Incidence of strains producing extended spectrum β-lactamases in Argentina

Summary The incidence of strains producing transferable -lactamases capable of hydrolyzing third generation cephalosporins or aminothiazole-oximino substituted monobactams in five Buenos Aires hospitals during a four month period was studied. These enzymes were categorized by 1) MIC 1 mg/l for third generation cephalosporins; 2) MIC < 0.06 mg/l for third generation cephalosporins combined with clavulanic acid or sulbactam; 3) sensitivity to imipenem or cephamycins (excluding permeability mutants); and 4) transferable resistance by conjugation. -lactamases hydrolyzing aminothiazole-oximino substituted monobactams were produced by 17.2% of Enterobacteriaceae from intensive care unit patients; 3.6% from inpatients of other units and 1.2% from outpatients. Producers were mainlyKlebsiella spp. (45/46) resistant to aminoglycosides (most of them AAC 3 — AAC 6 producers). Three strains had a an isoelectric point of 6.0, two of 6.5 and three of 7.7. TEM-1 -lactamase (isoelectric point 5.4) was detected in 6/8 strains. An inocolum effect was observed in 40/46 strains. AKlebsiella pneumoniae strain preserved since 1982 also produced a transferable -lactamase hydrolyzing aminothiazole-oximino substituted monobactams.

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Vorkommen von Bakterienstämmen mit erweitertem Breitspektrum -Laktamasen in Argentinien

Zusammenfassung Während einer Zeit von vier Monaten wurde an fünf Krankenhäusern in Buenos Aires nach dem Vorkommen von Stämmen mit Bildung übertragbarer -Laktamasen gesucht, die Cephalosporine der dritten Generation oder aminothiazol-oximino-substituierte Monobaktame hydrolysieren. Kriterien für die Selektion dieser Enzyme waren 1) MHK-Werte von 1 mg/l für Cephalosporine der dritten Generation; 2) MHK-Werte von < 0,06 mg/l für Drittgenerationscephalosporine in Kombination mit Clavulansäure oder Sulbactam; 3) Empfindlichkeit gegenüber Imipenem oder Cephamycinen; 4) Resistenz übertragbar. Zur Bildung von -Laktamasen mit hydrolytischer Aktivität gegen Aminothiazol-oximinosubstituierte Monobaktame waren 17,2% der Enterobacteriaceae-Isolate von Intensivpflegepatienten befähigt, 3,6% der Isolate von Patienten von anderen Stationen und 1,2% der Isolate von ambulanten Patienten. Die meisten der Stämme, die diese Enzyme bildeten (45/46), waren aminoglykosidresistenteKlebsiella spp. (Synthese von AAC 3-AAC 6). Der isoelektrische Punkt lag bei drei Enzymen bei 6,0, in zwei Fällen bei 6,5 und in drei Fällen bei 7,7. Sechs von acht Stämmen bildeten TEM-1 -Laktamase. Bei 40 der 46 Stämme wurde ein Inokulumeffekt festgestellt. Die Bildung übertragbarer -Laktamasen mit hydrolytischer Wirkung gegen Aminothiazolyl-oximino-substitutierte Monobaktame wurde in einem bereits seit 1982 gelagertenKlebsiella pneumoniae-Stamm nachgewiesen.

     

Indirect evidence of cell wall alterations in pseudomonas aeruginosa by immunoglobulin preparations

Summary Ten strains ofPseudomonas aeruginosa isolated from different clinical sources were incubated with immunoglobulin preparations for i.v. use. This resulted in the release of -lactamases normally cell-bound in the periplasmic space. The amount of released -lactamases — depending on the degree of cell wall alteration — was spectrophotometrically determined by the use of nitrocefin as an indicator substance. -lactamase release varies according to strain specificity and depends on the chemical modification of the immunoglobulins used.

