Normal values for blood immunoglobulins in the beagle dog

 The measurement of circulating immunoglobulins in laboratory animals may be a valuable tool to check their immune status and to ensure that the animals were not exposed to pathogens prior to their incorporation into preclinical safety studies. The aim of this study was to establish normal values for total immunoglobulins G, M and E in young beagle dogs purchased from an external breeder and to evaluate the possible variations between batches and the effect of a 3-week acclimatisation period under our own housing conditions. Immunoglobulins were measured by radial immunodiffusion using commercially available canine-specific assays on three successive batches at two sampling time points after their arrival in our facilities. There were no clear differences between batches and sampling time points (1 and 4 weeks after arrival). IgG was found to be the dominant class of immunoglobulin, followed by IgM and IgE. A slight sex-related difference was found for IgG and IgM, but not for IgE. Overall, the values we obtained were lower than those given in the literature for clinically normal companion animals of various strains and ages. This was interpreted to reflect the relative young age of our dogs and/or the strictly controlled environmental conditions of their housing. Based on our findings, comprehensive reference values are provided in this article. Received: 14 October 2002 / Accepted: 20 February 2003 Acknowledgements The authors wish to thank Mrs Anne Paquignon and Mr Damien Thierry for their outstanding technical assistance.     

Development of a fluorinated polyimide hollow fiber for medical devices

 We fabricated an asymmetric polyimide hollow fiber for medical devices. A dry/wet phase inversion process was applied to a spinning process to prepare the hollow fiber. The outer diameter was 330 μm with a wall thickness of 70 μm. Transfer rates of O₂ and CO₂ in the asymmetric polyimide fiber were 6.9 × 10⁻³ and 5.5 × 10⁻³ cm³ (STP)/(cm² s cmHg), respectively, which are approximately 10 times higher than those measured in the Menox and Si-polypropylene fibers of the presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber was evaluated in vivo, indicating that polyimide had excellent blood compatibility when compared with silicone-coated fiber. Additionally, we fabricated a novel porous membrane with three-dimensional fine structure from cylindrical microscale pores and examined possibility of a porous membrane for use in hemodialysis. Received: October 3, 2002 / Accepted: February 20, 2003 Acknowledgments This work was supported by a JSAO Grant. The authors wish to thank Mr. K. Kuwana of Senko Medical Instrument Mfg. Co., Ltd., for providing the Si-PP hollow fibers and Mr. K. Sakai of Dainippon Ink and Chemicals, Inc., for providing the Menox hollow fibers. Also the authors thank Dr. S. Nagaoka, Mr. M. Niwa, and Ms. Y. Taketani of Tokyo Metropolitan University for their useful comments.     

Development of a xenogeneic direct hemoperfusion method in a bioartificial liver system

 We devised a new method of xenogeneic direct hemoperfusion of a bioartificial liver support, which consists of a leukocyte adsorbent column, an immunoglobulin adsorbent column, and a substitute unit for hepatic function. Using this method in a hybrid bioartificial liver system incorporated with a nonwoven fabric bioreactor containing porcine hepatocytes, we successfully performed xenogeneic direct hemoperfusion in a canine liver failure model for 3 h without hyperacute rejection. Adequate ammonia detoxification was exhibited, and no findings of hepatocyte destruction by leukocytes or immunological proteins were detected in the canine blood analysis. Beneficial effects were also detected in a significant increase in Fischer's ratio and a decrease in intracranial pressure, indicating that our system could contribute to recovery from hepatic coma in patients with severe liver failure. Received: April 23, 2002 / Accepted: July 16, 2002 Acknowledgments We wish to thank Mr. Nobutaka Furuya from the Institute for Animal Experimentation, University of Tokyo, for his expert technical assistance in treating experimental animals. We also wish to thank Mr. Ken Shibata from Tokyo Medical Service for his excellent technical assistance in operating the perfusion equipment. We also wish to thank Dr. Wendy Gray, MD, for her excellent work in revising our English. This study is supported financially by grants in aid for scientific research (11357010, 1999–2001, and 12309003, 2000–2002) and a grant in aid for advanced forefront medical development (2000–2002). Correspondence to:K. Naruse     

