Rapid detection of −α 4.2 deletion of α-thalassemia-2 by polymerase chain reaction

Summary We sequenced part of the X boxes of-thalassemia-1 of Southeast Asia type (- -SEA) with– ^4.2,– ^3.7,– ^G-Taichung, and ^CS. We found the X box of– ^3.7 belonged to the X box of 2 globin gene and the X box of ^cs contained X boxes of both al and2 globin gene, whereas the X box of– ^4.2 and– ^G-Taichung was a hybrid of X boxes of 2 and 1 globin gene. We also found there are two types of– ^4.2 deletion; type 1 is a common type of– ^4.2 deletion and type 2 is linkage to– ^G-Taichung. We used a combination of two methods, the amplification refractory mutation system (ARMS) and the amplified created restriction sites (ACRS), to amplify the hybrids of X boxes specifically. The upstream primer for X box of2 globin gene was designed following the standard ARMS procedure to amplify the X segment of the-globin gene. The downstream primer was designed according to the ACRS method to check the specificity of PCR products. Using this approach, we can diagnose the different types of– ^4.2 deletion. This kind of approach can also be used to amplify the specific region from the cluster of highly homologous genes.     

Effects of 5-hydroxytryptamine and adenosine on the canine isolated cerebral resistance vessels

We investigated the effects of 5-hydroxytryptamine (5-HT), adenosine, and adenine nucleotides on the canine isolated cerebral resistance arteries ranging from 300 to 500 µm in diameter. Addition of 5-HT and 5-carboxami-dotryptamine (5-CT) produced concentration-dependent contractions in the resistance arteries. Methiothepin (10^–8 M-10^–6 M) caused a parallel shift to the right of the dose-response curves for 5-HT and 5-CT. Neither ketanserin (10^–7 M, 10^–6 M) nor [(1H, 5H)-8-methyl-8 azabicyclo [3,2,1] oct-3-y1]1H-indole-3-carboxylate hydrochloride (ICS 205–930 10^–7M, 10^–6M) caused any significant shift of the dose-response curve for 5-HT. Adenosine, ATP, ADP, and AMP caused concentration-dependent relaxations in the resistance arteries following contraction with PGF2. The adenosine analogs also caused a dose-dependent relaxation of the arterial segments contracted by PGF2. The decreasing order of potency of the agonists was 5-N-ethylcarboxamido adenosine (NECA)>adenosine L-diasteroisomers of phenylisopropyl adenosine (L-PIA). The relaxant response to NECA was competitively antagonized by 8-phenyltheophylline. The pretreatment with 3×10^–5 M N^G-monomethyl-L-arginine (L-NMMA) or 5×10^–5 M aspirin caused no significant effect on the adenosine-induced relaxation in the resistance arteries. These results suggest that 5-HT produces contraction of the canine isolated cerebral resistance arteries, which is mainly mediated via 5-HT₁-like receptors. Adenosine causes an endothelium-independent relaxation of the resistance arteries through the activation of adenosine A₂-receptors.     

