Growth In Experimental Animals Research Articles

Withaferin A-Encapsulated PEGylated Nanoliposomes Induce Apoptosis in B16F10 Melanoma Cells by Regulating Bcl2 and Bcl xl Genes and Mitigates Murine Solid Tumor Development.

Withaferin A (WA) is a natural steroidal lactone with promising pharmacological activities, but its poor solubility and bioavailability hinder its clinical application. The liposomal drug delivery system has attracted considerable attention to overcome the delivery limitations of pharmacological agents. The present study investigated the effect of WA-loaded pegylated nanoliposomes (LWA) on in vitro and in vivo B16F10 melanoma tumor models. In vitro results showed that LWA had significantly (P < 0.01) higher cytotoxicity than free WA and induced ROS-mediated apoptosis in B16F10 cells. Transwell cell migration and invasion studies demonstrated that LWA treatment significantly (P < 0.01) decreased the migratory and invasive capacities of melanoma cells compared with WA. In vivo study revealed that treatment significantly (P < 0.01) reduced tumor growth in experimental animals compared with WA or tumor control. Also, LWA administration remarkably inhibited tumor cell proliferation by downregulating the expression of Ki-67 and Cyclin D1 and induced apoptosis by regulating the expression of Bax, Bcl2, and Bcl xl levels. Our results strongly suggest that LWA could be a promising therapeutic formulation for treating malignant melanoma.

Comparative studies of the specific activity of soft dosage forms minoxidil for trichological practice

The treatment and prevention of hair loss have been a serious aesthetic problem for the population for a long time due to the widespread occurrence of the condition and its undesirable medical and social consequences. The pharmacotherapeutic options for trichological practice are very limited, and the effectiveness of many of these drugs is small or questionable. The peripheral vasodilator minoxidil is one of the few medicinal substances that has been proven to stimulate hair growth. However, the number of minoxidil preparations allowed by the Ministry of Health for use in Ukraine is minimal and mainly represented by rare dosage forms for external use. Considering the above, the employees of the Department of Medicines Technology at Zaporizhzhia State Medical University have proposed rational formulations and technologies for two new soft dosage forms of minoxidil – a 2 % cream for external use based on an emulsion, and a shampoo in gel form – based on complex physical-chemical, pharmaco-technological, rheological, and biopharmaceutical research. These formulations are intended to stabilize hair growth and restore hair coverage in men and women suffering from androgenic alopecia. The aim of this research is to study the specific activity of the proposed topical dosage forms of minoxidil. Materials and methods. Experimental cream with 2 % minoxidil on an emulsion carrier and a gel-like shampoo containing 1 % minoxidil combined with phytoextracts of hops and burdock, as well as corresponding placebo compositions, were used as objects of preclinical studies. The compositions of the topical minoxidil pharmacotherapeutic agents are scientifically based, ensuring their optimal pharmacotechnological, biopharmaceutical, and consistent consumer properties. The study of the specific activity of soft medicinal forms of minoxidil for external use was carried out by evaluating their growth-stimulating and follicle-protective activity. “Pilfood Bosnalek” spray for external use (Bosnalijek, Bosnia and Herzegovina), containing 2 % minoxidil, was used as a reference drug when studying the growth-stimulating effect of 2 % cream for external use with minoxidil. The reference drug in the study of the similar activity of the gel-like shampoo “Trichlomine” was the shampoo against hair loss “Dove” (Unilever, Germany). Results. It has been found that a 2 % minoxidil cream for topical use, when used together with the reference drug “Pilfood Bosnalek” (Bosnalijek, Bosnia and Herzegovina), has a significant hair growth-stimulating effect, which statistically significantly increases hair growth in experimental animals from as early as 3–4 days of use. The shampoo “Trichlomine” also has a significant effect on hair growth in experimental animals, with results observed from 3–4 days of use. Furthermore, the strength of the hair growth-stimulating effect observed from the use of “Trichlomine” shampoo significantly exceeds that of the reference “Dove” shampoo (Unilever, Germany). These results demonstrate the presence of significant follicle-stimulating activity in the tested minoxidil preparations for treating experimental alopecia and their positive effects on the weight and structure of laboratory animal fur. The follicle-protective activity of experimental minoxidil dosage forms (2 % cream and gel-like shampoo) was also experimentally confirmed, due to the regulation of various metabolic pathways in the dermis. The use of 2 % minoxidil cream and comparison preparations (“Dove” shampoo and “Pilfood Bosnalek” spray) did not result in the normalization of the investigated indicators of dermal energy metabolism. However, a positive effect of “Trichlomine” shampoo was found, which is associated with the presence of a plant antioxidant complex in the pharmacotherapeutic agent and its potentiating effect with minoxidil. Conclusions. It was found that the application of soft dosage forms of minoxidil (2 % emulsion-based cream and 1 % gel-based shampoo) exhibited pronounced growth-stimulating and follicle-protective activity in the model of experimental alopecia in white rats. At the same time, it was established that the cream with minoxidil 2 % for external use and the reference spray for external use “Pilfood Bosnalek” (Bosnalijek, Bosnia and Herzegovina) have a significant statistically comparable growth-stimulating effect. The strength of the growth-stimulating effect recorded from the use of the “Trichlomine” shampoo significantly exceeded the effect of the reference shampoo “Dove” (Unilever, Germany). It was found that both experimental medicinal forms of minoxidil (2 % cream and gel-like shampoo) had a follicle-protective effect by normalizing various links of dermis metabolism. The most pronounced effect in this regard was recorded in the shampoo “Trichlomine”, which contained not only minoxidil but also plant-antioxidant complexes. Application of 2 % cream with minoxidil, as well as reference drugs (spray “Pilfood Bosnalek” and shampoo “Dove”), did not have a reliable effect on the indicators of energy exchange of the dermis studied.

