The differential effects of pelvic and vagal inputs on the supraspinal cystitis viscero(noci)ceptive-related axis

Recently the development of the cyclophosphamide (CP, 100 mg/kg/i.p.) model has added an important element to the study of neural activities accompanying cystitis genesis. CP cystitis genesis results in the dual activation of the pelvic and vagal sensory afferent systems, which in turn activate a supraspinal network comprising the ventrocaudal bulbar reticular formation (vcBRF), the sensory subdivisions of the dorsal vagal complex (DVC) and its subcortical telencephalic targets, the dorsolateral subdivision of the bed nucleus of the stria terminalis (BSTLd) and the nucleus centralis of the amygdala (CeL). Altogether these structures form the sensory neural axis of the CP cystitis. However, both clinical and experimental observations have given evidence that only the pelvic afferents are at the origin of the painful sensation and related behaviour. Because of this, and for a better understanding of the nervous network that subserves cystitis painful information, we sought to determine whether the structures that constitute the cystitis sensory neural pathway have the same reactivity depending on the origin of the sensory afferent inputs, either pelvic or vagal. Using c-fos expression, which permits quantitative analysis of neural activity, we have demonstrated that the supraspinal CP cystitis responding structures do not form an homogeneous population in terms of sources of inputs. Although all structures are predominantly driven by vagal inputs, only the vcBRF, the DVC and the BSTLd respond to pelvic inputs. Consequently, and by referring to clinical observations, we have concluded that, it is these three areas, excluding the CeL, which constitute the main framework of the supraspinal pain sensory neural pathway of CP-induced cystitis. The activation of the vagus nerve would more probably relate to the other side effects that accompany CP injections such as nausea and headache attacks.     

Alpha-tocopherol, beta-carotene and melatonin administration protects cyclophosphamide-induced oxidative damage to bladder tissue in rats

Cyclophosphamide (CP) has potential urotoxicity such as hemorrhagic cystitis (HC). 2-Mercaptoethane sulfonate (mesna) has been widely used as an effective agent against CP-induced cystitis, but significant HC has still been encountered clinically. In recent studies, mesna was shown to be more effective if combined with antioxidants. The purpose of this study was to evaluate the effects of antioxidants, alpha-tocopherol, beta-carotene and melatonin on CP-induced bladder damage in rats, even if used without mesna administration. Male Sprague-Dawley rats weighing 180-210 g were divided into 5 groups. Four groups received a single dose of CP (100 mg/kg) intraperitoneally with the same time intervals. Group 2 received CP only, group 3 received beta-carotene (40 mg/kg/day), group 4 received alpha-tocopherol (40 mg/kg/day) and group 5 received melatonin (10 mg/kg/day) both before and the day after CP injection. Group 1 served as control. Bladder histopathology, as well as malondialdehyde (MDA) and iNOS levels, and excretion of nitrite-nitrates (NO(x)) in urine were evaluated. CP injection resulted in severe histological changes and macroscopic hematuria. alpha-Tocopherol and melatonin showed meaningful protection against bladder damage. Protection by beta-carotene was also significant but weaker. MDA levels increased significantly with CP injection and all antioxidants ameliorated this increase in bladder tissue. CP also elevated the NO(x) level in urine and iNOS activity in bladder. Only melatonin was able to decrease these parameters. In conclusion, there is no doubt that oxidants have a role in the pathogenesis of CP-cystitis. Antioxidants, especially melatonin and alpha-tocopherol, may help to ameliorate bladder damage induced by CP.     

Comparison of naloxonazine and beta-funaltrexamine antagonism of mu 1 and mu 2 opioid actions.

beta-Funaltrexamine (beta-FNA) irreversibly blocks morphine analgesia, lethality and its inhibition of gastrointestinal transit, confirming that these actions involve mu receptors. In dose-response studies, beta-FNA antagonized all the actions with similar potencies (ID50 values of 12.1, 11.3 and 12.3 mg/kg, respectively). beta-FNA also reduced intra-cerebroventricular and intrathecal DAMGO analgesia equally well (ID50 values of 6.09 and 7.7 mg/kg, respectively). Naloxanazine blocked systemic morphine analgesia (ID50 value 9.5 mg/kg) and supraspinal DAMGO analgesia (ID50 value 6.1 mg/kg) as potently as beta-FNA. However, against spinal DAMGO analgesia, morphine's inhibition of gastro-intestinal transit or lethality, naloxonazine (ID50 values 38.8, 40.7 and 40.9 mg/kg, respectively) was significantly less active than beta-FNA (p less than 0.05). beta-FNA remains a valuable tool in the classification of mu opioid actions. Within the mu category, actions can be defined as either mu 1 (naloxonazine-sensitive) or mu 2 (naloxonazine-insensitive).     