α-Adrenergic responses of isolated canine coronary microvessels

Although -adrenergic activation is known to increase coronary microvascular resistance in vivo, the magnitude of its segmental microvascular consequences is not well understood. Quantification of these effects in vivo is hindered by escape mechanisms that minimize the influences of constrictors, and alterations in flow and pressure, which effect microvascular tone by shear stress-dependent and myogenic mechanisms, respectively. To eliminate these confounding influences, we have studied responses in vitro under conditions with these variables controlled. We evaluated the diameter changes of isolated canine coronary arterioles (110±12 m, n=35) and venules (98±7 m, n=9) in response to -adrenergic activation by norepinephrine (10^–10 to 10^–4 M) in the presence of -adrenergic blockade by alprenolol (10^–6 M). In contrast to the situation in vivo, -adrenergic activation did not constrict isolated coronary arterioles, but constricted isolated coronary venules in a dose-dependent manner over a range of 10^–10 to 10^–4 M (–27 ±3% maximum diameter change). Coronary arteriolar -adrenergic constriction was not promoted by 1) subthreshold or vasoactive doses of the vasoconstrictors KCl, angiotensin II, U46619, endothelin-1, neuropeptide Y or arginine vasopressin, 2) inhibition of the presynaptic uptake of norepinephrine by imipramine (10^–6 M), 3) inhibition of EDRF synthesis by N^g-monomethyl-L-arginine (10^–5 M) or 4) inhibition of prostaglandin synthesis by indomethacin (10^–5 M). Furthermore, -adrenergic activation did not modify microvascular dilatation by adenosine (10^–9 to 10^–4 M) or nitroglycerin (10^–9 to 10^–4 M), suggesting that -adrenergic constriction in vivo is not due to attenuation of cAMP or cGMP-dependent mechanisms of coronary dilatation. In contrast to the lack of constriction in coronary arterioles, canine skeletal muscle arterioles exhibited significant -adrenergic constriction (–80±4%), maximum diameter change). The coronary venular -adrenergic constriction was significantly inhibited by both the ₁-and ₂-adrenergic receptor antagonists, prazosin (10^–8 M) and rauwolscine (10^–7 M), indicating a mixed population of ₁-and ₂-adrenergic receptors. These results suggest that coronary arterioles, but not venules, lose -adrenergic responsiveness during isolation and cannulation, or that the primary coronary microvascular response to -adrenergic activation is venular constriction.     

Rapid molecular characterization of Hb H disease in Chinese by polymerase chain reaction

Summary We have developed a rapid method to molecularly distinguish different types of Hb H disease. The study depended on (a) most of the Hb H disease in Taiwan having an-thalassemia-1 of the Southeast Asia type (-SEA) in one allele and (b) the differences of X box of-globin gene cluster in the other allele. To detect the -SEA allele, we utilized the primers located on either side of the breakpoint to do PCR, then characterized the amplified products. For the other allele, we sequenced part of the X box, and found that bases –2803 to –2461 of the X box of – ^3.7 belonged to the X box of ₂ globin gene. In – ^4.2, the bases belonged to the X box of ₁ globin gene, whereas in ^cs it contained both X boxes of ₁ and ₂ globin genes. There was anMboII site at this region of the X box of ₂ globin gene. We utilized PCR to amplify this region and digested it with restriction enzymeMboII, then combined it with another PCR of different primer pairs to molecularly diagnose different types of Hb H disease. One hundred and one cases of Hb H disease from different families were studied: all of the cases had one allele of -SEA deletion, while the other allele showed that 52/101 were – ^3.7, 41/101 were ^cs , 7/101 were – ^4.2, and 1/101 was – ^G.Taichung. Of 52 cases of Hb H with – ^3.7, 47 were type-I deletion and five were type-II deletion.     

Cytotoxic effect of a fusion protein from transforming growth factora andPseudomonas exotoxin on rat and human bladder carcinoma cells in vitro

A protein formed by fusion of transforming growth factor withPseudomonas exotoxin (TGF-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines. This study was designed to evaluate thein vitro cytotoxic effects of TGF-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells. The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory. Human cell lines used were RT4, T24, and 253J. As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1). We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGF-PE40 to bind EGFR, and the cytotoxic effect of TGF-PE40 and PE40. Rat cell lines, D44c, LMC19, and MYU3L (EGFR=4.9×10³–11.4×10³/cell) had ED₅₀ values (the concentration of TGF-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; forc ₁ (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGF-PE40 concentrations of 10⁴ pM, 10⁴ pM and higher than 10⁴ pM respectively were required. Human cell lines, RT4, T24, and 253J (EGFR=32×10³–126×10³/cell) had ED₅₀ values of 20 pM, 66 pM, and 330 pM respectively and T24 showedc ₁ values of 10³ pM. RU-P1 (EGFR =92.6×10³/cell) had the highest ED₅₀ value of 8000 pM. These data indicate that the susceptibility to TGF-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGF-PE40, and that normal urothelial cells are more resistant to TGF-PE40 than are cancer cells. The differential effect of TGF-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.Supported by National Institutes of Health Grant CA14649     

Lack of α2-Antiplasmin Enhances ADP Induced Platelet Micro-Aggregation through the Presence of Excess Active Plasmin in Mice

