Postchemotherapy and tumor-selective targeting with the La-specific DAB4 monoclonal antibody relates to apoptotic cell clearance

Early identification of tumor responses to treatment is crucial for devising more effective and safer cancer treatments. No widely applicable, noninvasive method currently exists for specifically detecting tumor cell death after cytotoxic treatment and thus for predicting treatment outcomes.

Differential effect of inbred mouse strain (C57BL/6, DBA/2, 129T2) on insulin secretory function in response to a high fat diet

The increasing production of genetically-modified mouse models has necessitated studies to determine the inherent physiological characteristics of commonly used mouse strains. In this study we examined insulin secretory function in response to an intravenous bolus of glucose or glucose plus arginine in anesthetized C57BL/6, DBA/2 and 129T2 mice fed either a control or high fat diet for 6 weeks. The results show that 129T2 mice had higher fasting plasma glucose levels and lower fasting plasma insulin levels compared with C57BL/6 and DBA/2 mice regardless of diet. Furthermore, 129T2 mice were glucose intolerant and secreted significantly less insulin in response to glucose and glucose plus arginine irrespective of diet compared with the other two strains of mice. DBA/2 mice hypersecreted insulin in response to glucose and glucose plus arginine compared with C57BL/6 and 129T2 mice. Moreover while first phase insulin secretion was appropriately increased in response to the high fat diet in C57BL/6 and 129T2 mice, this was not the case for DBA/2 mice. Mean islet area was decreased in response to a high fat diet in DBA/2 mice, while there was no dietary effect on the other two strains. This study highlights the inherent genetic differences that exist among seemingly normal strains of mice that are commonly used to make transgenic and knockout mice. Understanding these differences will provide researchers with the information to choose the appropriate genetic background on which to express their particular genetic alteration.

Angiotensin converting enzyme inhibition lowers body weight and improves glucose tolerance in C57BL/6J mice maintained on a high fat diet

The renin–angiotensin system (RAS) is functional within adipose tissue and angiotensin II, the active component of RAS, has been implicated in adipose tissue hypertrophy and insulin resistance. In this study, captopril, an angiotensin converting enzyme (ACE) inhibitor that prevents angiotensin II formation, was used to study the development of diet-induced obesity and insulin resistance in obesity prone C57BL/6J mice. The mice were fed a high fat diet (w/w 21% fat) and allowed access to either water or water with captopril added (0.2 mg/ml). Body weight was recorded weekly and water and food intake daily. Glucose tolerance was determined after 11–12 weeks. On completion of the study (after 16 weeks of treatment), the mice were killed and kidney, liver, epididymal fat and extensor digitorum longus muscle (EDL) were weighed. Blood samples were collected and plasma analysed for metabolites and hormones. Captopril treatment decreased body weight in the first 2 weeks of treatment. Food intake of captopril-treated mice was similar to control mice prior to weight loss and was decreased after weight loss. Glucose tolerance was improved in captopril-treated mice. Captopril-treated mice had less epididymal fat than control mice. Relative to body weight, captopril-treated mice had increased EDL weight. Relative to control mice, mice administered captopril had a higher plasma concentration of adiponectin and lower concentrations of leptin and non-esterified fatty acids (NEFA). The results indicate that captopril both induced weight loss and improved insulin sensitivity. Thus, captopril may eventually be used for the treatment of obesity and Type 2 diabetes.

Increased nicotinamide nucleotide transhydrogenase levels predispose to insulin hypersecretion in a mouse strain susceptible to diabetes

Aims/hypothesis Insulin hypersecretion may be an independent predictor of progression to type 2 diabetes. Identifying genes affecting insulin hypersecretion are important in understanding disease progression. We have previously shown that diabetes-susceptible DBA/2 mice congenitally display high insulin secretion. We studied this model to map and identify the gene(s) responsible for this trait.

Methods Intravenous glucose tolerance tests followed by a genome-wide scan were performed on 171 (C57BL/6 × DBA/2) × C57BL/6 backcross mice.

Results A quantitative trait locus, designated hyperinsulin production-1 (Hip1), was mapped with a logarithm of odds score of 7.7 to a region on chromosome 13. Production of congenic mice confirmed that Hip1 influenced the insulin hypersecretion trait. By studying appropriate recombinant inbred mouse strains, the Hip1 locus was further localised to a 2 Mb interval, which contained only nine genes. Expression analysis showed that the only gene differentially expressed in islets isolated from the parental strains was Nnt, which encodes the mitochondrial proton pump, nicotinamide nucleotide transhydrogenase (NNT). We also found in five mouse strains a positive correlation (r 2  = 0.90, p < 0.01) between NNT activity and first-phase insulin secretion, emphasising the importance of this enzyme in beta cell function. Furthermore, of these five strains, only those with high NNT activity are known to exhibit severe diabetes after becoming obese.