Prophylactic intravesical instillation of epinephrine prevents cyclophosphamide-induced hemorrhagic cystitis in rats

The objective of this study is to investigate the potential protective effects of intravesical instillation of epinephrine in cyclophosphamide-induced hemorrhagic cystitis. In an earlier study, we have shown that epinephrine promotes hemostasis on established hemorrhagic cystitis induced by cyclophosphamide. Female Sprague-Dawley rats were divided into seven groups as follows: group 1: positive control (150 mg/kg, cyclophosphamide, i.p.), group 2: negative control (10 microg/ml, epinephrine, intravesical), co-administration of cyclophosphamide (150 mg/kg, i.p.), group 3: saline (intravesical), groups 4-6: epinephrine (2.5, 5, and 10 mu g/ml, intravesical), and group 7: mesna (50 mg/kg, i.p.). Rats were sacrificed on 3 consecutive days and the urinary bladders were removed, weighed, and evaluated. The vesical vascular permeability was determined by wet bladder weight and Evan's blue dye absorbance. After 24 hours of cyclophosphamide administration, severe hemorrhagic cystitis was induced with marked edema, hemorrhage, and inflammation. In the epinephrine-treated groups, symptoms of hemorrhagic cystitis (such as edema, inflammation, and hemorrhage) were reduced significantly. Intravesical instillation of epinephrine prevents edema, hemorrhage, and inflammation in rats with cyclophosphamide-induced hemorrhagic cystitis.     

Delta9-tetrahydrocannabinol selectively acts on CB1 receptors in specific regions of dorsal vagal complex to inhibit emesis in ferrets

The aim of this study was to investigate the efficacy, receptor specificity, and site of action of Delta9-tetrahydrocannabinol (THC) as an antiemetic in the ferret. THC (0.05-1 mg/kg ip) dose-dependently inhibited the emetic actions of cisplatin. The ED50 for retching was approximately 0.1 mg/kg and for vomiting was 0.05 mg/kg. A specific cannabinoid (CB)1 receptor antagonist SR-141716A (5 mg/kg ip) reversed the effect of THC, whereas the CB2 receptor antagonist SR-144528 (5 mg/kg ip) was ineffective. THC applied to the surface of the brain stem was sufficient to inhibit emesis induced by intragastric hypertonic saline. The site of action of THC in the brain stem was further assessed using Fos immunohistochemistry. Fos expression induced by cisplatin in the dorsal motor nucleus of the vagus (DMNX) and the medial subnucleus of the nucleus of the solitary tract (NTS), but not other subnuclei of the NTS, was significantly reduced by THC rostral to obex. At the level of the obex, THC reduced Fos expression in the area postrema and the dorsal subnucleus of the NTS. The highest density of CB1 receptor immunoreactivity was found in the DMNX and the medial subnucleus of the NTS. Lower densities were observed in the area postrema and dorsal subnucleus of the NTS. Caudal to obex, there was moderate density of staining in the commissural subnucleus of the NTS. These results show that THC selectively acts at CB1 receptors to reduce neuronal activation in response to emetic stimuli in specific regions of the dorsal vagal complex.     

Effects of poly(ADP-ribose) polymerase inhibition in bladder damage caused by cyclophosphamide in rats

It was previously shown that nitric oxide produced by inducible nitric oxide synthase (iNOS) and peroxynitrite are responsible for cyclophosphamide (CP)-induced cystitis. Since endogenous production of peroxynitrite is known to lead to poly(ADP-ribose) polymerase (PARP) activation, in this study, the aim was to evaluate whether the PARP activation pathway is also included in the pathogenesis of CP-induced bladder ulceration in rats. A total of 48 male albino Wistar rats were divided into 5 groups. Group 1 served as control and was given 2 ml saline; four groups received a single dose of CP (200 mg/kg) with the same time intervals. Group 2 received CP only; Group 3, selective iNOS inhibitor 1400W (20 mg/kg); Group 4, peroxynitrite scavenger ebselen (30 mg/kg); and Group 5, PARP inhibitor 3-aminobenzamide (20 mg/kg). CP injection resulted in severe cystitis with continuous macroscopic hemorrhage, strong edema, inflammation, and ulceration. Moreover, bladder iNOS activation and urine nitrite-nitrate levels were dramatically increased. Histologically, 1400W protected bladder against CP damage and decreased urine nitrite-nitrate levels and bladder iNOS induction. Ebselen has shown similar histologic results with 1400W without changing urinary nitrite-nitrate level and iNOS activity. Furthermore in the 3-aminobenzamide group, beneficial effects had also occurred including decreased ulceration. These results suggest that PARP activation involves pathogenesis of CP-induced bladder ulceration. Furthermore, PARP is not only important for ulceration but also for bladder edema, hemorrhage, and inflammation because of broken uroepithelial cellular integrity.     