CSIG-17. IMMUNE MODULATORY ROLE OF TYPE I INTERFERON IN SYNGENEIC MOUSE GLIOMA MODELS

Abstract Glioblastoma is characterized by a poor prognosis and a challenging phenotype for drug development. Although multimodal treatment, including surgery, radio- and chemotherapy is applied, the overall survival remains just above one year. Numerous clinical trials have studied targeted therapies against commonly deregulated pathways, but an efficient targeted drug is yet to be discovered. Likewise, immunotherapy has not been shown to be active. A subset of glioma tumor cells demonstrates stem-like properties; these cells are commonly referred to as glioma initiating cells (GIC). These types of cells are pluripotent and can by definition initiate and recapitulate glioma growth in experimental animals in vivo. Furthermore, these cells are often resistant to conventional therapies. Interferon β (IFN-β) is an immunomodulatory molecule with anti-cancer properties. We have previously shown that IFN-β greatly reduces sphere-formation capability of GIC. It was also confirmed that IFN-β sensitized resistant GIC to irradiation or the chemotherapeutic agent, temozolomide (TMZ). IFN-β treatment significantly prolonged survival in a xenograft model with GIC cells. In the current project, we want to use syngeneic mouse models to study the immunomodulatory effects of type I IFNs. Preliminary results indicate that abrogation of IFN signalling in tumor cells by CRISPR/Cas9 technology prolonged survival in mice only in cell lines which have substantial baseline autocrine IFN signalling. On the contrary, we did not observe a difference in survival when wild-type tumor cells were implanted in either IFNAR1 deficient or proficient hosts. Flow cytometry analysis will elucidate changes in immune cell recruitment and infiltration upon IFN signalling disruption. Moreover, we explore different treatments in combination with IFN-β as there are indications that TMZ or radiotherapy can have synergistic effects with stimulation of interferon type I signalling.

A Novel Effect of Acyclovir on Hair Growth in BALB/c Mice: A Promising Future for Finding a New Topical Drug for the Treatment of Hirsutism.