Conclusions/interpretation Insulin hypersecretion is associated with increased Nnt expression. We suggest that NNT must play an important role in beta cell function and that its effect on the high insulin secretory capacity of the DBA/2 mouse may predispose beta cells of these mice to failure.

Vascular attack by 5,6-dimethylxanthenone-4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci

The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60–180 µg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2–6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 107 parental EL-4 tumor cells but not a challenge of 1 x 104 Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 108 splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 µg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients’ immune responses to their own tumors.

Comparison of tissue dosimetry in the mouse following chronic exposure to arsenic compounds

Several chronic bioassays have been conducted in multiple strains of mice in which various concentrations of arsenate or arsenite were administered in the drinking water without a tumorigenic effect. However, one study (Ng et al., 1999) reported a significant increase in tumor incidence in C57Bl/6J mice exposed to arsenic in their drinking water throughout their lifetime, with no tumors reported in controls. A physiologically based pharmacokinetic model for arsenic in the mouse has previously been developed (Gentry et al., 2004) to investigate potential differences in tissue dosimetry of arsenic species across various strains of mice. Initial results indicated no significant differences in blood, liver, or urine dosimetry in B6C3F1 and C57Bl/6 mice for acute or subchronic exposure. The current work was conducted to compare model-predicted estimates of tissue dosimetry to additional kinetic information from the (C57Bl/6 x CBA)F1 and TgAc mouse. The results from the current modeling indicate that the pharmacokinetic parameters derived based on information in the B6C3F1 mouse adequately describe the measured concentrations in the blood/plasma, liver, and urine of both the (C57Bl/6 x CBA)F1 and TgAc mouse, providing further support that the differences in response observed in the chronic bioassays are not related to strain-specific differences in pharmacokinetics. One significant finding was that no increases in skin or lung concentrations of arsenic species in the (C57Bl/6 x CBA)F1 strain were observed following administration of low concentrations (0.2 or 2 mg/L) of arsenate in the drinking water, even though differences in response in the skin were reported. These data suggest that pharmacodynamic changes may be observed following exposure to arsenic compounds without an observable change in tissue dosimetry. These results provided further indirect support for the existence of inducible arsenic efflux in these tissues.

Brain neuropeptide Y and CCK and peripheral adipokine receptors : temporal response in obesity induced by palatable diet

Objective:
Palatable food disrupts normal appetite regulation, which may contribute to the etiology of obesity. Neuropeptide Y (NPY) and cholecystokinin play critical roles in the regulation of food intake and energy homeostasis, while adiponectin and carnitine palmitoyltransferase (CPT) are important for insulin sensitivity and fatty acid oxidation. This study examined the impact of short- and long-term consumption of palatable high-fat diet (HFD) on these critical metabolic regulators.

Methods:
Male C57BL/6 mice were exposed to laboratory chow (12% fat), or cafeteria-style palatable HFD (32% fat) for 2 or 10 weeks. Body weight and food intake were monitored throughout. Plasma leptin, hypothalamic NPY and cholecystokinin, and mRNA expression of leptin, adiponectin, their receptors and CPT-1, in fat and muscles were measured.

Results:
Caloric intake of the palatable HFD group was 2–3 times greater than control, resulting in a 37% higher body weight. Fat mass was already increased at 2 weeks; plasma leptin concentrations were 2.4 and 9 times higher than control at 2 and 10 weeks, respectively. Plasma adiponectin was increased at 10 weeks. Muscle adiponectin receptor 1 was increased at 2 weeks, while CPT-1 mRNA was markedly upregulated by HFD at both time points. Hypothalamic NPY and cholecystokinin content were significantly decreased at 10 weeks.

Conclusion:
Palatable HFD induced hyperphagia, fat accumulation, increased adiponectin, leptin and muscle fatty acid oxidation, and reduced hypothalamic NPY and cholecystokinin. Our data suggest that the adaptive changes in hypothalamic NPY and muscle fatty acid oxidation are insufficient to reverse the progress of obesity and metabolic consequences induced by a palatable HFD.

Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic airway disease in mice

Background: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases.

Objective: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses.

Methods: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge.

Results: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective.

Conclusion: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.

'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Functional and metabolic remodelling in GLUT4-deficient hearts confers hyper-responsiveness to substrate intervention

Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying  mechanisms linking these conditions is yet to be determined. Using a ‘gene dose’ Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, > 95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8 ± 13.2 mmHg vs. WT, 146.4 ± 9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic β-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional ‘rescue’.