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Indirekter Nachweis der Zellwandalteration bei Pseudomonas aeruginosa durch Immunglobulinpräparate

Zusammenfassung Inkubation von zehn aus klinischem Material isoliertenPseudomonas aeruginosa-Stämmen mit i.v. applizierbaren Immunglobulinpräparaten führt zu einer Freisetzung sonst zellgebundener -Laktamasen aus den Bakterien. Die Menge der freigesetzten -Laktamasen ist abhängig vom Ausmaß der durch die Immunglobuline induzierten Zellwandalteration und läßt sich quantitativ spektrophotometrisch mit der Nitrocefin-Reaktion bestimmen; sie ist stammspezifisch unterschiedlich und abhängig von der chemischen Aufbereitung der Immunglobulinpräparate.

     

Epidemiology of extended spectrum β-lactamases

Summary -lactamases play a major part in resistance, as recently redemonstrated by the emergence of extended spectrum -lactamases. Since its discovery in FR Germany, SHV-2 has been reported from four continents and CTX-1 (TEM-3) was established in at least 26 French hospitals. More than 12 other enzymes have been individualized. The newest aspect of resistance was probably underestimated because most strains of enterobacteria (mainlyKlebsiella pneumoniae) appeared susceptible to oxyimino--lactams as suggested by MICs or diameters of inhibition zone sizes. The double-disk synergy test between amoxicillin/clavulanic acid and oxyimino--lactams was useful to easily detect two susceptibility patterns (CTX, CAZ). Extended spectrum -lactamases isolated among nosocomial isolates of enterobacteria (urines, blood, wound, sputum cultures) mostly from intensive care units have spread through hospitals. If outbreaks were described, numerous serotypes were identified inKlebsiella pneumoniae. In France the distribution of extended spectrum -lactamases showed that CTX-1 (TEM-3) was well distributed among ten species unlike SHV-type enzymes (SHV-2, SHV-3, SHV-4) preferentially detected inKlebsiella pneumoniae. A majority of strains produced CAZ-type enzymes inEscherichia coli. Some isolates produced two extended spectrum -lactamases. In Tunisia extended spectrum -lactamase producing strains were mainly identified among pediatric isolates ofKlebsiella pneumoniae, Salmonella andEscherichia coli; SHV-2 was predominant but recently CTX-1 and two other types with an isoelectric point of 6.35 and 5.4 (phenotype CTX) were individualized. Because plasmid-encoded, this mechanism was spreading in France among enterobacteria with other resistance markers (e.g. netilmicin, amikacin) for CTX-1 unlike SHV-2. The transferability of extended spectrum -lactamases appeared to occur less frequently from isolates of enterobacteria issued from Tunis and particularly for the first isolates ofSalmonella wien. The transmissible resistance to other antibiotics such as aminoglycosides was demonstrated in 1988. It seems highly likely that the use of newer antibiotics favors the appearance of extended spectrum -lactamases.

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Epidemiologie der Breitspektrum--Laktamasen