Background: The role of 2-antiplasmin (2-AP) on platelet aggregation was investigated using mice deficient in 2-AP (2-AP^–/–) or using wild type mice (2-AP^+/+). Methods: Blood samples were taken from each mouse under anesthesia with ether and platelet rich plasma (PRP) was prepared. Platelet aggregation induced by various doses of ADP (0.3–30 M) was detected using a laser-light scattering (LS) system. Aggregated forms were observed using a scanning electron microscopy (SEM). Results: Dose-dependent platelet aggregation was not different in both types of mice. However, platelet micro-aggregate formation in 2-AP^–/– mice induced by low dose of ADP (1.0 M) markedly increased compared to the situation in wild type mice. Aggregated form detected by SEM showed supported data from LS analysis. When washed platelets of 2-AP^+/+ mice were resuspended in plasma of 2-AP^–/– mice, platelet micro-aggregation was also increased. On the contrary, when washed platelets of 2-AP^–/– mice were suspended in plasma of 2-AP^+/+ mice, platelet micro-aggregation did not change. In separate experiments, tPA (1.0 g/ml) was added to PRP before the stimulation of ADP. tPA had no effect on platelet aggregation in 2-AP^+/+ mice, however platelet micro-aggregation in 2-AP^–/– mice was markedly increased by the treatment with tPA. Moreover, the amount of released ATP from stimulated platelets was increased in 2-AP^–/– mice treated with tPA. Conclusion: Lack of 2-AP increased platelet micro-aggregation, and plasmin plays an important role in the formation of platelet aggregation when 2-AP knockout mice are used. Consequently, the reduction of 2-AP could be a risk factor for the activation of platelets resulting in thrombus formation.     

Influence of recombinant tumour necrosis factor a on blood flow and antibody localisation in human tumour xenografts in nude mice

Summary Mice with s.c. grafts of gastric carcinoma MKN45 or osteosarcoma 788T were injected i.v. with recombinant tumour necrosis factor (rTNF) and tumour blood flow rates were determined 4 h later as a fraction of the cardiac output g tissue^–1. With MKN45, the tumour blood flow rate was significantly reduced from a mean of 1.86% cardiac autput g^–1 to 0.84% and 0.65% with 50 and 200 ug kg^–1 rTNF respectively. With 788T, the tumour blood flow rate was reduced at 50 g kg^–1 rTNF from 1.13% cardiac output g^–1 to 0.56%. There were essentially no changes in blood flow rates in other organs. The effect of rTNF on localisation of monoclonal antibodies into these xenografts were examined. When a single dose of rTNF (50 g kg^–1) was given at the same time as labelled NCRC-2 antibody there was a significant reduction in localisation into 788T osteosarcoma xenografts. In other tests, mice were injected daily for 3 days with 50 g kg^–1 rTNF. They were injected i.v. with monoclonal antibody 4 h after the first injection and dissected on the 4th day. With 788T there was a small but not statistically significant reduction in the absolute amount of NCRC-2 antibody localising in tumour, although this reduction was greater when results were expressed as tumour-to-blood ratios. With MKN45 xenografts, treatment with rTNF had little effect on tumour localisation of an anti-(carcinoembryonic antigen) monoclonal antibody (NCRC-24). These studies show that TNF can be administered so as to reduce tumour blood flow and with little effect on tumour localisation of antibody, suggesting that combination therapy with TNF and antibodies or their immunoconjugates is feasible. Other studies have suggested that TNF can increase antibody localisation into tumours, but this was not seen here, and in some cases administration could reduce tumour localisation. It appears that this method of enhancing antibody localisation may be critically dependent on scheduling, and therefore it may not be extensively applicable.Abbreviations rTNF recombinant tumour necrosis factor - CEA carcinoembryonic antigen - BSA bovine serum albumin     

Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

Different effects of α-human atrial natriuretic peptide and nitroglycerin on cardiac dimensions in humans