PurposeHirsutism (ie, terminal hair growth on the face and body in a male-like pattern in women) is a common dermatological disorder in women, with psychosocial implications. Consequently, there is demand for finding novel pharmacological treatments and agents that can safely reduce hair growth. This study aimed to investigate the potential effect of topical acyclovir on hair growth in mice.MethodsIn this study, twenty-four female BALB/c mice were randomly divided into three groups in order to evaluate the hair growth-reducing effects of acyclovir (control group, vehicle group, and acyclovir group). Topical acyclovir 5% was applied on the shaved denuded skin of mice. Topical application onto the backs of the animals was performed twice daily for 28 consecutive days. The time (in days) required for hair growth initiation as well as completion of hair growth in dorsal skin of animals were recorded. On day 28, horizontally cut biopsy samples were removed and the numbers of hair follicles were counted, and the diameter of hair follicles was measured under high-field microscopy by a specialist blinded to the treatments.ResultsHair growth initiation time was significantly increased with acyclovir, as compared to control and vehicle groups. The time required for complete hair growth in control and vehicle groups were 18±0.68 and 19±1.41 days, respectively; however, the hair growth completion in acyclovir-treated animals was not observed at the end of the experiment. Furthermore, the length of hairs in treatment group was significantly shorter than the control group at the end of the study (P < 0.001). In histologic examination, the count and the diameter of hair follicles in deep subcutis were significantly decreased.ConclusionThe results of this study, for the first time, showed that topical administration of acyclovir might have inhibitory effects on hair growth in experimental animals; however, further studies are required to understand its mechanism.

Are Platelet Aggregation Tests the Best Way to Assess New Drugs for Arterial Disease

Over many years, laboratory testing of platelet aggregability have been carried out in attempts to develop drugs that would prevent thrombosis in arteries. The problems encountered included the question of methodology. Blood samples have to be anticoagulated in order to study the platelets. Anti-coagulation with citrate and tests on derived platelet rich plasma did not correlate at all well with thrombus growth in the stenosed coronary arteries of experimental animals and citrate removes the calcium ions which are vital for platelet function. Anticoagulation with heparin also interfered with platelet function, so that now, hirudins are the preferred anticoagulant. However it was observed that if, instead of stimulating platelet aggregation with adrenaline or ADP, serotonin was applied to the preparation, very little aggregation took place in spite of serotonin 5HT2A antagonists being the most potent inhibitors of thrombus growth in experimental animals. Another indicator that primary platelet agggregation is not a predictor of in vivo efficacy was the finding that 5HT2A antagonism inhibited aggregate growth. In a stenosed artery the platelets are activated by increased shear stress and blood turbulence with release of platelet serotonin causing positive feedback activation of more platelets. At present, there does not seem to be a bench in vitro test that accurately predicts in vivo efficacy in stenosed artery occlusive thrombosis.

Perinatal and long-term effects of maternal uterine artery adenoviral VEGF-A165 gene therapy in the growth-restricted guinea pig fetus.

Uterine artery application of adenoviral vascular endothelial growth factor A165 (Ad.VEGF-A165) gene therapy increases uterine blood flow and fetal growth in experimental animals with fetal growth restriction (FGR). Whether Ad.VEGF-A165 reduces lifelong cardiovascular disease risk imposed by FGR remains unknown. Here, pregnant guinea pigs fed 70% normal food intake to induce FGR received Ad.VEGF-A165 (1×1010 viral particles, n = 15) or vehicle ( n = 10), delivered to the external surface of the uterine arteries, in midpregnancy. Ad libitum-fed controls received vehicle only ( n = 14). Litter size, gestation length, and perinatal mortality were similar in control, untreated FGR, and FGR+Ad.VEGF-A165 animals. When compared with controls, birth weight was lower in male but higher in female pups following maternal nutrient restriction, whereas both male and female FGR+Ad.VEGF-A165 pups were heavier than untreated FGR pups ( P < 0.05, ANOVA). Postnatal weight gain was 10-20% greater in female FGR+Ad.VEGF-A165 than in untreated FGR pups, depending on age, although neither group differed from controls. Maternal nutrient restriction reduced heart weight in adult female offspring irrespective of Ad.VEGF-A165 treatment but did not alter ventricular wall thickness. In males, postnatal weight gain and heart morphology were not affected by maternal treatment. Neither systolic, diastolic, mean arterial pressure, adrenal weight, nor basal or challenged plasma cortisol were affected by maternal undernutrition or Ad.VEGF-A165 in either sex. Therefore, increased fetal growth conferred by maternal uterine artery Ad.VEGF-A165 is sustained postnatally in FGR female guinea pigs. In this study, we did not find evidence for an effect of maternal nutrient restriction or Ad.VEGF-A165 therapy on adult offspring blood pressure.