Zusammenfassung -Laktamasen spielen in der bakteriellen Resistenz eine entscheidende Rolle; dies bestätigte sich erneut mit dem Auftreten von Breitspektrum--Laktamasen. Nach Erstentdeckung in der Bundesrepublik wurde SHV-2 in vier Kontinenten nachgewiesen; in mindestens 26 französischen Krankenhäusern trat CTX-1 (TEM-3) auf. Mehr als 12 weitere Enzyme wurden identifiziert. Die Bedeutung dieser neuesten Resistenz-Form wurde wahrscheinlich unterschätzt, da die meisten Stämme von Enterobakterien (vor allemKlebsiella pneumoniae nach Ergebnissen der MHK-Bestimmungen oder Hemmhofdurchmesser gegen Oxyimino--Laktame empfindlich zu sein schienen. Zwei Empfindlichkeitsmuster (CTX, CAZ) lassen sich mit dem Doppel-Blättchen-Synergismus-Test von Amoxicillin/Clavulansäure und Oxyimino--Laktamen ohne Schwierigkeiten nachweisen. In den Krankenhäusern haben sich Breitspektrum--Laktamasen ausgebreitet, die bei nosokomialen Enterobakterien-Stämmen (aus Urin-, Blut-, Wund- und Sputumkulturen), vorwiegend aus Intensivstationen, zu finden sind. Bei Ausbrüchen vonKlebsiella pneumoniae-Infektionen fanden sich zahlreiche Serotypen. Untersuchungen zur Verteilung der Breitspektrum--Laktamasen in Frankreich zeigten, daß CTX-1 (TEM-3) unter zehn Spezies verbreitet ist, während sich die Enzyme vom Typ SHV (SHV-2, SHV-3, SHV-4) vorwiegend inKlebsiella pneumoniae nachweisen lassen. BeiEscherichia coli bildeten die meisten Stämme Enzyme vom Typ CAZ. Manche Isolate bildeten zwei Breitspektrum--Laktamasen. Breitspektrum--Laktamasebildung durchKlebsiella pneumoniae, Salmonella undEscherichia coli wurde in Tunesien vor allem in Isolaten aus der Pädiatrie nachgewiesen; am häufigsten fand sich SHV-2, kürzlich wurden aber auch CTX-1 und zwei andere Typen mit isoelektrischem Punkt von 6,35 und 5,4 (Phänotyp CTX) identifiziert. Da diese Enzyme durch Plasmide kodiert sind, breitete sich dieser Resistenzmechanismus in Frankreich für CTX-1 zusammen mit anderen Resistenzmarkern aus (z. B. Resistenz gegen Netilmicin, Amikacin), was für SHV-2 nicht der Fall war. Bei Enterobakterien-Isolaten aus Tunis, vor allem den erstenSalmonella wien-Isolaten, war die Übertragbarkeit von Breitspektrum--Laktamasen offensichtlich seltener. 1988 wurde übertragbare Resistenz gegen andere Antibiotika wie Aminoglykoside festgestellt. Es scheint sehr unwahrscheinlich, daß die Verwendung neuerer Antibiotika das Auftreten von Breitspektrum--Laktamasen begünstigt.

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This study was supported in part by a grant to A. P. (N° 87-33403E) from the Caisse Nationale de l'Assurance Maladie des Travailleurs Salariés.

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supported by Pfizer GmbH, Karlsruhe     

Oligosaccharides accumulated in the bovineβ-mannosidosis kidney

Summary The phenotype of bovine-mannosidosis (-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbittet al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21µmol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man1-4GlcNAc and Man1-4GlcNAc1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man1-4GlcNAc1-4Man1-4GlcNAc, Man1-6Man1-4GlcNAc1-4GlcNAc and Man1-4GlcNAc1-4Man1-4GlcNAc1-4GlcNAc. As in caprine-mannosidosis, oligosaccharides with terminal-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.     

Interleukin-1β Inhibits Growth Factor-Stimulated Restoration of Wounded Rat Gastric Epithelial Cell Monolayers

We examined the effect of interleukin-1(IL-1) on spontaneous and enhanced restoration(cell migration and proliferation) using an in vitrowound model comprising a confluent monolayer of ratgastric epithelial RGM1 cells. Repair of an artificialwound in a cell monolayer was found to be time- andconcentration-dependent when the cells were incubatedwith epidermal growth factor (EGF) or transforming growth factor (TGF)- alone for up to 24hr. The growth factors also stimulated DNA synthesissignificantly for 24 hr in a concentration-relatedmanner. IL-1 had no effect on wound restoration in the absence of the growth factors. However, itmarkedly inhibited the restoration enhanced by EGF andTGF-, the inhibition being about 60% and 70%,respectively. In addition, IL-1 significantly reduced the DNA synthesis stimulated by thegrowth factors. The EGF- and TGF--enhancedrestoration was reduced by about 30% by mitomycin C,which potently inhibited the stimulated DNA synthesis.Mitomycin C had no effect on the spontaneous restoration.Even when treated with mitomycin C, the inhibitoryeffect of IL-1 on the enhanced wound repair wasstill observed; however, the extent of the inhibition was decreased. These results indicate thatIL-1 inhibits the migration as well as theproliferation of gastric epithelial cells enhanced byEGF and TGF-, resulting in a failure of woundhealing.     