Summary This study aimed to examine whether-human atrial natriuretic peptide (-hANP) alters cardiac dimensions in humans. Left atrial (LA) and left ventricular diastolic (LV) diameters were measured by echocardiography at control and with lower body negative pressure (LBNP) at –10 and –20 mmHg during intravenous (IV) infusion of saline or-hANP at a dose of 0.03–0.04 µg/kg per minute (n=8). Studies were also done during IV infusion of saline or nitroglycerin (NG) at a dose of 10–15 µg/kg per minute in another group of subjects (n=6). LBNP decreased central venous pressure (CVP) and the LA and LV diameter.-hANP lowered CVP at rest and with LBNP at –10 and –20 mmHg compared with corresponding values during saline infusion; NG produced comparable decreases in CVP, which suggests that decreases in venous return caused by the two drugs were similar. However, NG decreased, but-hANP did not alter the LA and LV diameter at rest or with LBNP. In another group of subjects (n=4), we observed that-hANP caused comparable decreases in CVP and pulmonary capillary wedge pressure. These data suggest that ANP may dilate the cardiac chambers in humans.     

Effects of PAF on cardiac function and eicosanoid release in the isolated perfused rat heart: comparison between normotensive and spontaneously hypertensive rats

The aim of this study was (a) in isolated perfused rat heart to characterize the effects of platelet-activating factor (PAF) on coronary flow, ventricular contractility, and eicosanoid release and (b) to determine whether PAF effects are altered in hearts from spontaneously hypertensive rats (SHR). PAF (10^–10–10^–7 mol) dose-dependently decreased coronary flow and ventricular contractility; concomitantly, coronary effluent concentrations of thromboxane (TX)B₂ and prostaglandin F₂ (PGF₂) were elevated but not those of prostacyclin. The PAF receptor antagonist WEB 2086 (10^–7–10^–5 mol/l) concentration-dependently antagonized these PAF effects. In addition, the cyclo-oxygenase inhibitor indomethacin (5×10^–5 mol/l) prevented PAF (10^–9–10^–7 mol) induced eicosanoid release; in the presence of indomethacin PAF caused coronary constriction and ventricular depression only at the highest dose (10^–7 mol) but had no effect at 10^–9 or 10^–8 mol. Moreover, the TXA₂ antagonist SQ 29,548 (10^–6 mol/l) completely inhibited 10^–8 mol PAF induced ventricular depression but did not effect coronary constriction. In SHR PAF (10^–9–10^–7 mol) evoked decreases in coronary flow and ventricular contractility did not differ from those in normotensive Wistar-Kyoto rats while PAF induced TXA₂ and PGF₂ release was markedly enhanced. In addition, decreases in coronary flow and ventricular contractility induced by the TXA₂ agonist U 46619 (10^–7 mol/l) were markedly depressed in SHR. We conclude that in isolated perfused rat heart PAF causes coronary constriction and depression of ventricular function mainly indirectly through released TXA₂ and/or PGF₂. Moreover, the fact that in SHR the PAF effects on coronary flow and ventricular function are not altered despite markedly enhanced TXA₂ and PGF₂ release supports the view that in the SHR the receptors mediating TXA₂ and/or PGF₂ effects are desensitized.     

Recombinant human interferonsα,β andγ reduce the antiproliferative action of cytarabine in K562 human myeloid leukaemia clonogenic cells

Summary The effects of recombinant human interferons , and (IFN) on the antiproliferative activity of cytarabine in K562 human myeloid leukaemia clonogenic cells were studied in an agar capillary microassay. The addition of IFN- did not affect the antiproliferative activity of cytarabine in K562 cultures treated with low concentrations of cytarabine (10–50 nM), whereas in those treated with high concentrations (100–150 nM) IFN increased the IC₅₀ of cytarabine on day 5 from 102 nM to 214 nM, i.e., cytarabine combined with IFN was about two-fold less potent than cytarabine alone. Similarly, low concentrations of IFN and IFN did not affect the antiproliferative activity of cytarabine on K562 colonies, but high concentrations of these two interferons: 4×10³ U/ml and 10⁴ U/ml respectively, increased the IC₅₀ of cytarabine on day 5 to 304 nM and to 316 nM respectively, i.e. cytarabine combined with IFN or IFN was about threefold less potent than cytarabine alone. The evaluation of the present negative interactions of interferons with cytarabine is warranted in fresh cells from myeloid leukaemia patients in primary culture.Abbreviation IFN interferon     

