Antimicrobial susceptibility monitoring of dermatological bacterial pathogens isolated from diseased dogs and cats across Europe (ComPath results).

AIMS: The ComPath project is a pan-European programme dedicated to the monitoring of antimicrobial susceptibility of pathogens from diseased dogs and cats using standardized methods and centralized minimum inhibitory concentration (MIC) determination. Here, the susceptibility of major pathogens is reported from antimicrobial nontreated animals with acute clinical signs of skin, wound or ear infections in 2008-2010. METHODS AND RESULTS: MICs were determined by agar dilution for commonly used antibiotics and interpreted using CLSI breakpoints, if available. Of the 1408 strains recovered, the main canine species was Staphylococcus pseudintermedius, followed by Pseudomonas and Streptococcus. In cats, Pasteurella multocida and Staph. pseudintermedius were most prevalent. For Staph. pseudintermedius, resistance was 18·4-25·2% for penicillin, clindamycin and chloramphenicol, but below 11% for ampicillin, amoxi/clav and fluoroquinolones. For Staphylococcus aureus, beta-lactam resistance was high (26·7-62·1%) but low (0·0-4·4%) for other antibiotics. 6·3% of Staph. pseudintermedius and 5·4% of Staph. aureus were confirmed mecA-positive. Gentamicin and fluoroquinolones exhibited moderate activity against Pseudomonas aeruginosa. For streptococci, resistance was absent/very low for penicillin, ampicillin, chloramphenicol and fluoroquinolones. For Escherichia coli, resistance was low to fluoroquinolones, chloramphenicol and gentamicin. No resistance was observed in Past. multocida. CONCLUSIONS: Overall, antimicrobial resistance was low in skin and soft tissue infections in dogs and cats. The results show the need for ongoing monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are a reference baseline for future surveillance. The paucity of clinical breakpoints underlines the need to set breakpoints for relevant antibiotics.

Dose-densified chemoimmunotherapy followed by systemic central nervous system prophylaxis for younger high-risk diffuse large B-cell/follicular grade 3 lymphoma patients: results of a phase II Nordic Lymphoma Group study.

BACKGROUND: Many patients with aggressive B-cell lymphomas and high clinical risk score still die of lymphoma after conventional R-CHOP chemoimmunotherapy. We hypothesized that intensified chemoimmunotherapy including systemic central nervous system (CNS) prophylaxis improves outcome and reduces the incidence of CNS-related events. PATIENTS AND METHODS: Inclusion criteria were age 18-65 years, primary diffuse large B-cell lymphoma or grade III follicular lymphoma without clinical signs of CNS disease and negative cerebrospinal fluid cytology, age-adjusted International Prognostic Index 2-3 and WHO performance score 0-3. Treatment consisted of six courses of R-CHOEP-14 followed by a course of high-dose cytarabine and a course of high-dose methotrexate. Primary end point was failure-free survival (FFS) at 3 years. RESULTS: A total of 156 eligible patients with a median age of 54 years (range 20-64) were included. Three toxic deaths were observed. Three-year overall survival (OS) and FFS rates (median observation time 52 months for survivors) were 81% and 65%, respectively. Seven patients experienced CNS relapse, all within 6 months. CONCLUSIONS: The results are promising with favorable 3-year OS and FFS rates, a low toxic death rate and a lower than expected number of CNS events. CNS progression might be further reduced by earlier CNS prophylaxis. CinicalTrials.gov. identifier NCT01502982.

Clinical efficacy of seasonal influenza vaccination: characteristics of two outbreaks of influenza A(H1N1) in immunocompromised patients.