The effect of the antipsychotic drug mosapramine on the expression of Fos protein in the rat brain: comparison with haloperidol, clozapine and risperidone

In this study, we examined the effect of the acute p.o. administration of the antipsychotic drug mosapramine, as well as the antipsychotic drugs clozapine, haloperidol and risperidone, on the expression of Fos protein in the medial prefrontal cortex, nucleus accumbens and dorsolateral striatum of rat brain. The administration of mosapramine (1 or 3 mg/kg) significantly increased the number of Fos protein positive neurons in the medial prefrontal cortex, but not in the dorsolateral striatum. In addition, mosapramine (1, 3 or 10 mg/kg) produced a dose-dependent increase in the number of Fos protein positive neurons in the nucleus accumbens. The acute administration of 10 mg/kg of mosapramine significantly increased the number of Fos protein positive neurons in all brain regions. The acute administration of clozapine (30 mg/kg), similarly to mosapramine at lower doses (1 or 3 mg/kg), significantly increased the number of Fos protein positive neurons in the medial prefrontal cortex and nucleus accumbens, but not dorsolateral striatum. In contrast, haloperidol (0.3 mg/kg) significantly increased the number of Fos protein positive neurons in the nucleus accumbens and dorsolateral striatum, but not medial prefrontal cortex. The acute administration of risperidone (0.3 or 1 mg/kg) did not affect the number of Fos protein positive neurons in the medial prefrontal cortex, nucleus accumbens or dorsolateral striatum of rat brain, whereas a 3 mg/kg dose of risperidone significantly increased the number of Fos protein positive neurons in all brain regions. These results suggest that the ability of mosapramine to enhance expression of Fos protein in the medial prefrontal cortex may contribute to a clozapine-like profile with respect to actions on negative symptoms in schizophrenia. Furthermore, the lack of effect of low doses of mosapramine on Fos protein expression in the dorsolateral striatum, an area believed to play a role in movement, suggests that it may have a lower tendency to induce neurological side effects.     

Development of spontaneous motility in chick embryos. Participation of the central cholinergic mechanism

Development of the effectivensess of the systemic administration of oxotremorin (200 micrograms/kg egg weight) on spontaneous motility in chick embryos was studied from the 11th to the 19th day of incubation. 1) Oxotremorin activated spontaneous motility from the 15th day of incubation; subsequently, its effect increased with the embryo's age. 2) Glycine (100 mg/kg e.w.) blocked the activating effect of oxotremorin, again in a direct correlation to the embryo's age. 3) The spontaneous motility of spinal embryos was activated significantly less, thereby demonstrating the importance of the supraspinal component of the effect of oxotremorin. 4) The results are evaluated primarily as evidence of the role of the cholinergic mechanism (Everett 1964) in embryonal development of supraspinal control of the spontaneous activity of spinal motoneurones.     

Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis

These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after cyclophosphamide (CYP)-induced bladder inflammation. In urethan-anesthetized Wistar rats with cystitis, intravesical saline distension significantly (P 相似文献   

Development of spontaneous motility in chick embryos. Interaction of reserpine and apomorphine

The effect of apomorphine (1 mg/kg egg weight) on a base of the pre-administration of reserpine (2.5 mg/kg e.w.) 30 min or 2 or 4 hours previously was studied in chick embryos from the 13th to 19th day of incubation. Only the depressant effect of apomorphine was manifested in 13-day embryos. In 15-day embryos summation of the depressant effect of both drugs was recorded for the first time. In 17-day and especially 19-day embryos apomorphine raised the frequency of spontaneous movements high above the level of reserpine-induced depression of motor activity. This activating effect of apomorphine depended upon the supraspinal parts of the CNS, as it disappeared in 17-day embryos after decentralization of the spinal cord (particularly after chronic decapitation). We consider these findings to be further experimental evidence of the participation of catecholaminergic (in particular dopaminergic) central systems in supraspinal control of embryonic motor activity.     