The calcineurin signal transduction pathway is essential for successful muscle regeneration in mdx dystrophic mice

Although mdx mice share the same genetic defect and lack dystrophin expression as in Duchenne muscular dystrophy (DMD), their limb muscles have a high regenerative capacity that ensures a more benign phenotype and essentially normal function. The cellular pathways responsible for this enhanced regenerative capacity are unknown. We tested the hypothesis that the calcineurin signal transduction pathway is essential for the successful regeneration following severe degeneration observed in the limb muscles of young mdx mice (2–4 weeks old) and that inhibition of this pathway using cyclosporine A (CsA) would exacerbate the dystrophic pathology. Eighteen-day-old mdx and C57BL/10 mice were treated with CsA for 16 days. CsA administration severely disrupted muscle regeneration in mdx mice, but had minimal effect in C57BL/10 mice. Muscles from CsA-treated mdx mice had fewer centrally nucleated fibers and extensive collagen, connective tissue, and mononuclear cell infiltration than muscles from vehicle-treated littermates. The deleterious effects of CsA on muscle morphology were accompanied by a 30–35% decrease in maximal force producing capacity. Taken together, these observations indicate that the calcineurin signal transduction pathway is a significant determinant of successful skeletal muscle regeneration in young mdx mice. Up-regulating this pathway may have clinical significance for DMD.

VVP808 is a novel agent that improves glucose tolerance in DIO mice

As the prevalence of diabetes mellitus continues to increase, there is an urgent need to discover new, effective treatment strategies to combat this disorder. In this study, we tested a novel agent, VVP808, which we previously demonstrated has insulin-sensitising properties (as measured by an increase in insulin-stimulated glucose uptake in 3T3-L1 adipocytes). A dose-ranging study was performed (10-100mg/kg/d) in C57BL/6J mice that had been fed a high-fat diet (45% of energy) for 12 weeks. VVP808 was administered by single daily oral gavage for a period of 16 days. Body weight, food intake and water intake were measured daily, whilst fasting blood glucose and plasma insulin levels were measured at the beginning and end of the study, with an intra-peritoneal glucose tolerance test (ipGTT) performed on day -1 and day 13. Administration of VVP808 to diet-induced obese (DIO) mice caused a strong dose-dependent improvement in glucose tolerance. There was a 34-42% reduction in the blood glucose area under the curve (AUC) at doses of 20mg/kg, 50mg/kg and 100mg/kg VVP808 (p=0.02-0.005). Administration of VVP808 resulted in a small but significant reduction in body weight in the 50mg/kg and 100mg/kg treated animals relative to vehicle (p=0.01 and 0.001 respectively). This decrease in body weight was associated with a reduction in food intake for the 100mg/kg treated animals only. Epididymal fat pad weight was significantly reduced in animals treated with 100mg/kg VVP808 (p=0.01). Furthermore, treatment with VVP808 for 16 days resulted in a highly significant dose-dependent reduction in fasting blood glucose levels relative to vehicle treated animals (p= 0.01-0.001). In conclusion, our data showed that VVP808 acts in a dose-dependent manner to reduce fasting blood glucose levels and improve glucose tolerance. These data suggest that VVP808 is an interesting new agent with potential for development as a novel therapeutic for type 2 diabetes.

Clustering of Th cell epitopes on exposed regions of HIV envelope despite defects in antibody activity

A long-standing question in the field of immunology concerns the factors that contribute to Th cell epitope immunodominance. For a number of viral membrane proteins, Th cell epitopes are localized to exposed protein surfaces, often overlapping with Ab binding sites. It has therefore been proposed that Abs on B cell surfaces selectively bind and protect exposed protein fragments during Ag processing, and that this interaction helps to shape the Th cell repertoire. While attractive in concept, this hypothesis has not been thoroughly tested. To test this hypothesis, we have compared Th cell peptide immunodominance in normal C57BL/6 mice with that in C57BL/6MT/MT mice (lacking normal B cell activity). Animals were first vaccinated with DNA constructs expressing one of three different HIV envelope proteins, after which the CD4 T cell response profiles were characterized toward overlapping peptides using an IFN- ELISPOT assay. We found a striking similarity between the peptide response profiles in the two mouse strains. Profiles also matched those of previous experiments in which different envelope vaccination regimens were used. Our results clearly demonstrate that normal Ab activity is not required for the establishment or maintenance of Th peptide immunodominance in the HIV envelope response. To explain the clustering of Th cell epitopes, we propose that localization of peptide on exposed envelope surfaces facilitates proteolytic activity and preferential peptide shuttling through the Ag processing pathway.

An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8+ T cells

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8 T cell responses to H2Db complexed with peptides from the viral nucleoprotein (NP366, ASNENMETM) and acid polymerase (PA224, SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of DbNP366- and DbPA224-specific acute and memory CD8 T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8 T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8DbNP366 T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.