Immunocytochemical identification of CD5 positive peripheral blood lymphocytes in four marsupial species

 Immunocytochemical analysis of peripheral blood mononuclear cells was undertaken using a streptavidin biotin–horseradish peroxidase method to detect CD5 positive lymphocytes from the blood of several marsupial species. A monoclonal antibody raised to a conserved peptide sequence of the human CD5 antigen positively labelled lymphocytes in freshly isolated peripheral blood mononuclear cells of the tammar wallaby (Macropus eugenii), the long-footed potoroo (Potorous longipes), the long-nosed potoroo (Potorous tridactylus) and the rufous hare-wallaby (Lagorchestes hirsutus). A polyclonal anti-CD3 antibody also positively labelled circulating lymphocytes from the tammar wallaby. Whereas previous studies using flow cytometry reported labelling of T cells in koala lymphocyte preparations using a polyclonal anti-CD3 antibody, there have been no other reports of marsupial blood immunophenotyping. The current study extends the known applications of monoclonal anti-CD5 and polyclonal anti-CD3 antibodies to blood lymphocytes of small wallaby species using an immunocytochemical slide technique that is simple, can be processed within a day and requires no dedicated large equipment. Received: 2 July 2002 / Accepted: 23 August 2002 Acknowledgements We thank Ron Claassens of Macquarie University Fauna Park (New South Wales, Australia) for assistance with Tammar wallabies; Veterinary Staff at Healesville Sanctuary (Victoria, Australia) for potoroo blood samples; Ro McFarlane of Alice Springs Veterinary Clinic (Northern Territory, Australia) for Mala samples and Margaret Jones of the Leukaemia Research Foundation (Oxford, UK) for the donation of the monoclonal CD3 and CD5 antibodies. Lauren Young was supported by an Australian Postgraduate Award during the period of this study.     

Nonsyndromic sensorineural deafness associated with the A1555G mutation in the mitochondrial small subunit ribosomal RNA in a Balinese family

 Sensorineural deafness associated with increased sensitivity to aminoglycoside antibiotics as the consequence of an A1555G mutation in the mitochondrial DNA (mtDNA) in a highly conserved region of the small (12S) rRNA gene has been reported in Caucasian, Chinese, and Japanese individuals. We report here a large family of Balinese Indonesian origin with progressive/congenital sensorineural deafness who carry the A1555G mutation. The pedigree shows a generally maternal inheritance pattern with some exceptions, which is the result of an unusual multiple entry of the mutation into the pedigree. A complete mtDNA genome sequence from three Balinese individuals revealed a relatively large number of single- nucleotide polymorphisms (20) not previously reported, and confirmed the genetic distance of Southeast Asian populations from those of Caucasians and Japanese. The biochemical expression of the A1555G mutation under the influence of this mtDNA background was investigated. Examination of respiratory enzyme activities showed a significant decrease in respiratory complex I activity, particularly in symptomatic family members. Received: July 12, 2002 / Accepted: November 22, 2002 Acknowledgments We thank all the family members that have participated in the present study. We would like to thank Dr. Helena Suryadi for her expert assistance in the fieldwork, and Ms. Neny Sitorus for tissue culture work. This work was supported by grants from PT Krakatau Steel and PT Inti through the Agency for Strategic Industries (Indonesia) and by a generous Development Fund from the National Development Planning Agency (BAPPENAS) of the Republic of Indonesia. Correspondence to:S. Marzuki     

