Antihyperglycaemic effect of saccharin in diabetic ob/ob mice

1. The effect of chronic saccharin (benzosulphimide) consumption on glucose homeostasis was examined in normal lean +/+ mice and genetically obese hyperglycaemic insulin-resistant ob/ob mice.
2. Consumption of a 5% (w/v) sodium saccharin solution for 7 weeks prevented the development of hyperglycaemia, improved glucose tolerance (area under curve decreased by 51%), reduced the extent of hyperinsulinaemia (by 21%), and reduced excessive weight gain (by 18%) in ob/ob mice.
3. Consumption of 5% (w/v) sodium saccharin temporarily decreased hyperphagia at the beginning of treatment, decreased hepatic glycogen content (by 47%), increased abdominal muscle glycogen content (by 82%), but did not significantly alter the hypoglycaemic response to exogenous insulin in ob/ob mice.
4. Consumption of a 1% (w/v) sodium saccharin solution did not prevent the development of hyperglycaemia in ob/ob mice.
5. Normal lean +/+ mice consuming 5% (w/v) sodium saccharin solution showed a marginal decrease (by 8%) in glycaemia, and glucose tolerance was improved (area under curve decreased by 30%) without a significant change in the insulin response to glucose or the hypoglycaemic effect of exogenous insulin.
6. The results suggest that chronic consumption of saccharin can defer the development of hyperglycaemia and improve glucose homeostasis in insulin-resistant ob/ob mice through a mechanism that is independent of insulin.

     

The cumulative normalised rim/disc area ratio curve

Background: The assessment of the cup of the optic disc depends, among other criteria, on the disc area. A small cup in a small optic disc can indicate an advanced glaucomatous lesion, while on the other hand a large cup in a large optic disc can be normal. Therefore, a cumulative normalised rim/disc area ratio curve could help to distinguish between glaucomatous and normal optic cups. The aim of our study was to calculate and to evaluate such a cumulative normalised rim/disc area ratio curve. Methods: Heidelberg Retina Tomograph examinations of the optic nerve head of 100 randomly selected eyes of 100 normal subjects were evaluated. We calculated the disc area-adjusted normalised rim/disc area ratio in sectors of 10°. The 95th, 90th and 50th percentiles of each of the 36 sectors were displayed in descending order. Results: In relation to the normal percentile curves, it is possible to display an individual normalised rim/disc area ratio curve. We obtained such curves for a normal optic disc, optic nerve heads with moderate and advanced lesions and a small optic disc with glaucomatous damage. Conclusion: We present a new display mode for the results of Heidelberg Retina Tomograph optic nerve head examination, which may be helpful for easy and reliable assessment of the local, diffuse and combined components of glaucomatous optic nerve head damage depending on optic disc size.     

Chronic (−)-isoprenaline infusion down-regulates β1- and β2-adrenoceptors but does not transregulate muscarinic cholinoceptors in rat heart