Role of Central Interleukin-1β in Gastrointestinal Motor Disturbances Induced by Lipopolysaccharide in Sheep

Cytokines are involved in the symptoms of theacute phase response induced by infectious diseases inhumans as well as in animals, and interleukin-1(IL-1 ) has a pivotal role in these changes. The role of central IL-1 in the gastrointestinalhypomotility and fever evoked by intravenousadministration of lipopolysaccharide (LPS) and themechanisms involved, were investigated in sheep as anexperimental model. LPS (0.1 g/kg, intravenously)induced gastrointestinal hypomotility and fever thatwere significantly reduced by priorintracerebroventricular administration of IL-1receptor antagonist protein (IL-1ra, 2 g/kg). The effects of LPS were mimickedby intracerebroventricular IL-1 (50 ng/kg),whereas IL-1 injected intravenously at the samedose only caused a slight and transient fever withoutmodifying the gastrointestinal motility. Priorintracerebroventricular administration of thecyclooxygenase inhibitor indomethacin (100 g/kg) butnot the corticotropin-releasing factor (CRF) receptorantagonist -helical CRF_9-41 (5 g/kg) blocked alleffects evoked by both LPS and IL-1. These resultssuggest that in sheep, LPS induces digestive motordisturbances through a central release of IL-1 andprostaglandins.     

Heterophilic interactions between cell adhesion molecule L1 and α<Subscript>v</Subscript> β<Subscript>3</Subscript>-integrin induce HUVEC process extension <Emphasis Type="Italic">in vitro</Emphasis> and angiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with _{v 3} to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab _{v 3} or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface _{v 3} and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab _{v 3} or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with _{v 3} suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of _{v 3} and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of _v and _{v 3} followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50–60% increased _v and _v3 protein expression and in vivo angiogenesis indicated by ~50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of _v3     

EGF and TGF-β1 Gene Expression in Chronically Rejecting Small Bowel Transplants

Long-term survival of small bowel transplants ishampered by chronic rejection. Epidermal growth factor(EGF) and transforming growth factor (TGF-)have opposing, regulatory roles in normal intestinal physiology and may be involved in thepathogenesis of chronic intestinal rejection. Our aimwas to investigate the expression of EGF andTGF-₁ in chronically rejecting smallbowel transplants. Orthotopic small bowel transplantation was performed inthe allogeneic DA-to-AS rat combination; Cyclosporin wasadministered temporarily to prevent acute rejection.Controls were DA isografts and normal DA rats. PreproEGF and TGF-₁ geneexpression was evaluated by northern blot analysis ofthe ileum RNA and standardized againstglyceraldehyde-3-phosphate-dehydrogenase expression.Allografts demonstrated functional impairment and histological features of chronicrejection, whereas isografts appeared normal. Allograftsdemonstrated a significant reduction of EGF mRNA whencompared to DA isografts. No significant changes were detected in TGF-₁expression in either allogeneic or syngeneic grafts. Inconclusion, this study demonstrates reduced preproEGFand preserved TGF-₁ gene expression inchronically rejecting small bowel transplants.     

Comparative in vitro activities of amoxicillin-clavulanate,ampicillin-sulbactam and piperacillin-tazobactam against strains of escherichia coli and proteus mirabilis harbouring known β-lactamases

Summary Strains ofEscherichia coli (N=124) andProteus mirabilis (N=29) harboring known -lactamases were analyzed as to their susceptibility to ampicillin, amoxicillin, and piperacillin alone and in combination with sulbactam, clavulanate, and tazobactam. With TEM 1-producingE. coli, a correlation between specific -lactamase activity and the MIC of piperacillin and ampicillin-sulbactam was observed. These strains also showed significant differences in susceptibilities to the various combinations, suggesting that, at least in strains resistant to one combination, several -lactam/-lactamase inhibitor combinations should be tested in the laboratory. All combinations tested enhanced the activity of the -lactam towards TEM 1-producingE. coli, piperacillin-tazobactam being the most active. The drugs were less active to OXA 1 enzymes; solely with piperacillin-tazobactam 90% of strains were within the therapeutic range of the drug. Sulbactam acted synergistically to chromosomally encoded -lactamases, whereas amoxicillin-clavulanate was inactive. Piperacillin and piperacillin-tazobactam inhibited all strains producing chromosomally encoded -lactamases at concentrations within the therapeutic range of the drugs. In contrast, TEM 2 ofP. mirabilis was not sensitive to ampicillin-sulbactam, but to the other combinations; here again piperacillin-tazobactam was the most active.