Interferon Alfacon-1 and Ribavirin Versus Interferon α-2b and Ribavirin in the Treatment of Chronic Hepatitis C

Despite advances in the therapy of chronic hepatitis C, a large number of patients do not respond to current therapies. The study objective was to assess whether a combination of interferon (IFN) alfacon-1 and ribavirin improves the response rate compared with a combination of INF -2b and ribavirin in chronic hepatitis C subjects. The study was designed as an open-label, prospective, randomized, controlled study; 128 subjects with chronic hepatitis C were randomized to INF alfacon-1, 15 g three times per week, plus ribavirin, 1 g/day, or IFN-2b, 3 million units three times per week, plus ribavirin, 1 g/day for 48 weeks. The end point of the study was a sustained viral response, defined as undetectable HCV RNA at 24 weeks post 48 weeks of treatment. Overall, 57% of subjects in the INF alfacon-1/ribavirin group achieved a sustained antiviral response, compared with 40% of subjects in the IFN-2b/ribavirin group (P = 0.052). In the subset of subjects with a high viral load, HCV RNA was successfully eradicated in more individuals who received INF alfacon-1/ribavirin than subjects who received IFN-2b/ribavirin (57 versus 31%; P = 0.025). Among individuals with genotype 1 and a high viral load, the sustained antiviral response was significantly higher with INF alfacon-1/ribavirin than with IFN-2b/ribavirin (46 versus 14%; P = 0.019). Adverse events were similar in both treatment groups. In conclusion, this study demonstrated that the combination of INF alfacon-1 and ribavirin provides a significantly better treatment response compared with the combination of IFN- 2b and ribavirin in chronic HCV subjects infected with genotype 1 and a high viral RNA load.     

A fluorimetric enzyme assay for the diagnosis of sanfilippo disease type A (MPS IIIA)

Summary 4-Methylumbelliferyl--d-N-sulphoglucosaminide (MU--GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulphamidase. Sanfilippo A patients' fibroblasts (n=42) and lymphocytes (n=1) showed 0–3% of mean normal heparin sulphamidase activity; in total leukocytes from patients (n=8) sulphamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulphamidase activity was reduced in 9 out of 10 cases. Heparin sulphamidase desulphates MU-GlcNS to MU-GlcNH₂ and further hydrolysis during a second incubation is required to liberate 4-methylumbelliferone, which can be measured. Yeast-glucosidase, which has low but sufficient-glucosaminidase activity, was used to hydrolyse the reaction intermediate MU-GlcNH₂ to release 4-methylumbelliferone and free glucosamine.     

Field stimulation-induced noradrenaline release from guineapig atria is modulated by prejunctional α2-adrenoceptors and protein kinase C

Summary Guinea-pig left atria were loaded with 10 Ci 7-[³H]noradrenaline, and noradrenaline release from sympathetic nerve endings was then elicited by refractory period field stimulation. When one pulse of 0.2 ms duration was applied during each refractory period, the resulting transmitter release was halved by 3×10^–7 mol/l of the ₂-adrenoceptor agonist clonidine and increased about 2.5-fold by either 3×10^–7 mol/l of the ₂-adrenoceptor antagonist idazoxan, 5×10^–3 mol/l of the potassium channel blocker tetraethylammonium chloride (TEA) or 3×10^–7 mol/l phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). Phorbol-12-myristate-13-acetate-4-O-methylether, a compound which does not stimulate PKC, was ineffective. The stimulatory effect of PMA was antagonized by 7×10^–5 mol/l of the PKC inhibitor polymyxin B. No significant transmitter release was observed when either PMA or TEA was applied together with 10^–7 mol/l of the sodium channel blocker tetrodotoxin. Combinations of either idazoxan and TEA or PMA and TEA caused greater increased of the noradrenaline release than any individual drug given alone. Thus, different mechanisms of action seem to mediate the increase of noradrenaline release by action potential prolongation on the one hand and activation of PKC or inhibition of ₂-adrenoceptors on the other hand. In contrast, the effects of idazoxan and PMA were not additive which suggests a common mechanism of action. In atria pretreated for 10 min with 10^–4 mol/l N-ethylmaleimide, an alkylating agent which inactivates G_i-proteins, neither idazoxan nor PMA caused a significant increase of the stimulation-induced transmitter release, while TEA was still effective. When a train of four pulses, lasting 0.05 ms each, was applied during each refractory period, the resulting transmitter release was not modified by idazoxan or PMA, but was significantly increased by TEA. From these results, a scheme is proposed which, links the regulation of noradrenaline release by prejunctional ₂-adrenoceptors and protein kinase C via an influence on a common inhibitory G_i-protein.     