Abstract A09: Development of epigenetic-based strategies for blocking breast cancer growth, invasion, and metastasis in vitro and in vivo

Abstract DNA hypomethylation has been implicated in the coordinated targeting of various signaling pathways involved in tumor growth and metastasis. In the current study, through various in vitro and in vivo assays, we have examined the plausibility of using universal methyl donor S-adenosyl methionine (SAM) to block breast cancer development, growth, and metastasis in our xenograft and transgenic models of breast cancer. Treatment of highly invasive human triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs578T with SAM resulted in a significant dose-dependent decrease in cell proliferation, invasion, migration, colony formation, and increased apoptosis in vitro. Affymetrix gene expression array and real-time PCR (qPCR) validated the ability of SAM to decrease the expression of several genes implicated in cancer progression in MDA-MB-231 cells. For the in vivo studies, MDA-MB-231 cells expressing green fluorescent protein (MDA-MB-231-GFP) were inoculated into female CD-1 nude mice via mammary fat pad. From day three post tumor cell inoculation, animals were treated with SAM (0.8-1.6 mg/day) or vehicle alone as control via daily oral gavage, and tumor volume was determined at weekly intervals for 10 weeks. SAM treatment caused a significant dose-dependent decrease in tumor volume and GFP-positive metastasis to lungs, liver, and spleen in experimental animals compared to vehicle-treated controls. Analysis of RNA from primary tumors by qPCR showed the ability of SAM to cause a marked decrease in the expression of several prometastatic and EMT pathway genes. Pyrosequencing of tumoral DNA from control and experimental animals showed that SAM treatment causes a significant increase in the percentage of CpG methylation at the promoter region of several cancer-related genes which were seen to be downregulated in the qPCR assay. We next determined the effect of SAM in MMTV-PyMT transgenic mouse model of breast cancer where SAM treatment resulted in a significant delay in the development of mammary tumors and decreased tumor growth in experimental animals as compared to vehicle-treated controls. SAM was found to bioavailable in the serum of experimental animals as determined by mass spectrometry and no notable adverse side effects were seen, including any change in animal behavior. Results from these studies provide compelling evidence for the therapeutic potential of SAM in breast cancer to provide the rationale for initiating clinical trials with SAM in patients with breast and other common cancers as monotherapy or in combination setting with current therapeutic agents to reduce cancer-associated morbidity and mortality. Note: This abstract was not presented at the conference. Citation Format: Niaz Mahmood, David Cheishvili, Ani Arakelian, William J. Muller, Imrana Tanveer, Haseeb Khan, Moshe Szyf, Shafaat A. Rabbani. Development of epigenetic-based strategies for blocking breast cancer growth, invasion, and metastasis in vitro and in vivo [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A09.

Abstract 4346: S-Adenosyl methionine (SAM) blocks breast cancer growth, invasion and metastasis in vitro and in vivo

