Interleukin-1 alpha protects against the toxicity associated with combined radiation and drug therapy.

A dose of total body irradiation sufficient to cause 70% mortality within 30 days (9.5 Gy) and maximally tolerated doses of either adriamycin (10 mg/kg) or cis-dichlorodiammine platinum (8 mg/kg) were administered to C57B1/6 mice and animal survival used as an index of toxicity. Whereas the nature of the toxicity resulting from the radiation alone was hematopoietic, the addition of either drug to the total body irradiation resulted in a pattern of animal death more consistent with that of gastrointestinal toxicity (100% dead within 7 days). However, if the radiation was delivered as a regional abdominal exposure, rather than total body, the gastrointestinal death observed following the combination of total body irradiation drug was not observed. The administration of 2.5 x 10(5) U IL-1 v24 hr prior to total body irradiation demonstrated significant protection against this dose of radiation (90% survival vs 30% survival). Similar protection was also observed when the IL-1 was administered 24 hr prior to the combination of total body irradiation with either drug. While these observations suggested that the IL-1 was protecting against gastrointestinal toxicity, subsequent studies demonstrated that IL-1, in addition to accelerating hematopoietic recovery following radiation insult, was equally effective in advancing the repopulation of the stem cell (CFU-GEMM) and progenitor cell (CFU-M and CFU-GM) compartments following drug treatment. Collectively, the data from these studies demonstrate that the lethal effects resulting from combined total body irradiation + drug treatment contain both a gastrointestinal and a hematopoietic component.

Altered radioprotective properties of interleukin I alpha (IL-1) in non-hematologic tumor-bearing animals.

The radioprotective properties of IL-1 were investigated in the respective murine hosts for the Lewis lung (LLca) and EMT-6 tumors. For these studies, doses of total body irradiation were selected for the C57B1/6 (9.5 Gy) and Balb/c (7.5 Gy) mice that resulted in a 60% mortality over a 30-day interval. When a "priming" dose of 2.5 x 10(5) U IL-1 was administered 24 hr prior to the radiation exposure, animal mortality was markedly reduced (60% vs 5-10%). Under identical experimental conditions, however, the presence of either the LLca or the EMT-6 tumors in their respective host strains was found to compromise the level of radioprotection conferred by this priming dose of IL-1. In Balb/c mice bearing the EMT-6 tumor, a priming dose of IL-1 resulted in only a modest level of radioprotection when compared to non-tumor-bearing control animals (median animal survival increased by 11.5 days). In C57B1/6 mice bearing the LLca tumor, IL-1 failed to demonstrate any evidence of radioprotection. Following a sublethal dose of total body irradiation, the appearance of an accelerated repopulation of the stem cell (8d CFUs and CFU-GEMM) and the myeloid progenitor (CFU-M) compartment in the marrow of the IL-1 primed EMT-6, but not the LLca, tumor-bearing animals was consistent with the hypothesis that the mechanism leading to radioprotection in IL-1 primed rodents involves an accelerated recovery of hematopoietic activity. It was also noted that the presence of the EMT-6 tumor was associated with an increase in the "radiosensitivity" of the Balb/c mouse. Collectively, these data suggest that the use of biological modifiers should be examined under altered physiological conditions prior to attempting to translate them into the clinic.

Residual adriamycin (AdR)-induced hematopoietic damage: a consideration for subsequent radiotherapy.

The long-term effect of adriamycin (AdR) on the radiation response of hematopoietic marrow was studied at 16 weeks after treatment with a MTD (10 mg/kg) for the BDF1 mouse. The radiation response was monitored in both the "stem cell" (CFUs-8) and myeloid (CFU-gm, granulocyte, WBC) compartments, as well as the erythroid (BFUe, CFUe, HcT) compartments of the marrow for 14 days following a whole body dose (TBI) of 4.5 Gy X ray. At the time of irradiation, animal and spleen weight of AdR treated animals were reduced while HcT and WBC remained at control levels. At the same time the granulocyte and CFUs-8d compartments were depressed, while the BFUe compartment was expanded. The CFUe and CFU-gm compartments remained at control levels. For all marrow compartments, treatment with AdR 16 weeks prior to 4.5 Gy resulted in a radiation response deficit determined from the temporal recovery curves. The data suggest that manifestation of long-term AdR injury, at least through 16 weeks following treatment, is dependent on a subsequent stress of sufficient magnitude to enhance the proliferative activity associated with hematopoietic cell production and differentiation. A comparison is made between these observations and previously reported long-term drug-induced hematopoietic injury.