Regulation of -adrenoceptor (-ar) subtypes and transregulation of muscarinic cholinoceptors (mAchr) was examined in regions of rat heart after chronic infusion of (–)-isoprenaline (450 /kg per hour) for 14 days. Following (–)-isoprenaline infusion systolic blood pressure was reduced for 10 days but then gradually returned to control levels, whereas heart rate was increased for 7 days before declining to a level significantly above control. Heart weight to body weight ratio was increased in (–)-isoprenaline treated rats. -ar subtype densities were measured by quantitative autoradiography with [¹²⁵I]-cyanopindolol (CYP) in sinoatrial node (SA), atrioventricular node (AV), bundle of His (BH), left (LB) and right (RB) bundle branches, interventricular (IVS) and interatrial (IAS) septa, right atria (RA), apex (AX) and mitral valve (MV). ₁-ars were reduced by 59.1–74.2% in the AV conducting regions, 53.4% in the SA node and 43.3–53.4% in myocardial areas. ₂-ars were markedly reduced in myocardial regions (93.2–98.5%) and in pacemaker and conducting regions (87.7–97.8%). No changes in mAchr densities measured using [³H]-N-methyl scopolamine (NMS) occurred in the AV node, BH, LB, RB, IVS and IAS following (–)-isoprenaline infusion.Densities of ₁- and ₂-ars and mAchrs were also measured in ventricular homogenates from control and (–)-isoprenaline treated animals. -ar levels were significantly reduced (P < 0.05) in treated animals and the ratio of ₁- to ₂-ars increased after treatment. mAchr density in ventricular homogenates measured using either [³H]-NMS or [³H]-quinuclidinyl [phenyl-4-³H]benzilate (QNB) was unchanged. Homogenates of left and right ventricle also showed no change using [³H]-NMS.Organ bath studies were used to investigate the effect of (–)-isoprenaline infusion on negative inotropic and chronotropic effects of the non-selective muscarinic receptor agonist bethanechol in left and right atria, respectively. Lower concentrations of bethanechol (3 × 10^–10 to 10^–6 M) produced a negative inotropic response in isolated electrically driven left atria from (–)-isoprenaline treated rats, but not from control rats, with the slope of the curves being significantly different between groups (ANCOVA, P = 0.037). At concentrations of bethanechol from 10^–6 to 3 × 10^–4 M the negative inotropic response was not changed between (–)-isoprenaline treated and control animals. Bethanechol also produced a negative chronotropic response at lower concentrations (10^–10 to 10^–6 M) in (–)-isoprenaline treated rats, but not in controls. A second, steeper phase of the negative chronotropic response occurred at concentrations of bethanechol greater than 10^–6 M and was also seen in control rats.Expression of M₂ (cardiac) mAchrs (m₂Achr) in left and right ventricular tissues measured using a quantitative non-competitive polymerase chain reaction (PCR) assay showed a significant (P = 0.001) 28.5% increase in expression in left ventricle and a significant (P = 0.003) 21.5% decrease in expression in right ventricle after (–)-isoprenaline treatment, compared to controls. There was no significant difference in total ventricular m₂Achr expression between the two groups of rats. The results suggest that chronic -ar stimulation down-regulates both ₁- and ₂-ars, and appears to differentially transregulate m₂Achr expression, but not mAchr protein. Following (–)-isoprenaline infusion, muscarinic receptor mediated responses were sensitised, with no change in receptor densities, suggesting changes occur in the cell signalling system beyond the level of the receptor.     

Active vs. passive resistance, dose-response relationships, high dose chemotherapy, and resistance modulation: a hypothesis

Summary With chemotherapy, the in vitro and clinical dose-response curve is steep in some situations, but is relatively flat in others, possibly due to the mechanism by which tumors are resistant to chemotherapy. For tumors with resistance due to factors that actively decrease chemotherapy efficacy (e.g., p-glycoprotein, glutathione, etc.), one would predict that high dose chemotherapy and therapy with some resistance modulating agents would increase therapeutic efficacy. Such active resistance would most likely generally arise from gene amplification or over expression, and would be characterized by a shoulder on the log response vs. dose curve, with eventual saturation of the protective mechanism. On the other hand, one would expect that high dose chemotherapy and most resistance modulating agents would be of little value for rumors with resistance due to defective apoptosis or due to a deficiency in or decreased drug affinity for a drug target, drug activating enzyme, drug active uptake system, or essential cofactor. Such passive resistance would most likely generally arise from gene down regulation, deletion, or mutation, and would probably be characterized by a relatively flat log response vs. dose curve, or by a curve in which a steep initial section is followed by a plateau, as target, etc., is saturated. (If response were plotted vs. log dose, then compared to the curve for a sensitive cell line, the curve for active resistance would be analogous to the pharmacodynamic curve seen with competitive antagonism [i.e., a sigmoid curve shifted to the right], and the curve for most types of passive resistance would be analogous to the pharmacodynamic curve seen with noncompetitive antagonism [i.e., a sigmoid curve with reduced maximal efficacy]. As such, one might also refer to active vs. passive resistance as competitive vs. noncompetitive resistance, respectively.) Many tumor types probably possess a combination of active and passive mechanisms of resistance. New in vivo strategies could be helpful in defining dose-response relationships, mechanisms of resistance, and targets for resistance modulation. Such in vivo studies would be conducted initially in animals, but might also be tested clinically if animal studies demonstrated them to be feasible and useful. These in vivo studies would be conducted by randomizing 5–25 subjects to one of 10–20 dose levels over a potentially useful therapeutic range. Nonlinear regression analysis would then be used to define the characteristics of a curve generated by plotting against dose the log percent tumor remaining after the first course of therapy. While this might offer insight into the nature of resistance mechanisms present initially, plotting further tumor shrinkage vs. dose-intensity vs. course number for each later treatment course (or plotting dose-intensity vs. time to tumor progression) might provide information on how tumors become increasingly resistant to drugs following treatment.     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: REGULATION OF RENAL TUBULAR SODIUM TRANSPORT BY ANGIOTENSIN II AND ATRIAL NATRIURETIC FACTOR