Liquid paraffin pneumonia — with chemical analysis and electronmicroscopy

Summary Liquid paraffin pneumonia was diagnosed after open lung biopsy in a woman age 73 with a hiatus hernia and rheumatoid arthritis who had been taking liquid paraffin nightly for fifty two years. Histological examination showed a lipid type pneumonia with involvement of alveoli, interstitial tissues and brochioles.Chemical analysis of the lung showed total lipids of 17.7% (w/w), 86% was liquid paraffin which was positively identified by infrared spectroscopy. Transmission electronmicroscopy showed macrophages in the alveoli filled by phagosomes. The alveoli were mainly lined by alveolar type II cells. Scanning electronmicroscopy showed alveoli filled by a mass of vacuoled material.I wish to thank Dr. G.C. Cheeseman, National Institute for Research in Dairying, Reading and Dr. D.A. Reaveley and Mr. R. Vaughan, Department of Chemical Pathology for the chemical analysis, Mrs. G. Miller, Mr. R. Barnett and Mr. T.B. Bull for technical assistance.     

A locus for congenital preauricular fistula maps to chromosome 8q11.1–q13.3

 The incidence of congenital preauricular fistula (CPF) is >1.1% in both Chinese and Caucasians, but it is even higher in Blacks. We mapped the locus for CPF to chromosome 8q11.1–q13.3 by linkage analysis of a family composed of 7 affected and 11 nonaffected members. The two-point LOD score was 2.40, shown by markers D8S285 and D8S1113 at a recombination fraction (θ) of 0.00. Results from three other markers (D8S1110, D8S260, and D8S1136) in the same region further support the linkage. Haplotype analysis for this family confined the locus to within an interval of approximately 26.7 cM, flanked by markers D8S532 and D8S279. A LOD score of <3 is likely due to the limitation of family size. Received: October 16, 2002; Accepted: November 25, 2002 Acknowledgments We would like to thank Shanghai Municipal Commission for Science and Technology, the National Natural Science Foundation of China, and the National 973 and 863 Projects for their generous financial support of this study. The first and third authors contributed equally to this work. Correspondence to:L. He     

Exercise-induced intravascular haemolysis in standardbred horses

 In human sports medicine a pathophysiological condition called `sports anaemia' is reported. This condition has been attributed to episodes of intravascular haemolysis induced by physical exercise. The occurrence of haemolytic episodes is indicated by the presence of high values of free plasma haemoglobin and lower plasma levels of haptoglobin after physical exercise. The literature regarding sports anaemia in horses, and in particular haemolysis induced by physical exercise, is rather limited. The purpose of this study was to evaluate the markers of intravascular haemolysis (plasma haemoglobin and haptoglobin) in Standardbred horses immediately after a race, in order to ascertain the presence of intravascular haemolysis, possibly induced by physical activity. We reported both the haptoglobin and free plasma haemoglobin values as a percentage of the total protein to avoid any influence of haemoconcentration induced by the exertion during the race. Free plasma haemoglobin concentration showed a significant increase both at 5 min (p < 0.01) and 10 min (p < 0.05) after exercise. A significant decrease in the haptoglobin occurred in both post-exercise blood samples, and was statistically significant 10 min after the race (p < 0.05). These data suggested that episodes of intravascular haemolysis may occur during physical activity in Standardbred horses. Received: 13 May 2002 / Accepted: 15 September 2002 Acknowledgements The authors are very grateful to Dr Lorenzo Berti (Florence, Italy) for his assistance in enrolling the subjects for the study; and to Dr David Marlin (Animal Health Trust, Newmarket, UK) for his advice.     

The C-terminal regions of the envelope glycoprotein gp2 of equine herpesviruses 1 and 4 are antigenically distinct

Summary.  The unusual mucin-like high molecular mass (Mr) glycoprotein 2 (gp2) has only been described in the equid alphaherpesviruses, among which there is considerable antigenic cross-reactivity. Equine herpesvirus 1 (EHV-1) gp2 is cleaved into a highly glycosylated N-terminal subunit and a 42 kDa C-terminal cleavage product. In order to investigate their antigenic recognition by horses naturally infected with EHV-1 and/or equine herpesvirus 4 (EHV-4), the C-terminal cleavage product and high Mr gp2 were affinity purified. Cross-reactivity between EHV-1 and EHV-4 was observed for the high Mr gp2 using Western blotting. In contrast only horses with antibodies to EHV-1 detected the 42 kDa EHV-1 gp2 C-terminal cleavage product. This phenomenon was evident in pooled sera from adult horses and also in foals that had demonstrated seroconversion due to EHV-1 infection. The results indicate that the C-terminal region of EHV-1 gp2 is antigenically distinct from that of EHV-4 gp2 and can be detected only after an EHV-1-specific immune response. Received June 23, 2001 Accepted November 7, 2001     