Abstract DNA hypomethylation has been implicated in the coordinated targeting of various signaling pathways involved in tumor growth and metastasis. In the current study through various in vitro and in vivo assays, we have examined the plausibility of using universal methyl donar S-adenosyl methionine (SAM) for its ability to block breast cancer development, growth and metastasis in our xenograft and transgenic models of breast cancer. Treatment of highly invasive human triple negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs578T with SAM resulted in a significant dose-dependent decrease in cell proliferation, invasion, migration, colony formation and increased apoptosis in vitro. Affymetrix gene expression array and real time PCR (qPCR) validated showed the ability of SAM to decrease the expression several genes implicated in cancer progression in MDA-MB-231 cells. For the in vivo studies, MDA-MB-231 cells expressing green fluorescent protein (MDA-MB-231-GFP) were inoculated into female CD-1 nude mice via mammary fat pad. From day three post tumor cell inoculation, animals were treated with SAM (0.8-1.6 mg/day) or vehicle alone as control via daily oral gavage and tumor volume was determined at weekly intervals for 10 weeks. SAM treatment caused a significant dose dependent decrease in tumor volume and GFP positive metastasis to lungs, liver and spleen in experimental animals compared to vehicle-treated controls. Analysis of RNA from primary tumors by qPCR showed the ability of SAM to cause a marked decrease in the expression of several pro-metastatic and EMT pathway genes. Pyrosequencing of tumoral DNA from control and experimental animals showed that SAM treatment causes a significant increase in the percentage of CpG methylation at the promoter region of several cancer-related genes which were seen to be downregulated in the qPCR assay. We next determined the effect of SAM in MMTV-PyMT transgenic mouse model of breast cancer where SAM treatment resulted in a significant delay in the development of mammary tumors and decreased tumor growth in experimental animals as compared to vehicle treated controls. SAM was found to bioavailable in the serum of experimental animals as determined by mass spectrometry and no notable adverse side effects were seen including any change in animal behavior. Results from these studies provide compelling evidence for the therapeutic potential of SAM in breast cancer to provide the rationale for initiating clinical trials with SAM in patients with breast and other common cancers as monotherapy or in combination setting with current therapeutic agents to reduce cancer associated morbidity and mortality. Citation Format: Niaz Mahmood, David Cheishvili, Ani Arakelian, William J. Muller, Moshe Szyf, Shafaat A. Rabbani. S-Adenosyl methionine (SAM) blocks breast cancer growth, invasion and metastasis in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4346. doi:10.1158/1538-7445.AM2017-4346

Tumor Control Index: a novel tool to assess and compare tumor growth in experimental animals

Abstract Measurement of tumor diameters, tumor volumes, or area under the curve has been traditionally used to quantitate the effects of experimental manipulations in immune competent as well as immune-compromised mice and rats. However, using these parameters to measure the kinetics of tumor growth fail to reveal several complexities of growing tumors. Here, by means of tumor growth data from a large number of mice challenged with live tumor cells, we describe the use of a new composite parameter, Tumor Control Index (TCI) as an alternative method to do the same. This index, comprised of three distinct values, the Tumor Progression Score, Tumor Rejection Score, and Tumor Stability Score, provides a complete picture of nearly every aspect of tumor growth in large numbers of animals, can be deduced automatically from tumor diameter or volume data, and can be used to compare several groups of animals in different experiments. This automatically derivable index also corresponds neatly to the use of complete and partial responses and tumor stability data generated in human tumors, and can be used to assess the efficacy of interventions to be used in clinical studies.

Tumor Control Index as a new tool to assess tumor growth in experimental animals

Measurement of tumor diameters, tumor volumes, or area under the curve has been traditionally used to quantitate and compare tumor growth curves in immune competent as well as immune-compromised mice and rats. Here, using tumor growth data from a large number of mice challenged with live tumor cells, we describe the use of a new composite parameter, Tumor Control Index (TCI) as an alternative method to do the same. This index, comprised of three distinct values, the Tumor Inhibition Score, Tumor Rejection Score, and Tumor Stability Score, provides a complete picture of nearly every aspect of tumor growth in large numbers of animals, can be deduced automatically from tumor diameter or volume data, and can be used to compare several groups of animals in different experiments. This automatically derivable index also corresponds neatly to the use of complete and partial responses and tumor stability data generated in human tumors, and can be used to assess the efficacy of interventions to be used in clinical studies.

Effect of two hydatid cyst antigens on the growth of melanoma cancer in C57/black mice.

