Ginkgo biloba leaf extract (EGb 761) enhances catalepsy induced by haloperidol and L-nitroarginine in mice

Ginkgo biloba extract EGb 761 has been reported to have therapeutic effects which have been attributed to anti-oxidant and free radical-scavenging activities, including a direct action on nitric oxide production. L G-nitro-arginine (L-NOARG), a nitric oxide synthase inhibitor, and haloperidol, a drug that blocks dopamine receptors, are both known to induce catalepsy in rodents. Nitric oxide has been shown to influence dopaminergic transmission in the striatum. The purpose of the present study was to evaluate the effect of the extract obtained from leaves of Ginkgo biloba tree EGb 761 on catalepsy induced by haloperidol or by L-NOARG. Albino Swiss mice (35-45 g, N = 8-12) received by gavage a single or repeated oral dose (twice a day for 4 days) of EGb 761 followed by ip injection of haloperidol or L-NOARG. After the treatments, the animals were submitted to behavioral evaluation using the catalepsy test. Acute treatment with 80 mg/kg EGb did not modify the catalepsy induced by L-NOARG but, the dose of 40 mg/kg significantly enhanced haloperidol-induced catalepsy measured at the 10th min of the test. After repeated treatment with 80 mg/kg EGb 761, a significant increase in the cataleptic effect produced by both haloperidol and L-NOARG was observed. These data show that repeated EGb 761 administration increases the effects of drugs that modify motor behavior in mice. Since the catalepsy test has predictive value regarding extrapyramidal effects, the possibility of pharmacological interactions between haloperidol and Ginkgo biloba extracts should be further investigated in clinical studies.

Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice

Nitric oxide (·NO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ·NO and oxidants leads to the generation of nitrogen dioxide (·NO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ·NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ·NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ·NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ·NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ·NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

A pyrazolyl-thiazole derivative causes antinociception in mice

The present study investigates the antinociceptive effect of the pyrazolyl-thiazole derivative 2-(5-trichloromethyl-5-hydroxy-3-phenyl-4,5-dihydro-1 H-pyrazol-1-yl)-4-(4-bromophenyl)-5-methylthiazole (B50) in mice. Male albino Swiss mice (30-40 g) were used in the acetic acid-induced abdominal writhes and tail-immersion tests. B50 caused dose-dependent antinociception (8, 23 and 80 µmol/kg, sc) in the acetic acid writhing assay (number of writhes: vehicle: 27.69 ± 6.15; B50 (8 µmol/kg): 16.92 ± 3.84; B50 (23 µmol/kg): 13.85 ± 3.84; B50 (80 µmol/kg): 9.54 ± 3.08; data are reported as means ± SEM for 9 animals per group). On the other hand, B50 did not cause antinociception in the tail immersion assay. Naloxone (2.75 µmol/kg, sc) prevented B50-induced antinociception (number of writhes: vehicle-saline: 31.11 ± 3.15; vehicle-naloxone: 27.41 ± 3.70; B50 (80 µmol/kg)-saline: 8.70 ± 3.33; B50 (80 µmol/kg)-naloxone: 31.84 ± 4.26; morphine-saline: 2.04 ± 3.52; morphine-naloxone: 21.11 ± 4.26; 8-9 animals per group). The removal of the methyl group of the thiazole ring of B50 or substitution of the bromo substituent with the methyl at position 4 of the phenyl group, which is attached to the thiazole ring of B50, resulted in loss of activity, suggesting that these substituents are important for antinociceptive activity. B50 had no effect on spontaneous locomotion or rotarod performance, indicating that the antinociceptive effect of B50 is not related to nonspecific motor effects. The antinociceptive profile of B50 seems to be closer to nonsteroidal anti-inflammatory drugs than to classic opioid agents, since it had no analgesic effect in a thermally motivated test.