BACKGROUND: Influenza A(H1N1) causes serious complications in immunocompromised patients. The efficacy of seasonal vaccination in these patients has been questioned. AIM: To describe two outbreaks of influenza A(H1N1) in immunocompromised patients. METHODS: Two outbreaks of influenza A(H1N1) occurred in our institution: on the kidney transplant ward in 2014 including patients early after kidney or simultaneous pancreas-kidney transplantation, and on the oncology ward in 2016 including patients receiving chemotherapy for malignant tumours. Factors leading to these outbreaks and the clinical efficacy of seasonal influenza vaccination were analysed. FINDINGS: Altogether 86 patients were exposed to influenza A(H1N1) during the outbreaks, among whom the seasonal influenza vaccination status was unknown in 10. Only three out of 38 vaccinated patients were infected with influenza A(H1N1), compared with 20 out of 38 unvaccinated patients (P = 0.02). The death of one out of 38 vaccinated patients was associated with influenza, compared with seven out of 38 unvaccinated patients (P = 0.06). Shared factors behind the two outbreaks included outdated facilities not designed for the treatment of immunosuppressed patients. Vaccination coverage among patients was low, between 40% and 70% despite vaccination being offered to all patients free of charge. Vaccination coverage of healthcare workers on the transplant ward was low (46%), but, despite high coverage on the oncology ward (92%), the outbreak occurred. CONCLUSION: Seasonal influenza vaccination was clinically effective with both a reduced risk of influenza infection and a trend towards reduced mortality in these immunocompromised patients. Several possible causes were identified behind these two outbreaks, requiring continuous awareness in healthcare professionals to prevent further outbreaks.

Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics.

Recent developments in instrumentation and bioinformatics show that the technique of synchrotron radiation circular dichroism spectroscopy can provide novel information on protein secondary structures and folding motifs, and has the potential to play an important role in structural genomics studies, both as a means of target selection and as a high-throughput, low-sample-requiring screening method. This is possible because of the additional information content in the low-vacuum ultraviolet wavelength data obtainable with intense synchrotron radiation light sources, compared with that present in spectra from conventional lab-based circular dichroism instruments.

Co-crystals of gramicidin A and phospholipid. A system for studying the structure of a transmembrane channel.

Single crystals of a complex of gramicidin A, a transmembrane channel-forming polypeptide, and dipalmitoyl phosphatidylcholine, a phospholipid, have been prepared and characterized by X-ray diffraction. They belong to space group P222(1), with unit cell dimensions a = 26.8 A, b = 27.5 A, c = 32.8 A. The asymmetric unit appears to be a complex of one gramicidin monomer and two phospholipid molecules. The unit cell dimensions, space group, and chemical composition are compatible with lipids packing in a bilayer-like motif, and an end-to-end association of gramicidin monomers to form a functional dimeric unit. The crystals diffract to 2 A and are suitable for structural studies by single-crystal X-ray analysis. This represents the first example of a well-ordered crystalline channel complexed with lipids, and solution of its structure may give insight into mechanisms of ion transport across membranes.

Preliminary crystallization and X-ray analysis of orthorhombic human endothelin.

Human endothelin (ET-1) is a highly potent, ubiquitous, endogenous 21 amino acid residue polypeptide, which acts as a vaso- and bronchoconstrictor. Crystals of the active, uncomplexed form of ET-1 have been grown from aqueous solutions. These crystals, which are long hexagonal prisms, diffract to 2.98 A and are apparently of the space group P222(1) with cell dimensions a = 33.5 A, b = 57.9 A, c = 59.6 A. The six molecules in the asymmetric unit are related by non-crystallographic symmetry operations, which result in a pseudo 6(1) appearance to the data.

Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein.

A random library of phage displayed peptides was screened for binding to a biotinylated derivative of paclitaxel (Taxol). Affinity-selected peptides were analyzed for similarity to human proteins. There was no significant similarity between the paclitaxel-selected peptides and tubulin. However, a subset of the peptides was identified that exhibits significant similarity to a non-conserved region of the anti-apoptotic human protein Bcl-2: ELISA assays confirmed binding of paclitaxel to Bcl-2, and circular dichroism spectroscopy demonstrated that a substantial conformational change accompanies this binding. In vivo, treatment with paclitaxel has been shown to lead to Bcl-2 inactivation with concomitant phosphorylation of residues in a disordered, regulatory loop region of the protein. Similarity between paclitaxel-selected peptides and this loop region implicate these residues in drug binding, and suggest that the apoptotic action of paclitaxel may involve the binding of paclitaxel to Bcl-2. These results demonstrate that peptides displayed on the surface of bacteriophage particles can mimic the ligand-binding properties of disordered regions of proteins.

A comparison of X-ray and NMR structures for human endothelin-1.