Hydatid cyst is the larval stage of Echinococcus granulosus. In previous studies inhibitory effect of this parasite on cancer cell growth in culture medium has been shown. In this study effect of hydatid cyst antigens on tumor growth in experimental animals has been investigated. Two antigens of hydatid cyst including protoscolices excretory secretory antigen and hydatid fluid absorbed on alum as adjuvant were injected to two groups of C57/black mice as case groups. Control groups were injected with only saline and alum. All mice then were injected with melanoma cells. Both antigens reduced the tumor size in mice in case groups. The difference of tumor size in mice in case groups and control group was statistically significant. In conclusion, anti-tumor effect of hydatid cyst antigens may be related to antigenic similarities which exist between hydatid cyst and cancer cells.

Modulation of scoliotic spine growth in experimental animals using intelligent metal bars

ObjectiveTo create an experimental structural scoliosis model in mice to evaluate the efficacy of shape-memory metals to gradually correct the deformity over time. Materials and methodsExperimental scoliosis was generated in 3 week-old mice by means of a suture between the left scapula and pelvis for 8 weeks. They were then randomized into two groups: a control group, in which the suture was cut, and another, in those that also had a Nitinol straight memory-wire implant fixed to the column. Serial X-rays were performed to determine the efficacy of the Nitinol in the correction of the scoliosis. In a second time, the histological changes at apical vertical body level and the adjacent discs were evaluated pre- and post-correction. ResultsA mean 81.5° kyphoscoliosis was gradually induced. In the control group, after cutting the suture, an initial reduction in the deformity was observed, but later it remained stable throughout the time (54° at two weeks). In the Nitinol group, a gradual reduction was observed in the scoliosis angle value, to a mean of 8.7° at two weeks. The curvature of the apical vertebral body and adjacent discs was partially corrected after two weeks of correcting the deformity. ConclusionThis scoliosis model has demonstrated the efficacy of a straight Nitinol wire fixed to the spinal column in the gradual correction of kyphoscoliosis and in the changes in its adjacent structures.

Manganese concentrations in maternal and umbilical cord blood: related to birth size and environmental factors

Manganese (Mn) is an essential element and a potential toxicant for developing organism. Deficiency and excess of it were both deleterious to fetal growth in experimental animals. However, literature on relationship between Mn status and birth outcome in humans is sparse. Mn concentrations were measured in mother whole blood (MWB) and umbilical cord blood (UCB) in 125 pairs of mother-infant; birth size was examined and relationship between them was analysed. Potentially environmental factors influencing Mn loads in maternal and fetal organisms were investigated through epidemiological method. Mn level in UCB was significantly higher than that in MWB (mean value: 54.98 vs. 78.75 µg/L), and a significant positive correlation was shown between them. There was a quadratic curvilinear (inverted U-shaped curve) relationship between MWB Mn and birth size, and between UCB Mn and birth size. Both univariate analysis and multiple linear regression analysis showed that exposure to harmful occupational factors during gestation remarkably increased maternal and fetal Mn levels. Living close to major transportation routes (<500 m) also increased the MWB Mn levels. Our results suggested that lower or higher Mn level in maternal and umbilical blood may induce adverse effect on birth size in humans. In addition, increased levels of Mn in MWB or UCB may be associated with exposure to some environmental hazard factors.

Evaluation of insulin-like growth factor acid-labile subunit as a potential biomarker of effect for deoxynivalenol-induced proinflammatory cytokine expression

Consumption of the trichothecene deoxynivalenol (DON) suppresses growth in experimental animals – an adverse effect that was used to establish the tolerable daily intake for this toxin. DON ingestion has been recently found to suppress plasma insulin-like growth factor acid-labile subunit (IGFALS), a protein essential for growth. Studies were conducted to explore the feasibility of using plasma IGFALS as a biomarker of effect for DON. In the first study, weanling mice were fed 0, 1, 2.5, 5 and 10ppm DON and weight and plasma IGFALS determined at intervals over 9 wk. Reduced body weight gains were detectable beginning at wk 5 in the 10ppm dose and wk 7 at the 5ppm dose. Plasma IGFALS was significantly depressed at wk 5 in the 5 and 10ppm groups at wk 9 in the 10ppm group. Depressed IGFALS significantly correlated with reduced body weight at wk 5 and 9. Benchmark dose modeling revealed the BMDL and BMD for plasma IGFALS reduction were 1.1 and 3.0ppm DON and for weight reduction were 2.1 and 4.5ppm DON. In the second study, it was demonstrated that mice fed 15ppm DON diet had significantly less plasma IGFALS than mice fed identical amounts of control diet. Thus DON's influence on IGFALS likely reflects the combined effects of reduced food intake as well as its physiological action involving suppressors of cytokine signaling. Taken together, these findings suggest that plasma IGFALS might be a useful biomarker for DON's adverse effects on growth.