A model of hemorrhagic cystitis induced with acrolein in mice

Acrolein is a urinary metabolite of cyclophosphamide and ifosfamide, which has been reported to be the causative agent of hemorrhagic cystitis induced by these compounds. A direct cytotoxic effect of acrolein, however, has not yet been demonstrated. In the present study, the effects of intravesical injection of acrolein and mesna, the classical acrolein chemical inhibitor, were evaluated. Male Swiss mice weighing 25 to 35 g (N = 6 per group) received saline or acrolein (25, 75, 225 µg) intravesically 3, 6, 12, and 24 h before sacrifice for evaluation of bladder wet weight, macroscopic and histopathological changes by Gray's criteria, and 3 and 24 h for assessment of increase in vascular permeability. In other animals, mesna was administered intravesically (2 mg) or systemically (80 mg/kg) 1 h before acrolein. Intravesical administration of acrolein induced a dose- and time-dependent increase in vascular permeability and bladder wet weight (within 3 h: 2.2- and 21-fold increases in bladder wet weight and Evans blue dye exuded, respectively, at doses of 75 µg/bladder), as confirmed by Gray's criteria. Pretreatment with mesna (2-mercaptoethanesulfonic acid), which interacts with acrolein resulting in an inactive compound, inhibited all changes induced by acrolein. Our results are the first demonstration that intravesical administration of acrolein induces hemorrhagic cystitis. This model of acrolein-induced hemorrhagic cystitis in mice may be an important tool for the evaluation of the mechanism by which acrolein induces bladder lesion, as well as for investigation of new uroprotective drugs.

Cytotoxic activity of the dichloromethane fraction from Vernonia scorpioides (Lam.) Pers. (Asteraceae) against Ehrlich's tumor cells in mice

Vernonia scorpioides has been widely used in Brazil to treat skin problems and chronic wounds, such as ulcers of the lower limbs and diabetic lesions. In the present study, we investigated the effect of a dichloromethane (DCM) fraction of V. scorpioides leaf extract on Ehrlich ascitic and solid tumor-bearing mice. The animals were treated once a day with the DCM fraction at a concentration of 5 mg/kg, administered ip during and after the development of the tumor. The lifespan, weight, number and type of leukocytes, number of tumor cells, volume of solid and ascitic tumors were measured. The development of the tumor with pre-treated tumor cells in vitro with the DCM fraction (5 mg/kg) was analyzed and the animals were sacrificed after 7 days. The DCM fraction (5 mg/kg) totally inhibited tumor development when in direct contact with tumor cells, and also ascitic tumor development with in vitro treatment or when administered ip, in loco (after 7 days). Animals treated with the DCM fraction increased their lifespan ca. 2 weeks and maintained their body weight for 30 days. When applied immediately after the inoculation of the tumor cells in vivo, it totally abolished tumor development, with tumor development only decreasing when treatment was started 3 days after the tumor challenge. These data suggest an antineoplastic activity of the fraction. Oral or ip administration of DCM fraction (5 mg/kg) for 7 days did not reduce the solid tumor volume. The cytotoxic activity described here differs from the conventional immune suppressing profile of standard chemotherapy because it increases neutrophil influx to the peritoneal cavity. These results show that, besides exhibiting a tumoricidal activity, the DCM fraction also exhibits inflammatory activity.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

Endothelin-1 receptors play a minor role in the protection against acute Trypanosoma cruzi infection in mice

Chagas' disease, caused by the protozoan Trypanosoma cruzi, is a major cause of cardiovascular disability in countries where it is endemic. Damage to the heart microvasculature has been proposed to be an important factor in the pathogenesis of heart dysfunction. Endothelin-1 (ET-1) is a potent vasoconstrictor and exerts its effects via specific ET A and ET B receptors. A few studies have suggested a role for ET-1 and its receptors in the pathogenesis of Chagas' disease. We investigated the effects of treatment with bosentan, an ET A/ET B receptor antagonist, on the course of T. cruzi infection (Y strain) in C57Bl/6 mice. Treatment with bosentan (100 mg kg-1 day-1) was given per os starting day 0 after infection until sacrifice. Bosentan significantly increased myocardial inflammation, with no effects on parasitemia. Although the total number of nests was similar, a lower number of intact amastigote nests was found in the heart of bosentan-treated animals. Bosentan failed to affect the infection-associated increase in the cardiac levels of the cytokines IFN-g and TNF-a and the chemokines CCL2/MCP-1, CCL3/MIP-1a and CCL5/RANTES. In vitro, pre-incubation with ET-1 (0.1 µM) 4 h before infection enhanced the uptake of the parasites by peritoneal macrophages, and this effect was abrogated when macrophages were pre-treated with bosentan (1 µM) 15 min before incubation with ET-1. However, ET-1 did not alter killing of intracellular parasites after 48 h of in vitro infection. Our data suggest that bosentan-treated mice have a delay in controlling parasitism which is compensated for exacerbated inflammation. Infection is eventually controlled in these animals and lethality is unchanged, demonstrating that ET-1 plays a minor role in the protection against acute murine T. cruzi infection.