Direct comparisons between the recently solved X-ray and NMR structures of human endothelin-1 with respect to secondary structure, RMS deviations, surface accessibilities, and side-chain conformers indicate important differences in conformation, especially in the C-terminus, but also in the central loop region, that are important for defining the specificity of binding. These differences are larger than seen for other X-ray and NMR structures that have been compared. Comparisons between the X-ray structure and the NMR NOE constraints highlight the regions of flexibility and environment-induced diversity in the endothelin structures.

Comparison of the structures of the endothelin A receptor antagonists BQ123 and N-methyl leucine BQ123 with the crystal structure of the C-terminal tail of endothelin-1.

The functionally important regions of the cyclic pentapeptide endothelin A receptor antagonist BQ123 are shown to correlate with the structure of the C-terminal tail of endothelin-1, as found in the recently-determined X-ray crystal structure. Residues 18 and 21 of endothelin-1 are spatially juxtaposed such that they superpose extremely well with D-Asp and D-Trp of the antagonist, consistent with the residues on this surface of the endothelin helix being important for binding. This study provides new information on the three-dimensional nature of the endothelin A receptor binding site which may prove useful for rational drug design.

A molecular model for human Big-Endothelin-1 (Big ET-1).

A molecular model has been developed for human Big Endothelin-1, which is the immediate precursor to the potent vasoconstrictor polypeptide endothelin-1 and the target of the highly specific endothelin converting enzyme. This model is produced by a threading algorithm protocol and is consistent with all the currently available structural and biochemical data for this molecule.

Solution structure of alpha-conotoxin SI.

The nuclear magnetic resonance solution structure of alpha-conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that alpha-conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of alpha-conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly-8 to Ser-12. This similarity is more surprising when considering that in SI a proline replaces the Arg-9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.

A model for Batten disease protein CLN3: functional implications from homology and mutations.

In an attempt to understand the molecular nature of Batten disease, we have examined the amino acid sequence of the affected CLN3 gene product (The International Batten Disease Consortium (1995) Cell 82, 949-957) and the site-specific mutations which give rise to the biological defect. Homology searches and molecular modeling have led to the development of a model for the folding and disposition of the protein, possibly within a mitochondrial membrane. High homology with a yeast protein of unknown function suggests a strong evolutionary conservation of function. We speculate that a possible role for the protein may be in chaperoning the folding/unfolding or assembly/ disassembly of other proteins, specifically subunit c of the mitochondrial ATP synthase complex.

Urinary 5-hydroxyindoleacetic acid (5-HIAA) excretion during multiple-day high-dose chemotherapy.

Highly emetogenic drugs such as cisplatin induce an increase in the urinary 5-hydroxyindoleacetic acid (5-HIAA) level, the main metabolite of serotonin (5-HT), within the first 24 h following a single infusion, thus providing a possible cause for acute emesis and an explanation for the action of 5-HT3 antagonists. No further excretion peaks have been observed, suggesting that additional or serotonin-independent mechanisms cause delayed emesis. Our aim was to study the mechanisms behind emesis seen during a highly emetogenic chemotherapy regimen given as a continuous infusion over several days. Seven women treated with a 4-day high-dose chemotherapy (HDCT) regimen for breast cancer entered the study. Pooled urine samples were collected prior to and during chemotherapy for determining 5-HIAA excretion. An excretion peak in the urinary 5-HIAA level was observed within the first 24 h with no further peaks thereafter. Thus, the mechanisms behind the emesis experienced during this highly emetogenic multiple-day chemotherapy regimen from days 2-3 onwards would appear to be at least partially serotonin independent and would not be expected to be completely relieved by 5-HT3 antagonists alone.

Anatomy of human extrinsic cardiac nerves and ganglia.