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Vergleich der In-vitro-Aktivitäten von Amoxicillin-Clavulansäure, Ampicillin-Sulbactam und Piperacillin-Tazobactam gegenüber Stämmen von Escherichia coli und Proteus mirabilis mit bekannten -Laktamasen

Zusammenfassung Wir untersuchten die Empfindlichkeit vonEscherichia coli (N=124) undProteus mirabilis-Stämmen (N=29) mit bekannten -Laktamasen gegenüber Ampicillin, Amoxicillin und Piperacillin, allein und in Kombination mit Sulbactam, Clavulansäure oder Tazobactam. Bei TEM 1-produzierendenE. coli-Stämmen fanden wir eine Korrelation zwischen der spezifischen -Laktamase-Aktivität und der MHK gegenüber Piperacillin oder Ampicillin-Sulbactam. Diese Stämme zeigten auch erhebliche Unterschiede in der Empfindlichkeit gegenüber den untersuchten Kombinationen. Daher ist zu empfehlen, wenigstens bei Stämmen, die gegenüber einer Kombination resistent sind, mehrere -Laktamase-Inhibitor-Kombinationen zu untersuchen. Alle eingesetzten Kombinationen zeigten Synergismus gegen TEM 1-produzierendeE.-coli-Stämme, dabei war Piperacillin-Tazobactam am aktivsten. Gegenüber OXA 1--Laktamasen waren alle Kombinationen weniger wirksam. Nur Sulbactam in der Kombination mit Ampicillin zeigte Synergismus gegen chromosomal kodierte Enzyme, Amoxicillin-Clavulansäure war nicht aktiv. Piperacillin und Piperacillin-Tazobactam waren wirksam gegenüber allen Stämmen mit chromosomal kodierten -Laktamasen, dabei lagen die minimalen Hemmkonzentrationen im therapeutisch nutzbaren Bereich. Demgegenüber war Ampicillin-Sulbactam inaktiv gegen TEM 2-produzierendeP. mirabilis-Stämme, die anderen Kombinationen waren wirksam, dabei zeigte Piperacillin-Tazobactam die höchste Aktivität.

     

Local and Systemic Liberation of Proinflammatory Cytokines in Ulcerative Colitis

Determination of plasma and tissue cytokinelevels in inflammatory bowel disease have frequentlyresulted in conflicting data. In the present study wedetermined in patients with ulcerative colitis (UC), the levels of the proinflammatory cytokinesinterleukin (IL)-1, IL-6, interferon(IFN)-, and tumor-necrosis factor (TNF)-liberated by peripheral blood mononuclear cells (PBMC)and lamina propria mononuclear cells (LPMC) after 48-hrculture with pokeweed mitogen (PWM). IL-1, IL-6,IFN- and TNF- in the supernatant weredetected by ELISA. Results show low basal levels ofIL-1 secretion by PBMC and LPMC, and a considerableincrease after mitogen stimulation. Basal IL-6production by PBMC was higher in UC patients than incontrols [2029 pg/ml, CI₉ (–165 to4223) vs 572 pg/ml (–383 to 1527) respectively, P = 0.05] and also afterPWM activation [14,995 pg/ml (7759 -22230) vs 6598 pg/ml(3240-9956), respectively, P = 0.05]. In LPMC, nodifferences in IL-6 secretion were observed. TNF- in activated PBMC of patients with UC was notsignificantly increased in relation to control (P =0.09). No constitutive secretion of IFN- wasobserved in mononuclear cells. IFN- levelssecreted by activated LPMC were lower in patients withUC than in controls [1571 pg/ml (–108 to 3251) vs7953 pg/ml (3851-12,055), respectively, P = 0.03]. Theseresults suggest that IL-6, IL-1, and TNF- participate as mediators in the inflammatoryphenomena observed in UC. Further studies are necessaryto evaluate the role of IFN- in thiscondition.     