The relationship between NMR spin-lattice relaxation times for human tumor tissue at 24 and 6.25 MHz.

The nuclear magnetic resonance (NMR) spin-lattice relaxation time (T1) of samples taken from human tumors was measured in vitro at Larmor frequencies of 24 and 6.25 MHz. It was found that on the average T1 at 6.25 MHz was linearly related to T1 at 24 MHz. An analogous set of measurements was performed on pieces of normal rat tissue. In this case, the relationship between T1 at the two frequencies was similar to that found for the human tumor tissue.

Biochemical modulation of combined radiation and 5-fluorouracil treatment of murine tumors by d,l-leucovorin.

Experiments were designed to establish whether the response of the Lewis lung tumor (LLca) to combined 5-Fluorouracil (5-FU) and radiation could be enhanced by the addition of the reduced folate leucovorin (LV) to the treatment protocol. Twenty-four hr after the tumors received a single dose of from 2.0 to 8.0 Gy of X-rays, the tumor-bearing animals were treated with a 5 day schedule of 10 mg/kg LV + 30 mg/kg 5-flourouracil (q 24 hr) in which LV preceded the 5-FU by 60 minutes. Under these time/dose configurations, no evidence of treatment-limiting gastrointestinal or hematopoietic toxicity was observed with the LV + 5-FU treatment. Following radiotherapy alone, tumor growth delays (TGDs) ranging from 1 to 6.5 days were observed with X-ray doses of from 2.0 to 8.0 Gy, respectively. When the radiotherapy was followed by the administration of 5-FU (q24 hr x 5) alone, a modest increase in the tumor response was observed; TGD increased from 1.0 to 2.0 days with 2.0 Gy and from 6.5 to 8.5 days with 8.0 Gy of X-rays. The addition of LV to the 5-FU schedule, however, resulted in a significant enhancement of the response of the tumor to the combined radiation + 5-FU treatment. TGDs increased from 1.0 to 8.0 days with a radiation dose of 2.0 Gy and from 6.5 to 34.5 days with a dose of 8.0 Gy. When administered as single agents, 5-FU and 2.0 Gy resulted in only a modest response by the LLca tumor (TGDs = 1.0 day), while LV had no effect on tumor growth. These observations, therefore, suggest that biochemical modulation of 5-FU by LV can be realized in vivo and that this biochemical modulation may be valuable in more conventional clinical schedules using combined radiation and 5-FU treatment.

Interleukin 1 alpha (IL-1) and macrophage colony-stimulating factor (M-CSF) accelerate recovery from multiple drug-induced myelosuppression.