• 1 The effects of angiotensin II (AngII) on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations (10^?12 to 10^?9 mol/L) stimulate reabsorption and high concentrations (10^?7 to 10^?6 mol/L) inhibit reabsorption. Similar dose-response relationships have been obtained for luminal and peritubular addition of AngII.
• 2 The cellular responses to AngII are mediated via AT₁ receptors coupled via G-regulatory proteins to several possible signal transduction pathways. These include the inhibition of adenylyl cyclase, activation of phospholipases A₂, C or D and Ca²⁺ release in response to inositol-1,4,5,-triphosphate or following Ca²⁺ channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. In the brush border membrane, transduction of the AngII signal involves phospholipase A₂, but does not require second messengers.
• 3 Angiotensin II affects transepithelial sodium transport by modulation of Na⁺/H⁺ exchange at the luminal membrane and Na⁺/HCO₃ cotransport, Na⁺/K⁺-ATPase activity and K⁺ conductance at the basolateral membrane.
• 4 Atrial natriuretic factor (ANF) does not appear to affect proximal tubular sodium transport directly, but acts via specific receptors on the basolateral and brush border membranes to raise intracellular cGMP levels and inhibit AngII-stimulated transport.
• 5 It is concluded that there is a receptor-mediated action of ANF on proximal tubule reabsorption acting via elevation of cGMP to inhibit AngII-stimulated sodium transport. This effect is exerted by peptides delivered at both luminal and peritubular sides of the epithelium and provides a basis for the modulation by ANF of proximal glomerulotubular balance. The evidence reviewed supports the concept that in the proximal tubule, AngII and ANF act antagonistically in their roles as regulators of extracellular fluid volume.

     

In vivo modulation of vincristine-induced neurotoxicity in Lymnaea stagnalis, by the ACTH(4–9)analogue Org 2766

Summary The use of the cytostatic agent vincristine (VCR) is limited by the occurrence of peripheral neuropathy. This side-effect is probably caused by interference with axonal microtubules. VCR depolymerizes microtubules and reacts with tubulin to form paracrystals. The potential of a neurotrophic ACTH_(4–9) analogue, Org 2766, to counteract peripheral neuropathy caused by cytostatic agents is being investigated. In the present ultrastructural study, modulatory effects of Org 2766 on VCR-induced neurotoxicity were studied in vivo in neurons of the pond snail Lymnaea stagnalis, which has been shown previously to be a suitable test system to investigate neurotoxic side-effects of cytostatic agents. 24 h after treatment with VCR (25 M), 68.4 ± 34.7 paracrystals were counted per cross-section of the cerebral commissure and the number of microtubules in the axons had been lowered to 46% of the control level. After a survival period of two weeks all paracrystals had disappeared. By that time, no recovery of the axonal microtubular system could be observed. However, post-treatment with Org 2766 (10^–6 M) on day 6 after VCR treatment had induced a significant increase in the number of microtubules (+ 55%) on day 7. This beneficial effect lasted for the rest of the experimental period (14 days). These results suggest that post-treatment with Org 2766, i.e. after VCR clearance, can induce long-lasting beneficial effects on VCR-induced neurotoxicity in vivo.     

INT2 and ERBB2 amplification and ERBB2 expression in breast tumors from patients with different outcomes

Summary The relationships of INT2 and ERBB2 amplification and of ERBB2 overexpression in primary breast tumors to prognostic factors, recurrence, and survival have generated considerable controversy. The rationale for this study is that long-term, recurrence-free survival is a more direct criterion for testing the validity of a tumor marker than correlation either with prognostic factors or with short-term recurrence and survival. We examined the association of recurrence with INT2 and ERBB2 amplification and ERBB2 expression by comparing primary breast tumors from patients surviving without recurrence for 8.5 years after diagnosis. the LTS group, to tumors from patients recurring within two years, the RR group. The RR (N = 63) and LTS (N = 61) samples were coded and examined for amplification by Southern blotting and for expression by immunohistochemistry. Comparison between the RR and LTS groups demonstrated that INT2 amplification was associated with a significantly (P = 0.018) higher (5.6-fold) risk of recurrence, an association that remained significant after controlling for lymph node (LN), tumor size (TS), and histograde (HG) status. ERBB2 amplification and expression were not associated with a higher recurrence risk. Survival analyses within the RR group, however, demonstrated significantly shorter survival time among cases with than without ERBB2 amplification (P = 0.018, median survival 16 vs 25 months), or ERBB2 expression (P = 0.019, median survival 15 vs 25 months), but not INT2 amplification. Univariate Cox proportional hazards regression models also demonstrated significantly shorter survival among cases with ERBB2 amplification (P = 0.016) or expression (P = 0.049), that remained significant in multivariate analyses (P = 0.022) for ERBB2 amplification. These results indicate a significant positive association between INT2 amplification and risk for tumor recurrence in the RR as compared to the LTS group. The relationship of ERBB2 amplification or overexpression to patient outcome is more complex. ERBB2 amplification and expression have a significant relationship with shorter survival among patients recurrent within two years, but their occurrence in tumors from women surviving without recurrence for 8.5 years suggests that ERBB2 status is not predictive of shorter survival for all breast cancers.     