Presence of Two Signaling TGF-β Receptors in Human Pancreatic Cancer Correlates with Advanced Tumor Stage

Transforming growth factor- (TGF-)signal transduction is mediated via specific cellsurface signaling TGF- receptors, most notably thetype I ALK5 (TR-I_ALK5)and the type II(TR-II). We evaluated TR-I_ALK5 andTR-II expression in 41 human pancreatic cancertissue samples and correlated these findings withclinical data of the patients. Northern blot analysisindicated that, in comparison with the normal pancreas,pancreatic adenocarcinomas exhibited 8.0-fold and4.5-fold increases (P < 0.01), respectively, in mRNAlevels encoding TR-I_ALK5 andTR-II. In situ hybridization showed that both TR-I_ALK5 mRNAwere highly expressed in the majority of pancreaticcancer cells. Immunohistochemical analysis ofTR-I_ALK5 and TR-II revealedpositive immunostaining in 73% and 56% of the tumors, respectively. Both receptorswere concomitantly present in 54% of the pancreaticcancer samples. The presence ofTR-I_ALK5 or TR-II and theconcomitant presence of TR-I_ALK5 and TR-II in the cancer cells was associatedwith advanced tumor stage (P < 0.01). These findingsshow that in many human pancreatic cancers, increasedlevels of the two signaling TRs are present. The presence of the signaling TRs inadvanced tumor stages indicates a role in diseaseprogression.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Role of β-Adrenergic Receptor Subtypes in Lipolysis

In vitro lipolysis stimulated by low (-)-isopre-naline concentrations (30 nM) in epididymal white adipo-cytes from Sprague-Dawley rats was inhibited at least 60–80% by the specific ₁-antagonists LK 204-545 and CGP 20712A (1 M), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via ₁-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for ₁-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, ₃-adrenergic receptors were fully activated and were the dominant -adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the ₁-antagonists, demonstrating that the ₃-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively ₂-selective agonist formoterol in the presence of ₁-blockade (1 M CGP 20712A) demonstrated the inability of the ₂-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 1 M. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (500-fold lower than its ₂-adrenergic receptor pA ₂, 7.80 ± 0.21), suggesting that formoterol was not acting via ₂-adrenergic receptors. These data are consistent with ₁-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that ₂-adrenergic receptors play no obvious direct role in mediating -adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a ₃-selective antagonist), was found to be a non-selective antagonist at the three -adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.     

B cell adrenoceptors and sulphonylurea-induced insulin release in mouse islets

Summary Interactions of tolbutamide and glibenclamide with B cell adrenoceptors have been reported. This study evaluated the possible role of such interactions in the stimulation of insulin release. Mouse islets were incubated in the presence of 10 mmol/l glucose alone or with tolbutamide (10 mol/l) or glibenclamide (0.02 mol/l). At 0.01–10 mol/l, blockers of ₂-adrenoceptors (yohimbine, idazoxan) or ₁-adrenoceptors (prazosin) had practically no effect on glucose-induced insulin release and did not affect its potentiation by sulphonylureas, except for a slight increase by 10 mol/l prazosin and idazoxan. Nonspecific -blockers (phentolamine, dihydroergotamine) increased control release at 10 mol/l, but only the latter amplified the response to tolbutamide. Blockers of -adrenoceptors were tested at 0.1–100 mol/l: propranolol (₁, ₂), metoprolol (₁) and compound ICI 118-551 (₂). They increased glucose-induced insulin release at 100 mol/l but variably altered the effect of sulphonylureas. Blockers of adrenoceptors have, thus, no effect on insulin release in vitro at therapeutic concentrations. At high concentrations, they non-specifically affect the action of sulphonylureas. We conclude that an interaction with B cell adrenoceptors is not involved in the insulinotropic action of sulphonylureas.     