Area under the curve as a tool to measure kinetics of tumor growth in experimental animals

Measurement of tumor diameters or tumor volumes has been traditionally used to quantitate and compare tumor growth curves in mice and rats. Here, we describe the use of area under the curve (AUC) as an alternative method to do the same. This single numerical value (a) is easy to obtain for individual curves, (b) reflects the entire tumor growth curve through a single number, (c) can be easily modified to obtain data for defined sections of the growth curve (for example, to determine stable disease), and (d) allows easier comparisons between groups. Using tumor growth data from a large number of mice challenged with live tumor cells, we demonstrate the utility of this tool over the conventional methods for these purposes.

The contribution of pulmonary‐to‐systemic cardiac shunting to growth, metabolism, and recovery from exercise in Alligator mississipiensis

All non-avian reptiles have the capacity to mix oxygenated and deoxygenated blood (cardiac shunt), but the adaptive significance of cardiac shunting has not been tested. The fully divided ventricle of crocodilians allows for surgical elimination of the capacity for pulmonary-to-systemic (R-L) shunting by occluding the left aortic arch (LAo) outside of the heart. With the experimental removal of pulmonary-to-systemic shunt capacity we can make inferences about its physiological function in reptiles. We surgically tied off the left aortic arch (LAo) of hatchling American alligators (Alligator mississipiensis) both proximal and distal to the foramen of Panizza, thereby eliminating shunting. The alligators were divided into three groups: experimental (occluded LAo), sham-operated and non-operated control. We studied the consequences of the elimination of shunt capacity on SDA, resting metabolism, lactate clearance following exhaustive exercise and the growth parameters of body mass, head length, snout-vent length and total length. In the first nine months following surgery, growth shows a non-significant trend of decreased growth in experimental animals. Supported by NSF IOB grant #04445680 to JWH.

Effects of peat feeding on the performance and health status of fattening pigs and environmentally derived mycobacteria

The purpose of the present study was to investigate the effects of feeding peat as a supplement in the period after weaning on the performance and health status of pigs. Also to assess the risk of the development of tuberculous lesions in the lymph nodes and parenchymatous organs, caused by conditionally pathogenic mycobacteria present in peat. Twenty Large White&amp;nbsp;&amp;times;&amp;nbsp;Landrace pigs in equal numbers of barrows and gilts (mean live weight 18.0&amp;nbsp;&amp;plusmn;&amp;nbsp;1.7&amp;nbsp;kg) were used in the experiment. The experimental group was fed a diet containing commercial underground peat in the dose of 80&amp;nbsp;g peat/kg dry matter for 30 days. Subsequently, they were fed an identical diet with the control group without peat for 60 days. A short-time feeding peat did not significantly affect the growth and performance of pigs. From day 21, a statistically highly significant (P &amp;lt; 0.01) increase in the consumption of the experimental diet was recorded, however, without a positive effect on the growth of experimental animals. The conversion of the peat containing diet was comparable to the conversion of the control diet. It follows from the results of biochemical analysis of blood that peat feeding for 30 days did not adversely affect the metabolic profile and health status of experimental animals. No tuberculous or tuberculoid lesions in lymph nodes or parenchymatous organs were detected in any of 20 slaughtered animals. Despite that, mycobacteria were isolated from 10 (25.0%) tissues of 5 (50.0%) pigs from the experimental group. One isolate was identified as Mycobacterium avium subsp. hominissuis (IS901&amp;ndash; and IS1245+). Seven of nine isolates were determined as conditionally pathogenic atypical mycobacteria: M.&amp;nbsp;fortuitum (n&amp;nbsp;=&amp;nbsp;2) and M.&amp;nbsp;xenopi (n&amp;nbsp;=&amp;nbsp;5). It follows from the present results that feeding of a peat supplemented diet to pigs may be considered as economically non-effective and due to the findings of mycobacteria as risky.