Heterogeneity ofin vitro chemosensitivity in perioperative breast cancer cells to mitoxantroneversus doxorubicin evaluated by a microplate ATP bioluminescence assay

Summary Apart from clinical trials, mitoxantrone (MX) is rarely used in breast cancer (BC) due to the anticipated anthracycline cross-resistance. We have examined this drug versus doxorubicin (DOX) using data obtained fromin vitro microplate ATP tumor chemosensitivity assays (ATP-TCA) of BC cells which were derived from 55 chemotherapy-naive patients at time of primary surgery. Both drugs were tested at 6 different concentrations ranging from 6.25% to 200% peak plasma concentrationin vivo (PPC). Differences between DOX and MX observed for mean IC₅₀, IC₉₀, and a sensitivity index (SI) were not statistically significant.In vitro response rates were 44% for DOX and 52% for MX. 34 of 52 eligible assays (65%) showed comparable activity of both drugs whereas a lack of cross-resistance was observed in the remaining 18 (35%) tumors as indicated by differences for SI. Cumulative concentration-response plots of tumors respondingin vitro with a 50 percent or 90 percent tumor cell inhibition showed a strong dose-dependence for both DOX and MX at concentrations which normally can be achieved within clinical tumors (i.e. 6.25%-50% PPC). At higher concentrations, however, cytotoxicity of DOX and MX could not be improved by furtherin vitro dose escalation. Moreover, a substantial proportion of BC specimens (DOX: 48.1%; MX: 40.4%) did not experience a 90 tumor cell inhibition at 200% PPC. In conclusion,in vitro results obtained by ATP-TCA indicate that there is no cross-resistance between MX and DOX in a substantial proportion of BC patients. This may be clinically useful and suggests that combinations including MX should be tested in patients clinically resistant to DOX containing regimens. Since both drugs produced sigmoidal concentration-response curves, dose escalation beyond a certain point may not produce increased sensitivity.     

Cell, tissue and organ culture as in vitro models to study the biology of squamous cell carcinomas of the head and neck

In vitro models are currently being used to study head and neck squamous cell carcinoma (HNSCC). Several hundred HNSCC cell lines have been established by various investigators and used to study a broad spectrum of questions related to head and neck cancer. The head and neck model with respect to multistage carcinogenesis is now complete. Several techniques exist for the culture of normal epithelial cells from the upper aerodigestive tract (UADT). The biology of these UADT cells (oral cavity, oropharynx, hypopharynx and larynx) is being studied. Successful culture of premalignant lesions (dysplastic mucosa, leukoplakia, erythroplakia) has resulted in establishment of a limited number of premalignant cell lines and cell cultures. HPV infection of normal oral epithelial cells for immortalization ( premalignant cells) coupled with transformation with carcinogens (malignant cells) has established an experimental model for progression. Two in vivo models for oral carcinogenesis, the 7,12 dimethylbenz(a)anthracene-induced hamster cheek pouch model and the 4-nitroquinoline-N-oxide rat oral model, have been established in culture. Thus, multistage carcinogenesis models have been established from both human tissues and animal models and include cultures of normal, premalignant and malignant cells. Culture techniques for growing dissociated primary tumor cells for short term experimental analysis are being used. The culture of normal or tumor tissue as organ/explant cultures allows for the maintenance of normal cell-cell and cell-matrix interactions, but limits experimentation since these cultures cannot be propagated. Several three dimensional model systems are being used to obtain this histological complexity but allow for experimentation. The ability to culture normal, premalignant and malignant cells coupled with the use of a variety of culture techniques, should allow for the continued growth and experimentation in head and neck cancer research.     

nm23-H1 expression in squamous cell carcinoma of the head and neck

Archival material from 47 patients with primary squamous cell carcinoma of the head and neck (SCCHN) was studied immunohistochemically for the presence of nm23-H1 protein. Our data indicate that nm23-H1 protein expression is a common event in SCCHN and that there is a trend toward correlation of increased expression of nm23-H1 with increasing tumor size (p = 0.072). The results also show that when adjusting for age and cause of death, there tended to be an inverse relationship between overall survival and the expression of nm23-H1 gene in the primary tumor (p = 0.088).