Interactive influences of peroxisome proliferator-activated receptor α activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat

Aims/hypothesis We sought to elucidate whether excess glucocorticoids and increased dietary lipids act synergistically to impair glucose tolerance and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR) has an adverse or beneficial effect on glucose tolerance.Methods Dexamethasone (100 g kg^–1 body weight day^–1; 5 days) was administered to insulin-resistant rats fed a high-saturated-fat (HF) diet for 4weeks. The PPAR agonist WY14643 was administered (50 mg kg^–1 body weight intraperitoneally) 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed in vivo after an acute glucose bolus injection, and in vitro using step-up and step-down islet perifusions.Results Although neither PPAR activation nor dexamethasone alone affected fasting glycaemia in the HF group, dexamethasone in combination with PPAR activation elicited marked postabsorptive hyperglycaemia. Dexamethasone treatment of HF rats had little effect on GSIS after an acute glucose challenge in vivo, but induced glucose intolerance. PPAR activation augmented GSIS in dexamethasone-treated HF rats in vivo, restoring glucose tolerance. Contrasting with data obtained in vivo, greatly enhanced peak rates of GSIS were observed ex vivo in perifusions of islets from dexamethasone-treated HF rats compared with those from untreated HF rats, an effect attenuated by antecedent PPAR activation.Conclusions/interpretation The study demonstrates that glucocorticoid excess precipitates the development of glucose intolerance in rats maintained on a high-saturated-fat diet. It does this by interrupting the negative feedback loop between insulin sensitivity and secretion in vivo, such that further enhancement of compensatory insulin secretion is not possible. PPAR activation restores the coupling between insulin secretion and action.     

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts

Summary Treatment of diploid human fibroblasts with stereoisomeric benzo[a]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5–12)×10^-4] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1–8)×10^-4] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (<1.6×10^-7 frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.Abbreviations used s⁶G^r 6-thioguanine-resistant - anti diol epoxide, racemic mixture of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - syn diol epoxide, racemic mixture of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene - DME Dulbecco's modified Eagle medium - hprt hypoxanthineguanine phosphoribosyltransferase     

Ameliorative effect of IDS 30, a stinging nettle leaf extract, on chronic colitis

Background and aims Anti-TNF- antibodies are very effective in the treatment of acute Crohns disease, but are limited by the decline of their effectiveness after repeated applications. The stinging nettle leaf extract, IDS 30, is an adjuvant remedy in rheumatic diseases dependent on a cytokine suppressive effect. We investigated the effect of IDS 30 on disease activity of murine colitis in different models.Methods C3H.IL-10–/– and BALB/c mice with colitis induced by dextran sodium sulphate (DSS) were treated with either IDS 30 or water. Mice were monitored for clinical signs of colitis. Inflammation was scored histologically, and faecal IL-1 and mucosal cytokines were measured by ELISA. Mononuclear cell proliferation of spleen and Peyers patches were quantified by ³H-thymidine.Results Mice with chronic DSS colitis or IL-10–/– mice treated with IDS 30 clinically and histologically revealed significantly (p<0.05) fewer signs of colitis than untreated animals. Furthermore, faecal IL-1 and mucosal TNF- concentrations were significantly lower (p<0.05) in treated mice. Mononuclear cell proliferation after stimulation with lipopolysaccharide was significantly (p<0.001) reduced in mice treated with IDS 30.Conclusions The long-term use of IDS 30 is effective in the prevention of chronic murine colitis. This effect seems to be due to a decrease in the Th1 response and may be a new therapeutic option for prolonging remission in inflammatory bowel disease.     