Quantitative EEG in elderly depressives

Summary Thirty-one elderly depressive patients were evaluated with topographic, quantitative EEG using relative measures, absolute measures, and computations of interhemispheric asymmetry and interhemispheric coherence in four frequency bands: delta, theta, alpha and beta. Patients were found to have lower than normal delta, higher than normal theta, higher than normal alpha and lower than normal beta values. EEG values were greater over the left than the right hemisphere in theta, alpha and beta bands. Lower than normal anterior interhemispheric coherence was found in all four frequency bands.Research supported, in part, by DA06728, MH12507, GCRC MO1-RR00349, BSRG SO7-RR05417 and by the Einstein Society. We wish to thank Mrs. Anna Cornwell, Mrs. Eleanor Dixon, Mr. Stephen Slepner and Dr. Denise Sharon for assistance in this study.     

A double mutation (G11778A and G12192A) in mitochondrial DNA associated with Leber's hereditary optic neuropathy and cardiomyopathy

 We report a male patient with Leber's hereditary optic neuropathy (LHON) and hypertrophic cardiomyopathy. Besides a G11778A mutation in the ND4 gene of the mitochondrial DNA (mtDNA), one of the most common mutations in LHON patients, sequencing of total mtDNA revealed a G12192A mutation in the tRNA (His) gene that was recently noted to be a risk factor for cardiomyopathy. Because no case of LHON presenting with cardiomyopathy has been reported, the present finding suggests that the G12192A mutation caused cardiomyopathy as an additional symptom. In the present case, the double pathogenic mtDNA mutations may be associated either synergistically or concomitantly with two different clinical manifestations. Received: July 4, 2002 / Accepted: October 28, 2002 Acknowledgments We thank Mayuko Kato, Yoko Murase, and Munemitsu Yuasa for technical assistance. This work was supported in part by a Research Grant (13B-1) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare of Japan (Y.G.). Correspondence to:Y. Goto     

Detection of skeletal muscle fatigue using an accelerometer in dynamic cardiomyoplasty

 An animal experiment was done using six mongrel dogs that weighed 28 ± 3 kg to show that an accelerometer could detect skeletal muscle fatigue in dynamic cardiomyoplasty. Through left-side thoracotomy, the heart was exposed and an electrode to sense the heartbeat was positioned on the left ventricle. A left latissimus dorsi muscle flap (LDMF) was inserted into the left chest cavity and rolled around the heart. An accelerometer was put on the rolled LDMF to sense the ventricular acceleration by contraction of the LDMF and the heart. The LDMF was stimulated under these settings: pulse width, 210 μs; stimulation output, 6 V; burst frequency, 30 Hz; burst duration, 200 ms; synchronous ratio, 1 : 4; and synchronous delay, 66 ms. Output voltage from the accelerometer was recorded 1, 3, 5, 10, and 15 min after the beginning of stimulation. Percentages of the amplitude in all dogs after 3, 5, 10, and 15 min were 81 ± 10%, 63 ± 12%, 48 ± 11%, and 45 ± 14% of the values after 1 min, respectively. Significant differences were found between the values after 1 min and those after 3 min, between the values after 3 min and those after 5 min, and between the values after 5 min and those after 10 min. This study suggests that muscle fatigue is detectable with an accelerometer in actual dynamic cardiomyoplasty. Received: May 11, 2001 / Accepted: September 10, 2002 Acknowledgments This work was financially supported in a part by a Grant in Aid for Scientific Research (05671113) from the Ministry of Education, Science, and Culture of Japan. Correspondence to:H. Kuroda     