The anatomy of the human extrinsic cardiac nerves and ganglia was reinvestigated because descriptions of human cardiac innervation vary, detailed analyses of subhuman mammalian cardiac innervation reveal considerable similarities among species and the anatomic pattern of cardiac innervation observed in subhuman mammals differs significantly from those described for humans. The presence of a consistent pattern of cardiac innervation in subhuman mammals raised the question as to whether a similar pattern exists in humans. To investigate this, the cervical and thoracic autonomic nerves and ganglia were dissected in 13 embalmed and 10 autopsy cadavers. All major sympathetic cardiopulmonary nerves were found to arise from the stellate ganglia and the caudal halves of the cervical sympathetic trunks below the level of the cricoid cartilage. These sympathetic cardiopulmonary nerves usually consisted of 3 nerves on the right side and 4 on the left. In contrast to widely accepted reports, no sympathetic cardiopulmonary nerves were found to arise from the superior cervical ganglia or the thoracic sympathetic trunks inferior to the stellate ganglia. Parasympathetic cardiopulmonary nerves were found to arise from the recurrent laryngeal nerves and the thoracic vagi immediately distal to them. These nerves interconnected with sympathetic cardiopulmonary nerves anterior and posterior to the main pulmonary artery to form the ventral and dorsal cardiopulmonary plexuses. These plexuses contained relatively large discrete nerves as well as smaller interconnections. Emerging from these plexuses to innervate the ventricles were 3 distinct relatively large cardiac nerves, the right and left coronary cardiac nerves and the left lateral cardiac nerve. In addition to these 3 major nerves, small cardiac nerves arose from the plexuses and the thoracic vagi. Histologic examination of representative dissections confirmed the presence of neural tissue and identified the locations of neuronal cell bodies in these structures. Cell bodies were located in the nodose, superior cervical, middle cervical, stellate and thoracic sympathetic ganglia. The middle cervical ganglia varied in size and number. Neuronal cell bodies were found in the cervical and thoracic sympathetic trunks and in small mediastinal ganglia located along the courses of the cardiopulmonary and cardiac nerves. Marked similarities exist between the anatomy of the cardiopulmonary nerves and ganglia of humans and baboons.

Complement activation in circulation and central nervous system after rituximab (anti-CD20) treatment of B-cell lymphoma.

Rituximab (IDEC-C2B8, Mabthera, Rituxan), a chimeric monoclonal antibody against the B-cell specific CD20-antigen, has been demonstrated to be effective in the treatment of non-Hodgkin's B-cell lymphoma (B-NHL). Previous in vitro studies have shown that direct complement-dependent cytotoxicity (CDC), ADCC and apoptosis are important in the rituximab-induced killing of lymphoma cells. It is, however, unknown whether rituximab penetrates the blood-brain barrier. Therefore, we studied rituximab levels and complement (C) activation in blood and cerebrospinal fluid (CSF) following intravenous rituximab therapy in a patient with relapsing non-Hodgkin's lymphoma with central nervous system (CNS) involvement. Longitudinal samples from blood and CSF were taken at 13 time-points during the treatment period. The results show that the C cascade becomes activated in blood during the first mAb infusion (C3a-desArg concentration rose from 55 to 138 microg/ml during the first 2 hours). After the first infusion the proportions of lymphocytes positive for the CD19- and CD20-antigens in the peripheral blood were reduced from 41% and 35%, respectively, to a level of 2% (for both). In CSF the rituximab concentration increased after successive infusions, but remained below 0.55 microg/ml (compared to a Cmax of 400 microg/ml in peripheral blood). Although a minor and delayed C activation response was seen in the CSF the treatment did not clear CD20-positive cells away from the CNS. Thus, it appears that an intact blood-brain barrier restricts the entry of rituximab into the CNS. Possible options to circumvent this would be dose escalation or intrathecal rituximab treatment.

Initial 300 consecutive stereotactic core-needle breast biopsies by a surgical group.

BACKGROUND: Stereotactic localization and breast biopsy by fine needle and 14-gauge core needle is a new technique for diagnosing nonpalpable breast lesions. The procedure employs a device that affords extremely accurate localization and sampling of nonpalpable breast abnormalities. METHODS: We are a 5-man surgical group reporting on the experience of our initial 300 consecutive stereotactic core-needle biopsies (SCNB). RESULTS: The procedures, conducted over a 13-month period, revealed 37 cancers, for a malignancy rate of 12%. Seven percent were infiltrating ductal carcinoma and 4% ductal carcinoma in situ. There were 2 cases of adenocarcinoma, and 1 case of mucinous carcinoma. Benign microscopic diagnoses included 193 categorized as "fibrocystic change," 34 "fibroadenomas," 19 "benign breast tissue," and 5 lesions that were suspected of being malignant but were proven to be benign. There were 12 "others." CONCLUSION: We conclude that SCNB is an essentially painless, short outpatient procedure with a reduced cost compared to open biopsy. It can be easily mastered by surgeons. Results are comparable to controlled series in the literature, and rates of malignant diagnosis are similar to our group's experience in previous years.