Spontaneous interferon-α antibodies in a patient with pure red cell aplasia and recurrent cutaneous carcinomas

Summary High-titered spontaneous interferon (IFN) antibodies were detected in a patient with pure red cell aplasia (PRCA), a benign mediastinal tumor, and recurrent cutaneous carcinomas. The circulating IFN antibodies reacted broadly with various human IFN- subtypes (20–140×10³ neutralizing units/ml serum) but not with IFN- or IFN-, and they neutralized the antiviral activity of the patient's endogenous IFN-. The IFN-binding activity was restricted to the IgG₁ subclass in a nonmonoclonal manner. Whereas the PRCA repeatedly responded to immunosuppression with high-dose cyclosporin A (CSA) and CSA plus plasmapheresis, IFN antibody production continued during treatment with cyclophosphamide and CSA. Serological analysis revealed past infection with parvovirus B19 and persistent B19 IgM titers. Antibody-mediated impairment of the IFN- system might have favored the development of both PRCA and the various cutaneous carcinomas in this patient.     

Luminal dopamine modulates canine ileal water and electrolyte transport

Previous studies have suggested that dopamine stimulates active ileal ion absorption via ₂-adrenergic or dopaminergic receptor activation. Identification of a dopamine 1a receptor on rat enterocytes located in intestinal crypts prompted this investigation of the effect of luminally administered dopamine on water and ion transport in the canine ileum. Absorption studies (n=27) were performed in dogs with 25-cm ileal Thiry-Vella fistulas. Perfusion with [¹⁴C PEG was used to calculate absorption of water and electrolytes from the Thiry-Vella fistula. Experiments consisted of three 1-hr periods: basal, luminal drug infusion at 10^–4 M, and recovery. Agonists used included dopamine (DOP: -adrenergic, D₁ and D₂ receptor) and SKF 38393 (D₁ receptor). Antagonists used included terazosin (TZ: ₁) and yohimbine (YOH: ₂). DOP caused significant increases in water and electrolyte absorption. TZ and YOH prevented the dopamine-induced proabsorptive response. Luminal DOP may serve as a proabsorptive modulator of ileal transport, acting via ₁, ₂, and dopaminergic receptors. The development of more potent proabsorptive dopamine analogs, which maintain the ability to broadly activate mucosal receptors, may be useful in such clinical situations as diabetic diarrhea, short gut syndrome, or following small bowel transplantation.Presented in part as a poster presentation at the Annual Meeting of the American Gastroenterological Association, New Orleans, May 14–18, 1994, and published in abstract form inGastroenterology 106:A432, 1994.Supported in part by National Institutes of Health grant R29-DK 41178 (C.J.Y.).     

The Role of MIP-1α in Experimental Hypersensitivity Pneumonitis

S. rectivirgula (SR) causes Farmers Lung Disease, a classic example of hypersensitivity pneumonitis (HP). We utilized a model of experimental hypersensitivity pneumonitis (EHP), antibody to MIP-1 and MIP-1^–/– mice, to test the hypothesis that MIP-1 is essential in the development of EHP. Treatment of C57BI/6 mice with anti-MIP-1 antibody did not change the extent of pulmonary histology abnormalities, BALF cell number or characteristics, or BALF concentration of IL12p40, TNF, IL1 and IL6, after an i.t. challenge with SR. MIP-1^–/– animals responded similarly to wild-type (wt) animals in the extent and nature of pulmonary histologic changes and BALF cell number and type after a single i.t. injection of SR There was a dose-response relationship between the amount of SR and BALF IL12p40, MCP-1 and IL6 in both strains, and MIP-1 in wild-type animals. We next transferred SR cultured spleen cells from SR sensitized mice (both wt and MIP-1^–/–) to naïve recipients. Lung histology and BALF characteristics after SR i.t. challenge of the recipients were used to determine if adoptive transfer had occurred. Cultured cells from MIP-1^–/– animals were fully capable of transferring EHP to recipients. There was no difference of BALF TNF, IL6 and IL1 between the strains, but there was more MCP-1 and IL12p40 in the MIP-1^–/– mice than in the control mice. MIP-1 is not necessary for the recruitment of cells into the lung and BALF after i.t. administration of SR, or the development of cells able to adoptively transfer EHP.