IL-1β and IL-6 in community-acquired pneumonia: Bacteremic pneumococcal pneumonia versusMycoplasma pneumoniae pneumonia

Summary Interleukin-1 (IL-1) and interleukin-6 (IL-6) levels in 20 patients with bacteremicStreptococcus pneumoniae community-acquired pneumonia (CAP) were compared with these cytokine levels in 20 patients withMycoplasma pneumoniae CAP. All 40 patients survived hospitalization and underwent a follow-up examination one month later. Serum IL-1 and IL-6 levels were determined by the enzyme immunoassay (EIA) method using commercial kits. In the acute phase of CAP, IL-6 levels were significantly higher in theS. pneumoniae group (p=0.014), while IL-1 levels were higher in theM. pneumoniae group (p=0.046). In the convalescence phase, the two cytokines were detected in a considerable number of patients in both groups. In this phase, only the level of IL-1 was significantly higher in theM. pneumoniae group than in theS. pneumoniae group (p=0.03). We conclude that the levels of IL-1 and IL-6 are different between patients withS. pneumoniae-CAP andM. pneumoniae-CAP during the acute phase. In the convalescence phase, cytokine levels remain high in some of the CAP patients, but a significant difference between the groups exists only for IL-1. Further studies are required.     

Estrogen Receptor Beta mRNA Expression in Normal and Adenomatous Pituitaries

Estrogen (E2), acting via its nuclear receptors, has been implicated in tumor development and growth, particularly in the pathogenesis of breast cancer. E2 also modulates anterior pituitary hormone production and is a potent cell mitogen. Until recently, the actions of E2 were thought to be mediated by a single estrogen receptor (ER) isoform (ER), and currently little is known of the pathophysiological relevance of the ER isoform. The presence of ER mRNA has been demonstrated by RT-PCR in the normal human pituitary, although expression of ER mRNA in human pituitary tumors has not been described. We have used semiquantitative RT-PCR to determine the relative levels of expression of ER mRNA in normal human pituitaries, non-functioning pituitary adenomas and GH-secreting tumors. ER mRNA was detected in normal pituitaries and all pituitary tumors examined. The ratio of ER mRNA to -actin mRNA expression was significantly reduced in non-functioning pituitary tumors (NFTs; 0.92 ± 0.09; mean ± SE; n=23) compared with findings in normal pituitaries (1.56 ± 0.21; mean ± SE; n=5; p<0.05 Student's t-test). Studies of ER protein expression are required to determine the functional significance of reduced ER mRNA expression in NFTs.     

β-Endorphin-like immunoreactivity in normal mucosa,muscle layer,adenocarcinoma, and polyp of the colon

-Endorphin-like immunoreactivity was detected in the mucosa and muscle layer of normal colon, adenocarcinomas derived from the colon mucosa, and colon polyps which were histologically confirmed to be adenoma without a focus of carcinoma or with in situ carcinoma. The contents of -endorphin-like immunoreactivity in adenocarcinomatous tissue (11.94± 1.77 pmollg wet wt) and colon polyps without focus of carcinoma (10.71 ±1.50 pmollg wet wt) were found to be significantly higher than those in the mucosal layer (6.86± 0.64 pmollg wet wt) and muscle layer (8.30 ±0.68 pmollg wet wt) of normal colon. These data suggest that the production of -endorphin-like immunoreactivity is specifically increased in some adenocarcinomas and adenomatous polyps and may be related to the alteration of bowel habits. Gel exclusion chromatography of - endorphinlike immunoreactivity revealed three peaks corresponding to -endorphin, -lipotropin, and an immunoreactive form between the two. In the mucosal layer and muscle layer of the colon, a broad major peak was eluted at the position of -endorphin, and minor peaks were eluted at the position of -lipotropin and between -endorphin and -lipotropin. In adenocarcinoma and polyp, the peak size corresponding to authentic -lipotropin was greater than that of -endorphin. This study demonstrated that -endorphin-like immunoreactivity existed at a high concentration in some colon adenocarcinomas and polyps whose elution patterns were different from those of normal colon tissue.     