Uroprotective Effect of Pentoxifylline in Cyclophosphamide‐Induced Hemorrhagic Cystitis in Rats

The role of phosphodiesterase inhibitor, pentoxifylline, in the prevention of cyclophosphamide‐induced hemorrhagic cystitis was evaluated in a rat model. Hemorrhagic cystitis was induced in rats by an intraperitoneal (i.p.) injection of a single dose of cyclophosphamide (150 mg/kg). Pentoxifylline (150 mg/kg/day/ip) was administered for 10 days followed by cyclophosphamide. Hemorrhagic cystitis was well characterized macroscopically, microscopically, and biochemically. Cyclophosphamide induced bladder injury including acute severe inflammation, vascular congestion, severe edema, hemorrhage, inflammatory cell infiltration in the lamina propria, and epithelial denudation; as well as it notably elevated serum inflammatory cytokines (tumor necrosis factor‐α, interleukin‐6, and interleukin‐1β), bladder content of malondialdehyde and total nitrate, accompanied with depletion of bladder antioxidant enzymes activities (glutathione peroxidase, superoxide dismutase, glutathione‐S‐transferase, and catalase). Prior administration of pentoxifylline improved all biochemical and histologic alterations induced by the cytotoxic drug cyclophosphamide. In conclusion, pentoxifylline has proven uroprotective efficacy in the cyclophosphamide‐induced hemorrhagic cystitis model, possibly through modulating the release of inflammatory cytokines and nitric oxide and restoring the oxidant/antioxidant balance. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:343‐350, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21494     

Protective effects of lipoic acid and mesna on cyclophosphamide‐induced haemorrhagic cystitis in mice

The protective roles of lipoic acid (LA)/vitamin C (VC) and mesna on preventing cyclophosphamide (CYP)‐induced haemorrhagic cystitis (HC) were investigated. Swiss mice were divided into five groups randomly. HC was induced by a single dose of CYP injection (150‐mg kg^?1 bodyweight). Group I was injected with saline (four times in total) throughout as control group. Group II received CYP and three equal doses of saline. Group III received CYP and three doses of mesna, whereas Group IV (or Group V) received CYP, mesna + two doses of VC (or LA). All injections were performed intraperitoneally. After 24 h of cystitis induction, the bladders were collected for all the experiments. Histological characterization showed that CYP injection resulted in severe HC. Reactive oxygen species (ROS) and thiobarbituric acid reactive substances' levels were increased in CYP group. The activities of antioxidant enzymes, e.g. superoxide dismutase, catalase, glutathione S‐transferase and glutathione peroxidase, were inhibited significantly in CYP groups, respectively. In addition, activation of c‐jun N‐terminal kinases (JNK) and p38 mitogen‐activated protein kinase (MAPK) may be involved in the mechanism of CYP‐induced HC but not extracellular signal regulated kinases (ERK). Significant suppression of p38 phosphorylation on Group V suggests that LA and mesna may have synergistic beneficial effect. In Groups III–V, all the parameters of HC and oxidative stress were inhibited significantly. Taking together, we found that these results illustrated that ROS play an important role on CYP‐induced HC and the administration of LA/VC with mesna may have therapeutic potential against CYP‐induced bladder HC. Copyright © 2013 John Wiley & Sons, Ltd.     

Intracisternal TRH analog induces Fos expression in gastric myenteric neurons and glia in conscious rats

Activation of gastric myenteric cells by intracisternal injection of the stable thyrotropin-releasing hormone (TRH) analog RX-77368, at a dose inducing near maximal vagal cholinergic stimulation of gastric functions, was investigated in conscious rats. Fos immunoreactivity was assessed in gastric longitudinal muscle-myenteric plexus whole mount preparations 90 min after intracisternal injection. Fos-immunoreactive cells were rare in controls (~1 cell/ganglion), whereas intracisternal RX-77368 (50 ng) increased the number to 24.8 +/- 1.8 and 26.8 +/- 2.2 cells/ganglion in the corpus and antrum, respectively. Hexamethonium (20 mg/kg sc) prevented Fos expression by 90%, whereas atropine (2 mg/kg sc) had no effect. The neuronal marker protein gene product 9.5 and the glial markers S-100 and glial fibrillary acidic proteins showed that RX-77368 induced Fos in both myenteric neurons and glia. Vesicular ACh transporter and calretinin were detected around the activated myenteric neurons. These results indicated that central vagal efferent stimulation by intracisternal RX-77368 activates gastric myenteric neurons as well as glial cells mainly through nicotinic ACh receptors in conscious rats.     

