Clonal complex Pseudomonas aeruginosa in horses

Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated.

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.

N-terminal glycation of cholecystokinin-8 abolishes its insulinotropic action on clonal pancreatic B-cells

Monoglycated cholecystokinin octapeptide (Asp(1)-glucitol CCK-X) was prepared under hyperglycaemic reducing conditions and purified by reverse phase-high performance liquid chromatography. Electrospray ionisation mass spectrometry and automated Edman degradation demonstrated that CCK-8 was glycated specifically at the amino-terminal Asp(1) residue. Effects of Asp(1)-glucitol CCK-8 and CCK-8 on insulin secretion were examined using glucose-responsive clonal BRIN-BD11 cells. In acute (20 min) incubations, 10(-10) mol/l CCK-8 enhanced insulin release by 1.2-1.5-fold at 5.6-11.1 mmol/l glucose. The stimulatory effect induced by 10(-10) mom CCK-8 was abolished following glycation. At 5.6 mmol/l glucose, CCK-8 at concentrations ranging from 10(-11) to 10(-7) mol/l induced a significant 1.6-1.9-fold increase in insulin secretion. Insulin output in the presence of Asp(1)-glucitol CCK-8 over the concentration range 10(-11)-10(-7) mol/l was decreased by 21-35% compared with CCK-8, and its insulinotropic action was effectively abolished. Asp(1)-glucitol CCK-8 at 10(-8) mol/l also completely blocked the stimulatory effects of 10(-11)-10(-8) mol/l CCK-8. These data indicate that structural modification by glycation at the amino-terminal Asp(1) residue effectively abolishes and/or antagonises the insulinotropic activity of CCK-8. (C) 1999 Elsevier Science B.V. All rights reserved.

Differential evolution combined with clonal selection for dynamic economic dispatch

Dynamic economic load dispatch (DELD) is one of the most important steps in power system operation. Various optimisation algorithms for solving the problem have been developed; however, due to the non-convex characteristics and large dimensionality of the problem, it is necessary to explore new methods to further improve the dispatch results and minimise the costs. This article proposes a hybrid differential evolution (DE) algorithm, namely clonal selection-based differential evolution (CSDE), to solve the problem. CSDE is an artificial intelligence technique that can be applied to complex optimisation problems which are for example nonlinear, large scale, non-convex and discontinuous. This hybrid algorithm combines the clonal selection algorithm (CSA) as the local search technique to update the best individual in the population, which enhances the diversity of the solutions and prevents premature convergence in DE. Furthermore, we investigate four mutation operations which are used in CSA as the hyper-mutation operations. Finally, an efficient solution repair method is designed for DELD to satisfy the complicated equality and inequality constraints of the power system to guarantee the feasibility of the solutions. Two benchmark power systems are used to evaluate the performance of the proposed method. The experimental results show that the proposed CSDE/best/1 approach significantly outperforms nine other variants of CSDE and DE, as well as most other published methods, in terms of the quality of the solution and the convergence characteristics.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

The detection of contaminating clonal cells in apheresis products is related to response and outcome in multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

In the present paper, we report on the use of the heteroduplex PCR technique to detect the presence of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) in the apheresis products of patients with multiple myeloma (MM) undergoing autologous peripheral blood stem cell (APBSC) transplantation. Twenty-three out of 31 MM patients undergoing APBSC transplantation with VDJH segments clonally rearranged detected at diagnosis were included in the study. Samples of the apheresis products were PCR amplified using JH and VH (FRIII and FRII) consensus primers and subsequently analyzed with the heteroduplex technique, and compared with those obtained at diagnosis. 52% of cases yielded positive results (presence of clonally rearranged VDJH segments in at least one apheresis). The presence of positive results in the apheresis products was not related to any pretransplant characteristics with the exception of response status at transplant. Thus, while no one patient with positive apheresis products was in complete remission (CR), negative immunofixation, before the transplant, five cases (46%) with negative apheresis were already in CR at transplant (P = 0.01). The remaining six cases with heteroduplex PCR negative apheresis were in partial remission before transplant. Patients with clonally free products were more likely to obtain CR following transplant (64% vs 17%, P= 0.02) and a longer progression-free survival, (40 months in patients transplanted with polyclonal products vs 20 with monoclonal ones, P = 0.03). These results were consistent when the overall survival was considered, since it was better in those patients with negative apheresis than it was in those with positive (83% vs 36% at 5 years from diagnosis, P= 0.01). These findings indicate that the presence of clonality rearranged VDJH segments is related to the response and outcome in MM transplanted patients.

Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin

AIMS/HYPOTHESIS: This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide.

METHODS: Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice.

RESULTS: In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small.

CONCLUSIONS/INTERPRETATION: These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n¼56), mantle cell lymphoma (n¼54), marginal zone lymphoma (n¼41) and follicular lymphoma (n¼109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Chromosomal rearrangements affecting biofilm production and antibiotic resistance in a Staphylococcus epidermidis strain causing shunt-associated ventriculitis.