Differential-Display Analysis of Gene Expression in Livers From Normal and Partially Hepatectomized Mice

Partial hepatectomy, resulting in the removal of approximately 70% of the liver, is widely utilized for studies of liver growth in experimental animals. The regenerative response is proportional to the amount of liver removed. Knowing when and where genes are expressed provides a strong clue as to its biological role. The RNA differential-display (DD) technique facilitates monitoring the differential expression of a large number of activated or suppressed genes under various biological conditions. To reveal mechanisms of liver regeneration, we performed a comparative analysis of gene expression during liver regeneration using DD. We sacrificed male Balb/c mice, aged 10 to 12 weeks, at 0, 24, 48, and 72 hours, and 1 and 2 weeks after PHx. The livers were weighed, and the amount of glutamic-oxaloacetate transaminase in serum measured. We extracted the total RNA from frozen liver tissue and confirmed the RNA quality using a lab-chip system. DD analysis was performed essentially as described by Liang and Pardee. Semiquantitative reverse-transcription polymerase chain reaction was performed to confirm the results of DD analysis. Of the 56 fragments that exhibited changed expression levels during PHx, 39 were cloned and sequenced. There were 31 known genes, 13 unknown genes, and 9 expressed-sequence tags. These results indicated that DD is a powerful approach for monitoring molecular events in the regenerating liver.

Mechanical strain leads to condylar growth in adult rats

Mechanical strain produced by forward mandibular positioning was found to enhance mandibular condylar growth in experimental animals and in patients. This study was designed to identify the changes in number and rate of the proliferating mesenchymal cells in mandibular condyles of adult rats and to correlate these changes to the expression of SOX9 and type II collagen under mechanical strain. Seventy-eight 120-day-old female Sprague-Dawley rats were randomly allotted to six groups, nine animals in each experimental group according to different time points. Cell kinetic studies for expression of PCNA were used to identify number and rate of proliferating mesenchymal cells. Immunostaining of SOX9 and in situ hybridization of Col2a1 gene were carried out. Results showed a significant increase in number of replicating mesenchymal cells and proliferation rate. The expression of SOX9 was enhanced and Col2a1 gene transcript was then activated. The proliferative layer became thicker on experimental day 21. The thickness of chondroblast layer and chondrocyte layer showed significant increase from experimental day 14 to day 30. In conclusion, mechanical strain produced by mandibular advancement in adult rats promotes the proliferation of mesenchymal cells. Under control of transcription factor SOX9, these mesenchymal cells are then committed to enter the chondrogenic route leading to condylar growth.

Technical Note: Measurement of Total Estrone Content in Foods. Application to Dairy Products

Estrone is a powerful growth-inducing hormone that is present in milk, mainly in the form of fatty acid esters, at concentrations that promote growth in experimental animals. We present here a method useful for the measurement of this natural hormone in foods and applied it to several common dairy products. Samples were frozen, finely powdered, and lyophilized then extracted with trichloromethane/methanol; the dry extract was saponified with potassium hydroxide. The free estrone evolved was extracted with ethyl acetate and was used for the estimation of total estrone content through radioimmunoassay. Application of the method to dairy products showed high relative levels of total estrone (essentially acyl-estrone) in milk, in the range of 1μM, which were halved in skimmed milk. Free estrone levels were much lower, in the nanomolar range. A large proportion of estrone esters was present in all other dairy products, fairly correlated with their fat content. The amount of estrone carried by milk is well within the range, where its intake may exert a physiological response in the sucklings for which it is provided. These growth-inducing and energy expenditure-lowering effects may affect humans ingesting significant amounts of dairy products.