Therapeutic effects of Sophora moorcroftiana alkaloids in combination with albendazole in mice experimentally infected with protoscolices of Echinococcus granulosus

The objective of the present study was to determine if the combination of alkaloids from Sophora moorcroftiana seeds and albendazole might be effective in the treatment of experimental echinococcosisin female NIH mice (6 weeks old and weighing 18-20 g, N = 8 in each group) infected withprotoscolices of Echinococcus granulosus. Viable protoscolices (N = 6 x 103) were cultured in vitro in 1640 medium and mortality was calculated daily. To determine the in vivo efficacy, mice were inoculated intraperitoneally with viable protoscolices and then treated once daily by gavage for three months with the alkaloids (50 mg kg-1 day-1) and albendazole (50 mg kg-1 day-1), separately and in combination (both alkaloids at 25 mg kg-1 day-1 and albendazole at 25 mg kg-1 day-1). Next, the hydatid cysts collected from the peritoneal cavity of the animals were weighed and serum IL-4, IL-2, and IgE levels were analyzed. Administration of alkaloids to cultured protoscolices showed significant dose- and time-dependent killing effects. The weight of hydatid cysts was significantly decreased upon treatment with each drug (P < 0.01), but the decrease was more prominent and the rate of hydatid cyst growth inhibition was much higher (76.1%) in the group receiving the combined treatments (18.3 ± 4.6 mg). IL-4 and total IgE were decreased (939 ± 447 pg/mL and 2.03 ± 0.42 IU/mL, respectively) in serum from mice treated with alkaloids and albendazole compared with the untreated control (1481 ± 619 pg/mL and 3.31 ± 0.37 IU/mL; P < 0.01). These results indicate that S. moorcroftiana alkaloids have protoscolicidal effects and the combination of alkaloids and albendazole has significant additive effects.

Leishmania (Leishmania) chagasi-infected mice as a model for the study of glomerular lesions in visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL we have detected new immunopathological elements in the glomeruli such as T cells and adhesion molecules. Although Leishmania (Leishmania) chagasi-infected dogs and hamsters are considered to be good models for VL, their use is limited for immunopathologic studies. The use of isogenic mouse strains susceptible to L. (L.) chagasi infection was an alternative but, on the other hand, the renal lesions of these animals have not yet been characterized. Thus, our purpose in the present study was to characterize mice infected with L. (L.) chagasi as a suitable model to study VL nephropathy. Kidney samples were obtained from control mice (N = 12) and from BALB/c mice (N = 24) injected intraperitoneally with 20 million L. (L.) chagasi amastigotes 7, 15, and 30 days after injection and processed for histopathological studies and detection of IgG deposits. Glomerular hypercellularity was clearly visible and, upon Mason's trichrome and periodic acid methenamine silver staining, a pattern suggestive of mesangial proliferative glomerulonephritis was observed in mice with VL. Time-dependent IgG deposits were also seen in infected mice. We consider L. (L.) chagasi-infected mice to be a suitable model for studies of the immunopathogenesis of glomerular lesions in VL.

Influence of He-Ne laser therapy on the dynamics of wound healing in mice treated with anti-inflammatory drugs

We determined the effects of helium-neon (He-Ne) laser irradiation on wound healing dynamics in mice treated with steroidal and non-steroidal anti-inflammatory agents. Male albino mice, 28-32 g, were randomized into 6 groups of 6 animals each: control (C), He-Ne laser (L), dexamethasone (D), D + L, celecoxib (X), and X + L. D and X were injected im at doses of 5 and 22 mg/kg, respectively, 24 h before the experiment. A 1-cm long surgical wound was made with a scalpel on the abdomens of the mice. Animals from groups L, D + L and X + L were exposed to 4 J (cm²)-1 day-1 of He-Ne laser for 12 s and were sacrificed on days 1, 2, or 3 after the procedure, when skin samples were taken for histological examination. A significant increase of collagen synthesis was observed in group L compared with C (168 ± 20 vs 63 ± 8 mm²). The basal cellularity values on day 1 were: C = 763 ± 47, L = 1116 ± 85, D = 376 ± 24, D + L = 698 ± 31, X = 453 ± 29, X + L = 639 ± 32 U/mm². These data show that application of L increases while D and X decrease the inflammatory cellularity compared with C. They also show that L restores the diminished cellularity induced by the anti-inflammatory drugs. We suggest that He-Ne laser promotes collagen formation and restores the baseline cellularity after pharmacological inhibition, indicating new perspectives for laser therapy aiming to increase the healing process when anti-inflammatory drugs are used.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Dose-response effects of systemic anandamide administration in mice sequentially submitted to the open field and elevated plus-maze tests