Functional integrity of sympathetic efferent postganglionic axons in a region of stunned myocardium.

In order to investigate whether brief, repeated episodes of acute myocardial ischemia impair the function of the intrinsic sympathetic efferent post ganglionic cardiac nerves, 12 cycles of left anterior descending coronary artery occlusions (5 mins) and reperfusions (10 mins) were successfully performed in 10 dogs. Following each brief occlusion percentage systolic shortening in the ventral myocardium was reduced, consistent with myocardial stunning. Stellate ganglion stimulation increased intramyocardial pressures in the ventral and lateral regions of the left ventricle before and after each of the artery occlusions in which stimulations were performed. Isoproterenol and tyramine also augmented intramyocardial pressures in those regions. When the coronary artery was occluded permanently in seven of the dogs tested, stellate ganglion stimulation continued to augment intramyocardial pressure in the ventral region of the left ventricle for up to 35 mins. These results indicate that following repetitive episodes of brief occlusion of a coronary artery that are reported to 'stun' the involved myocardium in a transmural distribution, sympathetic efferent post ganglionic axons can augment inotropism in the affected zone. Furthermore, when a coronary artery is totally occluded thereafter for up to 35 mins these efferent sympathetic post ganglionic nerves continue to function. It is concluded that sympathetic efferent post ganglionic neurons innervating the ventricle can augment cardiac inotropism in a region of stunned myocardium.

Tryptophans in membrane proteins. X-ray crystallographic analyses.

While tryptophans are generally found in low abundance in soluble proteins, in many integral membrane proteins they comprise a significantly higher proportion of the amino acid composition. Now that crystal structures are available for a number of membrane proteins, it has been possible to examine the distribution and disposition of the tryptophans within these structures. The tryptophan locations with respect to the lipid bilayer (along the direction normal to the membrane surface) are strikingly non-uniform in nearly all of the membrane proteins examined. They tend to cluster at the interface between the polar head group region and the hydrophobic interior, in a relatively uniform layer just below the surface. In many cases, their distributions with respect to the extra- and intra-cellular surfaces tend to be asymmetric. These observations provide evidence for possible structural roles for tryptophans in transmembrane sheets and helices, where they may play a part in the stabilization of the transmembrane segments and perhaps in the orientation and bilayer insertion processes.

Rural hospitals in New Zealand.

AIM: To produce a list of New Zealand rural hospitals, including information on acute bed numbers, population served, average driving time to base hospital, and number and payment of rural hospital doctors. METHODS: Information about rural hospitals was obtained from local doctors and/or administrators, and reflected conditions at June 15, 1998. A 'rural hospital' was defined as a facility with no resident medical specialists, where acutely ill patients are admitted and cared for solely by generalist doctors, either general practitioners or medical officers of special scale. RESULTS: Varying definitions for acute 'facilities' and 'beds' made analysis difficult. There were a total of 36 'rural hospitals' in New Zealand at June 15, 1998 containing 293 acute beds and serving a population of about 340000. Patient care was provided by a total of 131 generalists (general practitioners or medical officers of special scale) equivalent to 40 full-time rural hospital doctors. CONCLUSIONS: Approximately 10% of the New Zealand population are served by rural hospitals. Discrepancies exist between the list of rural hospitals provided in this study and that provided by the Government's recent 'Hospital Services Plan'.

Assessment of rubella immunity in women born in 1959-74 in a solo rural general practice.

AIM: To evaluate the rubella immunity of all women in the practice born 1959-74. METHOD: A chart audit followed by offering testing to all women whose rubella status was unknown. RESULTS: According to their records, of 203 female patients born 1959-74, 149 (73%) were immune to rubella and 54 were not. All of the 54 nonimmune women agreed to rubella testing and only three were found to be susceptible. These three women had never been pregnant before, none were from the higher risk cohort born 1965-7 and all accepted vaccination. CONCLUSION: For general practitioners it is important to assess the rubella immunity of all women of childbearing age in their practice, especially those who have never been pregnant and not just the high risk cohort born 1965-7.