Ziebuhr W, Dietrich K, Trautmann M, Wilhelm M. Institut für Molekulare Infektionsbiologie, Würzburg, Germany. w.ziebuhr@mail.uni-wuerzburg.de During two clinical courses of shunt-associated meningitis in a 3-month-old child, five multiresistant S. epidermidis isolates were obtained and analyzed with regard to biofilm production and antibiotic susceptibility. Three S. epidermidis strains, which were initially isolated from the cerebrospinal fluid, produced biofilms on polystyrene tissue culture plates. Following antibiotic treatment and subsequent exchange of the shunt system, sterilization of the CSF was achieved. However, after three weeks a relapse of the infection occurred. The two S. epidermidis isolates obtained now were biofilm negative, but showed an identical resistance pattern as those from the previous infection, except that resistance to rifampicin and increased mininal inhibitory concentrations of aminoglycoside antibiotics had emerged. DNA fingerprinting by PFGE indicated the clonal origin of all isolates. However, some DNA rearrangements and differences in the IS256-specific hybridization patterns could be identified in the isolates from the second infection period that led to altered biofilm formation and increased expression of aminoglycoside resistance traits. The data evidence that variation of biofilm expression occurs in vivo during an infection and highlight the extraordinary genome flexibility of pathogenic S. epidermidis.

T-cell-rich histiocyte-rich B-cell lymphoma of parotid gland

We describe a malignant lymphoma of the parotid gland arising in a patient with Sjögren's syndrome. Diagnosis was established on needle biopsy which showed a mixed population of lymphoid cells. Immunohistochemistry revealed B-lymphoid cells, T-lymphoid cells and histiocytes. Clonal immunoglobulin heavy chain gene rearrangement was demonstrated using the polymerase chain reaction. Within the confines of the small biopsy, the lesion qualifies for the designation T-cell-rich histiocyte-rich B-cell lymphoma. The value of molecular techniques in the diagnosis of malignant lymphoma on limited tissue samples is highlighted by this case.

Antagonistic effects of two novel GIP analogs, (Hyp(3))GIP and (Hyp(3)) GIPLys(16)PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

This study examines the actions of the novel enzyme- resistant, NH2- terminally modified GIP analog ( Hyp(3)) GIP and its fatty acid- derivatized analog ( Hyp(3)) GIPLys(16)PAL. Acute effects are compared with the established GIP receptor antagonist ( Pro(3)) GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor- transfected fibroblasts and in clonal pancreatic BRIN- BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulinreleasing effects of native GIP. Administration of once daily injections of ( Hyp(3)) GIP or ( Hyp(3)) GIPLys(16)PAL for 14 days resulted in significantly lower plasma glucose levels ( P <0.05) after ( Hyp3) GIP on days 12 and 14 and enhanced glucose tolerance ( P <0.05) and insulin sensitivity ( P <0.05 to P <0.001) in both groups by day 14. Both ( Hyp(3)) GIP and ( Hyp(3)) GIPLys(16)PAL treatment also reduced pancreatic insulin ( P <0.05 to P <0.01) without affecting islet number. These data indicate that ( Hyp3) GIP and ( Hyp(3)) GIPLys(16)PAL function as GIP receptor antagonists with potential for ameliorating obesity- related diabetes. Acylation of ( Hyp(3)) GIP to extend bioactivity does not appear to be of any additional benefit.

DPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser(2)-Asp(13))GIP

Structure-function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser(2)-Asp(13))GIP, containing a negatively charged Asp residue in place of an Ala in position 13, seas synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser(2)-Asp(13))GIP to be 2.3 and >4 h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser(2)-Asp(13))GIP (10(-12) to 10(-7) mol/l) was significantly less potent (60-90%; P

Cyclic AMP production and insulin releasing activity of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide

Synthetic fragment peptides of glucose-dependent insulinotropic polypeptide (GIP) were evaluated for their ability to elevate cellular cAMP production and stimulate insulin secretion. In GIP receptor transfected CHL cells, GIP(4-42) and GIP(17-30) dose-dependently inhibited GIP-stimulated cAMP production (40 +/- 8%; p <0.01 and 15 +/- 6%; p <0.05, respectively), while GIP(1-16) exerted very weak agonist effects on cAMP production. In the clonal pancreatic beta-cell line, BRIN-BD11, GIP(1-16) demonstrated weak insulin releasing activity compared with native GIP. In contrast, GIP(4-42) and GIP (17-30) weakly antagonized the insulin releasing activity of the native peptide (23 +/- 6%; p <0.05 and 11 +/- 3%, respectively). These data demonstrate the critical role of the N-terminus and the involvement of regions of the C-terminal domain in generating full biological potency of GIP.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala(8)-substituted GLP-1 analogues

The hormone glucagonlike peptide-1(736)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucosedependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the epsilon-amino group in the side chain of the Lys(26) residue and to combine this modification with substitutions of the Ala(8) residue, namely Val or aminobutyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val(8),Lys(pal)(26)]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal beta-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val(8),Lys(pal)(26)]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucoselowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.