Association of the –381T/C promoter variation of the brain natriuretic peptide gene with low bone-mineral density and rapid postmenopausal bone loss

 Osteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone-mineral density (BMD). Recent quantitative trait locus analysis in human suggested a possible involvement of chromosomal region 1p36.2–p36.3 for determination of BMD. The brain natriuretic peptide (BNP, also named NPPB) gene lies within this candidate region for BMD determination. Overexpression of the BNP resulted in skeletal overgrowth in transgenic mice. Association analysis between nucleotide variations of the BNP gene and radial BMD in 378 Japanese postmenopausal women revealed a significant association of the –381T/C variation of the BNP gene with radial BMD (r = 0.17, P = 0.01). Homozygous T-allele carriers had the lowest BMD values (0.395 ± 0.056 g/cm²), homozygous C-allele carriers had the highest (0.429 ± 0.051 g/cm²), and heterozygous individuals had intermediate radial BMD values (0.405 ± 0.048 g/cm²), indicating a dosage effect. Accelerated bone loss also correlated with the –381 T allele in a 5-year follow-up study (r = 0.21, P = 0.017). These results suggest that variation of BNP may be an important determinant of postmenopausal osteoporosis, in part through the mechanism of accelerated postmenopausal bone loss. Received: November 8, 2002 / Accepted: November 12, 2002 Acknowledgments This work was supported in part by a special grant for Strategic Advanced Research on “Cancer” and “Genome Science” from the Ministry of Education, Science, Sports and Culture of Japan; by a Research Grant for Research from the Ministry of Health and Welfare of Japan; and by a Research for the Future Program Grant of The Japan Society for the Promotion of Science. Correspondence to:M. Emi     

The interleukin 6 −174G/C Polymorphism is associated with indices of obesity in men

 Obesity represents an expansion of adipose tissue (AT) mass and is closely related to insulin resistance and cardiovascular disease. Several hormonal signals have been shown to originate from AT, one of them being interleukin 6 (IL6), for which one third of circulating levels is accounted for by AT. To study the impact of the IL6 −174G/C polymorphism on obesity-related phenotypes, we genotyped a cohort of 270 French-Canadian men from the greater Quebec City area selected to cover a wide range of body fatness values. The IL6 −174G allele was more commonly observed among lean subjects (body mass index <25 kg/m², χ² = 7.27, P = 0.007 or waist-line <100 cm, χ² = 6.63, P = 0.01). When men were subdivided according to insulin and glucose levels at 180 min following the glucose load, using 160 pmol/l and 4.6 mmol/l, respectively, as cutoff points, the −174G allele was more frequently observed in groups with low concentrations of either insulin or glucose, P = 0.03 and P = 0.01, respectively. When comparisons between genotype groups were performed, −174G/G homozygotes presented the lowest waist circumference (P < 0.05). In summary, this study showed that, in men, the IL6 −174G/C polymorphism is associated with some indices of body composition and parameters of glucose and insulin homeostasis. Received: November 1, 2002 / Accepted: November 6, 2002 Acknowledgments We are indebted to the subjects involved in this study. We want to acknowledge Mr. Alain Houde for his technical support. Marie-Claude Vohl and André Tchernof are recipients of a scholarship from the Fonds de la Recherche en Santé du Québec (FRSQ). Jean Bergeron is a clinical scholar from the FRSQ. Jean-Pierre Després is chair professor of human nutrition, lipidology, and prevention of cardiovascular disease supported by Pfizer Canada and Provigo. Part of this work was supported by the Canadian Institutes for Health Research (operating grant: MOP 44074) and the Heart and Stroke Foundation of Canada. Correspondence to:M.-C. Vohl     

A novel splice site mutation in neonatal carnitine palmitoyl transferase II deficiency