Elevated Serum Levels of Astroglial S100β in Patients with Liver Cirrhosis Indicate Early and Subclinical Portal-Systemic Encephalopathy

Portal-systemic encephalopathy is the prototype among the neuropsychiatric disorders that fall under the term Hepatic Encephalopathies. Ammonia toxicity is central to the pathophysiology of Portal-systemic encephalopathy, and neuronal ammonia toxicity is modulated by activated astrocytes. The calcium-binding astroglial key protein S100 is released in response to glial activation, and its measurement in serum only recently became possible. Serum S100 was determined by an ultrasensitive ELISA in patients (n=36) with liver cirrhosis and transjugular intrahepatic portosystemic stent-shunt. Subclinical portal-systemic encephalopathy and overt portal-systemic encephalopathy were determined by age-adjusted psychometric tests and clinical staging, respectively. Serum S100 was specifically elevated in the presence of subclinical or early portal-systemic encephalopathy, but not arterial ammonia. S100 levels elevated above a reference value (S100 110pg/ml) or the cut off value determined in our group of patients (112pg/ml) predicted subclinical portal-systemic encephalopathy with a specificity and sensitivity of 100 and 56.5%, respectively. Serum S100 was significantly dependent on liver dysfunction (Child-Pugh score), but was more closely related to cognitive impairments than the score. Serum S100 seems to be a promising biochemical surrogate marker for mild cognitive impairments due to portal-systemic encephalopathy.     

Thalassemia intermedia: compound heterozygous β∘/β+-thalassemia and co-inherited heterozygous α+-thalassemia

Summary The relative excess of - over -globin chains in the erythroid precursors is the chief pathophysiological factor of homozygous -thalassemia. The clinical picture is usually characterized by a transfusion-dependent dyserythropoietic anemia (thalassemia major). However, some patients present with moderate anemia that does not require regular blood transfusions (thalassemia intermedia). The molecular heterogeneity of -thalassemia mutations and changes of - and -globin gene expression play an important role in modifying the clinical phenotype. We report here on a female Greek patient with homozygous -thalassemia but normal growth and development, excellent exercise tolerance, and no need of blood transfusions. She is thus mildly affected clinically, although there is marked pallor, jaundice, and hepatosplenomegaly. These signs correspond to her marked hypochromic, microcytic anemia with erythroid hyperplasia of the bone marrow. -Globin genotyping shows her to be compound heterozygous for the codon 39 C T -nonsense mutation and for the T C ⁺-mutation at position 6 of the splice consensus at the exon 1/intron 1 junction (CD39 C T/IVS 1–6 T C). -Globin gene mapping demonstrates the presence of a 3.7-kb ⁺-thalassemia deletion on one allele (–^3.7/). Taken together, this study identifies a complex interaction of genetic factors that do not significantly alter the clinical phenotype when present alone but ameliorate the course of homozygous -thalassemia when inherited in combination.Abbreviations Hb hemoglobin - Hct hematocrit - HPFH hereditary persistence of fetal hemoglobin - IVS intervening sequence - MCH mean corpuscular hemoglobin - MCV mean corpuscular volume - PCR polymerase chain reaction     

Pharmacological evidence for beta3 adrenoceptors in the control of rat gastric acid secretion

The effect of the ₃-adrenoceptor agonist BRL37344 on gastric acid secretion evoked by different secretory stimuli was investigated in anaesthetized rats with lumen-perfused stomachs in comparison with the ₂-adrenoceptor agonist clenbuterol. Intravenous injections of BRL37344 (1–10 mol/kg) and clenbuterol (0.01–1 mol/kg) dose-dependently reduced 2-deoxy-D-glucose-induced acid secretion, with BRL37344 about forty times less potent than clenbuterol. BRL37344 (0.1–3 mol/kg) inhibited pentagastrin-induced acid output, whereas clenbuterol was effective only at high doses (10–100 mol/kg). The inhibitory effect of BRL37344 on pentagastrin-induced acid secretion was not modified by the nonselective –adrenoceptor antagonist propranolol, but it was prevented by bupranolol, a ₃-adrenoceptor antagonist. Furthermore, neither BRL37344 (10 mol/kg) nor clenbuterol (100 mol/kg) modified the acid secretion induced by histamine. These data suggest that ₃ adrenoceptors have an inhibitory role in the control of rat gastric acid secretion induced by indirect stimuli.