The endocannabinoid system is involved in the control of many physiological functions, including the control of emotional states. In rodents, previous exposure to an open field increases the anxiety-like behavior in the elevated plus-maze. Anxiolytic-like effects of pharmacological compounds that increase endocannabinoid levels have been well documented. However, these effects are more evident in animals with high anxiety levels. Several studies have described characteristic inverted U-shaped dose-response effects of drugs that modulate the endocannabinoid levels. However, there are no studies showing the effects of different doses of exogenous anandamide, an endocannabinoid, in animal models of anxiety. Thus, in the present study, we determined the dose-response effects of exogenous anandamide at doses of 0.01, 0.1, and 1.0 mg/kg in C57BL/6 mice (N = 10/group) sequentially submitted to the open field and elevated plus-maze. Anandamide was diluted in 0.9% saline, ethyl alcohol, Emulphor® (18:1:1) and administered ip (0.1 mL/10 g body weight); control animals received the same volume of anandamide vehicle. Anandamide at the dose of 0.1 mg/kg (but not of 0.01 or 1 mg/kg) increased (P < 0.05) the time spent and the distance covered in the central zone of the open field, as well as the exploration of the open arms of the elevated plus-maze. Thus, exogenous anandamide, like pharmacological compounds that increase endocannabinoid levels, promoted a characteristic inverted U-shaped dose-response effect in animal models of anxiety. Furthermore, anandamide (0.1 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze (P < 0.05) after exposing the animals to the open field test.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Stem cell factor protects against neuronal apoptosis by activating AKT/ERK in diabetic mice

Neuronal apoptosis occurs in the diabetic brain due to insulin deficiency or insulin resistance, both of which reduce the expression of stem cell factor (SCF). We investigated the possible involvement of the activation of the MAPK/ERK and/or AKT pathways in neuroprotection by SCF in diabetes. Male C57/B6 mice (20-25 g) were randomly divided into four groups of 10 animals each. The morphology of the diabetic brain in mice treated or not with insulin or SCF was evaluated by H&E staining and TUNEL. SCF, ERK1/2 and AKT were measured by Western blotting. In diabetic mice treated with insulin or SCF, there was fewer structural change and apoptosis in the cortex compared to untreated mice. The apoptosis rate of the normal group, the diabetic group receiving vehicle, the diabetic group treated with insulin, and the diabetic group treated with SCF was 0.54 ± 0.077%, 2.83 ± 0.156%, 1.86 ± 0.094%, and 1.78 ± 0.095% (mean ± SEM), respectively. SCF expression was lower in the diabetic cortex than in the normal cortex; however, insulin increased the expression of SCF in the diabetic cortex. Furthermore, expression of phosphorylated ERK1/2 and AKT was decreased in the diabetic cortex compared to the normal cortex. However, insulin or SCF could activate the phosphorylation of ERK1/2 and AKT in the diabetic cortex. The results suggest that SCF may protect the brain from apoptosis in diabetes and that the mechanism of this protection may, at least in part, involve activation of the ERK1/2 and AKT pathways. These results provide insight into the mechanisms by which SCF and insulin exert their neuroprotective effects in the diabetic brain.

Oral rapamycin attenuates atherosclerosis without affecting the arterial responsiveness of resistance vessels in apolipoprotein E-deficient mice

The objective of the present study was to assess the effects of the immunosuppressant rapamycin (Rapamune®, Sirolimus) on both resistance vessel responsiveness and atherosclerosis in apolipoprotein E-deficient 8-week-old male mice fed a normal rodent diet. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (ACh)- and sodium nitroprusside (SNP)-induced vasorelaxation of isolated mesenteric bed, and atherosclerotic lesions were evaluated. After 12 weeks of orally administered rapamycin (5 mg·kg-1·day-1, N = 9) and compared with untreated (control, N = 9) animals, rapamycin treatment did not modify either NE-induced vasoconstriction (maximal response: 114 ± 4 vs 124 ± 10 mmHg, respectively) or ACh- (maximal response: 51 ± 8 vs 53 ± 5%, respectively) and SNP-induced vasorelaxation (maximal response: 73 ± 6 vs 74 ± 6%, respectively) of the isolated vascular mesenteric bed. Despite increased total cholesterol in treated mice (982 ± 59 vs 722 ± 49 mg/dL, P < 0.01), lipid deposition on the aorta wall vessel was significantly less in rapamycin-treated animals (37 ± 12 vs 68 ± 8 µm² x 10³). These results indicate that orally administered rapamycin is effective in attenuating the progression of atherosclerotic plaque without affecting the responsiveness of resistance vessels, supporting the idea that this immunosuppressant agent might be of potential benefit against atherosclerosis in patients undergoing therapy.