 Mitochondrial β-oxidation of long-chain fatty acids requires the concerted action of three tightly integrated membrane-bound enzymes (carnitine palmitoyltransferase I and II and carnitine/acylcarnitine translocase) that transport them into mitochondria. Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of this transport. We describe a novel splice-site mutation in the CPT II gene, found in a Moroccan family, of which four out of five children have died from the neonatal form of CPT II deficiency. Mutation detection studies at the mRNA level in the CPT II gene implied that the affected children were homozygous for the previously reported 534T insertion followed by a 25-bp deletion (encompassing bases 534–558). Studies of genomic DNA, however, revealed all patients to be compound heterozygous for this 534T ins/del 25 mutation, and for a new g→a splice-site mutation in the splice-acceptor site of intron 2. Because of these findings, prenatal diagnosis was performed in chorionic villi of three new pregnancies. This did not reveal new compound heterozygous genotypes, and, after uneventful pregnancies, all children appeared to be healthy. The new mutation is the first splice-site mutation ever identified in CPT II deficiency. The fact that it was not discovered in the patient's cDNA makes this study another example of the incompleteness of mutation detection at the mRNA level in cases where a mutation leads to aberrant splicing or nonsense-mediated messenger decay. Received: September 18, 2002 / Accepted: October 29, 2002 Acknowledgments We thank the patients and their family for participation, and Anouk Hanstede and Marloes Siers for technical assistance. Correspondence to:J.A.M. Smeitink     

The genomic sequence of cowpea aphid-borne mosaic virus and its similarities with other potyviruses

Summary.  The genomic sequence of a Zimbabwe isolate of Cowpea aphid-borne mosaic virus (CABMV-Z) was determined by sequencing overlapping viral cDNA clones generated by RT-PCR using degenerate and/or specific primers. The sequence is 9465 nucleotides in length excluding the 3′ terminal poly (A) tail and contains a single open reading frame (ORF) of 9159 nucleotides encoding a large polyprotein of 3 053 amino acids and predicted Mr of 348. The size of the genome and the encoded polyprotein is in agreement with other potyviruses and contains nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. The P1 and P3 were the most variable proteins while CI, NIb and CP were the most conserved. Received August 2, 2001; accepted January 15, 2002     

Development and in vitro validation of a device for measuring non-shunt cardiac output by nitrous oxide throughflow

A system has been developed for measuring non-shunt cardiac output by the throughflow technique, using nitrous oxide in patients undergoing general anaesthesia. The throughflow measurement technique is a non-invasive method based on inert gas throughflow theory. In vitro validation of the measurement system was performed using a lung gas exchange simulator. The accuracy and precision of the throughflow measurement system was assessed by comparing measured and target values for five simulated values of non-shunt cardiac output, from 2.88 to 9.86 l min⁻¹. This showed an overall mean bias of −0.03l min⁻¹ (range −0.00 to −0.10 l min⁻¹), with a mean coefficient of variation of the difference of 1.39% (1.20–1.93%). These results indicate that the measurement system is suitable for monitoring the non-shunt cardiac output in patients undergoing general anaesthesia using nitrous oxide throughflow.     

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat

Summary.  Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 × 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 × 10⁶). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 ×  10⁶. The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5′ co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear. Received February 22, 2000 Accepted June 23, 2000     

Normal blood chemistry and age-related changes in the white-bellied bustard ( Eupodotis senegalensis ), with some clinical observations

Blood samples were obtained from adult (τ;1 year) and juvenile (4–8 weeks, 9–16 weeks, 17–24 weeks) captive white-bellied bustards (Eupodotis senegalensis) to determine normal blood chemistry reference values and to study age-related changes. Twelve different tests were conducted using a Dupont Dimension wet chemistry analyser. A comparison of the values obtained was made between adult and juvenile white-bellied bustards and from the literature with other bustard species. Significant differences between adult and juvenile bustards were found for glucose, uric acid, total protein, alkaline phosphatase, aspartate amino transferase, lactate dehydrogenase, magnesium and calcium. The results obtained from this study provide blood chemistry reference values for this species and demonstrate age-related differences between adult and juvenile birds.