Vagal modulation of intestinal afferent sensitivity to systemic LPS in the rat

The central nervous system modulates inflammation in the gastrointestinal tract via efferent vagal pathways. We hypothesized that these vagal efferents receive synaptic input from vagal afferents, representing an autonomic feedback mechanism. The consequence of this vagovagal reflex for afferent signal generation in response to LPS was examined in the present study. Different modifications of the vagal innervation or sham procedures were performed in anesthetized rats. Extracellular mesenteric afferent nerve discharge and systemic blood pressure were recorded in vivo before and after systemic administration of LPS (6 mg/kg iv). Mesenteric afferent nerve discharge increased dramatically following LPS, which was unchanged when vagal efferent traffic was eliminated by acute vagotomy. In chronically vagotomized animals, to eliminate both vagal afferent and efferent traffic, the increase in afferent firing 3.5 min after LPS was reduced to 3.2 +/- 2.5 impulses/s above baseline compared with 42.2 +/- 2.0 impulses/s in controls (P < 0.001). A similar effect was observed following perivagal capsaicin, which was used to eliminate vagal afferent traffic only. LPS also caused a transient hypotension (<10 min), a partial recovery, and then persistent hypertension that was exacerbated by all three procedures. Mechanosensitivity was increased 15 min following LPS but had recovered at 30 min in all subgroups except for the chronic vagotomy group. In conclusion, discharge in capsaicin-sensitive mesenteric vagal afferents is augmented following systemic LPS. This activity, through a vagovagal pathway, helps to attenuate the effects of septic shock. The persistent hypersensitivity to mechanical stimulation after chronic vagal denervation suggests that the vagus exerts a regulatory influence on spinal afferent sensitization following LPS.     

Experimental autoimmune prostatitis induces chronic pelvic pain

Pain is the hallmark of patients with chronic prostatitis (CP) and chronic pelvic pain syndrome (CPPS). Despite numerous hypotheses, the etiology and pathogenesis remain unknown. To better understand CP/CPPS, we used a murine experimental autoimmune prostatitis model to examine the development, localization, and modulation of pelvic pain. Pelvic pain was detected 5 days after antigen instillation and was sustained beyond 30 days, indicating the development of chronic pain. The pain was attenuated by lidocaine treatment into the prostate, but not into the bladder or the colon, suggesting that pain originated from the prostate. Experimental autoimmune prostatitis histopathology was confined to the prostate with focal periglandular inflammatory infiltrates in the ventral, dorsolateral, and anterior lobes of the mouse prostate. Inflammation and pelvic pain were positively correlated and increased with time. Morphologically, the dorsolateral prostate alone showed significantly increased neuronal fiber distribution, as evidenced by increased protein gene product 9.5 expression. Pelvic pain was attenuated by treatment with the neuromodulator gabapentin, suggesting spinal and/or supraspinal contribution to chronic pain. These results provide the basis for identifying mechanisms that regulate pelvic pain and the testing of therapeutic agents that block pain development in CP/CPPS.     

Effects of clopheline on impulse activity of neurons in the midbrain and dorsal horn of the spinal cord

Clophelin (1-5 mg/kg) suppresses spinal dorsal horn neuronal nociceptive responses but does not change their touch stimuli reactions in unanesthetized curarized cats. This effect is steady to naloxone (1 mg/kg), yohimbine (5 mg/kg) and removal by prazosin (1 mg/kg). Clophelin depresses nociceptive responses of the central gray matter neurones which generalized pain impulses in the supraspinal structures.     

Functional vagal input to gastric myenteric plexus as assessed by vagal stimulation-induced Fos expression

Immunohistochemical detection of c-Fos expression was used to identify gastric myenteric plexus neurons that receive excitatory input from vagal efferent neurons activated by electrical stimulation of the cervical vagi in anesthetized rats. Vagal stimulation-induced Fos expression increased with higher pulse frequency, so that with 16 Hz (rectangular pulses of 1 mA/0.5 ms for 30 min) approximately 30% and with 48 Hz 90% of all neurons near the lesser curvature were Fos positive. In sham-stimulated rats there was no Fos expression. The percentage of Fos-activated neurons was only slightly smaller (85% with 48 Hz) near the greater curvature. Prior atropine administration (1 mg/kg ip) had little effect on vagal stimulation-induced Fos expression, and in unilaterally stimulated rats there was no Fos expression on the contralateral (noninnervated) side of the stomach, ruling out mediation by gastric motility or secretory responses. However, polysynaptic recruitment of third- and higher-order neurons cannot be ruled out completely. These results support the idea that, at least in the stomach, functional excitatory innervation of myenteric plexus neurons by the efferent vagus is profuse and widespread, refuting the notion of only a few vagal "command neurons."     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.