Interleukin 1 alpha (IL-1) and macrophage colony-stimulating factor (M-CSF) interact synergistically to enhance the restoration of stem and progenitor subpopulations in murine marrow and, vis-a-vis, to accelerate hematopoietic recovery in 5FU myelosuppressed mice. Similarly, IL-1 is reported to accelerate recovery following myelosuppressive treatment with doxorubicin (AdR), cis-platinum (DDP) and cyclophosphamide (CTx). Studies were carried out in C57Bl/6 mice in order to determine whether IL-1 (+/- M-CSF) intervention was as effective against the myelosuppression experienced following 5FU-based multiple drug combinations. Maximal-tolerated doses (MTD) of AdR (10 mg/kg), DDP (8 mg/kg) or CTx (250 mg/kg) were administered either alone or in combination with 150 mg/kg 5FU. Cytokine intervention (q24 hours x 2) was initiated 24 hours later. Hematopoietic recovery was assessed by measuring the femoral content of the more primitive [IL-1 + IL-3 + M-CSF-responsive] HPP-CFC and the total granulocyte levels in the animals over a ten-day interval following treatment. MTDs of AdR, DDP and CTx, when compared with 5FU, produced only marginal levels of myelosuppression. As a result, cytokine intervention in animals treated with AdR, DDP or CTx resulted in only a modest, transient increase in the HPP-CFC and total granulocyte subpopulations when compared with their effect on 5FU--treated animals. Neither AdR, DDP nor CTx interacted with 5FU to significantly increase the cytotoxic effects of 5FU on the HPP-CFC or granulocyte subpopulations, and both IL-1 and IL-1 + M-CSF effectively stimulated hematopoietic recovery in all animals that received the 5FU--based drug combinations. However, the significant advantage (p < 0.05) achieved by combining IL-1 + M-CSF (vs. IL-1 alone) was only observed in animals that were treated with 5FU and either AdR or DDP. Furthermore, the initial stimulation of HPP-CFC recovery by IL-1 + M-CSF in animals that received DDP + 5FU, when compared with 5FU alone, was subsequently dampened. Although there were subtle, drug-related differences in the temporal response of the more primitive HPP-CFC and granulocyte populations to cytokine therapy, the data from this study demonstrated that abbreviated cytokine interaction can effectively accelerate hematopoietic recovery after combination drug therapy.

Antioxidant enzyme activity in murine hematopoietic bone marrow following treatment with interleukin 1 alpha: influence of tumor.

To determine whether non-hematologic tumors influence the bone marrow's antioxidant enzyme response to the radioprotective cytokine interleukin 1 alpha (IL-1), studies were undertaken using BDF1 and Balb/c mice bearing small, medium or large Lewis lung carcinoma (LLCa) or EMT6 mammary carcinoma tumors, respectively. Results demonstrated that, similar to nontumor-bearing mice, treatment of tumor-bearing animals with IL-1 was associated with a significant increase in marrow MnSOD activity. However, the duration of this elevated activity was reduced as tumor burden increased, and this reduction may have an impact on IL-1's ability to radioprotect tumor bearing animals, especially when tumor burden is large. In addition to cytokine-mediated responses, significant tumor-related influences on the marrow's antioxidant enzyme status were seen. Notably, it was observed that the presence of tumor was correlated with a marked suppression of antioxidant enzyme activity. Surprisingly, however, the pattern of enzyme suppression was found to differ between the two tumor models studied both in temporal onset and in the number of enzymes involved. In conclusion, the data obtained from these studies on tumor-bearing animals demonstrate that there are both cytokine-related and tumor-related influences which can effect the antioxidant enzyme status of the hematopoietic marrow-influences which may have the potential to alter the marrow's ability to tolerate free radical-generating events, both endogenous (i.e inflammation, infection) and exogenous (i.e. radiation, certain chemotherapeutic drugs) in origin.

Determination of plasma trace elements in tumor-bearing animals by proton-induced X-ray emission spectroscopy.

Although altered levels of circulating essential trace elements are known to accompany malignant disease, the lack of sensitivity of conventional detection methods has generally limited their study to clinical conditions involving extensive disease (i.e., significant tumor burden). As such, the application of altered trace element levels as potential prognostic guides or as response indicators subsequent to treatment has been of limited use. During this study, proton-induced X-ray emission spectroscopy was evaluated as a tool to determine trace element imbalances in a murine tumor model. Using plasma from C57B1/6 mice bearing the syngeneic Lewis lung carcinoma (LLCa), levels of Fe, Cu, and Zn, as well as changes in the Cu /Zn ratio, were measured in animals carrying an increasing primary tumor burden. The plasma levels of Fe, Cu, and Zn were found to decrease significantly 7 d following implants of LLCa cells with no significant change observed in the Cu/Zn ratio. By d 21, however, an increase in the Cu/Zn ratio was found to accompany increased growth of the LLCa tumor; the plasma levels of Cu had returned to normal levels, whereas both the Fe and Zn plasma levels remained lowered. Collectively, the results suggest that although a net change in individual plasma trace element concentrations might not be accurately associated with tumor growth, a clear relationship was established between the Cu/Zn ratio and tumor size.