bluuebunny/crossref_metadata_2025 huggingface dataset:
T‐Cell Activation and the Development of an Apoptosis‐Resistant CD45RO+ T‐Cell Population
Authors: Müller M. , Grunewald J. , Gigliotti D. , Eklund A. , Stridh H.
Published: December 2004

Growing experimental evidence supports a broadening role for the caspases; not only do they participate in the process of apoptosis but also in the control of the cell cycle and cellular proliferation. The biological role of the caspases in the process of T‐cell activation and proliferation is still not defined. In the present study, we propose a potential role, by demonstrating an association of T‐cell receptor‐mediated caspase activity with the development of an apoptosis‐resistant memory CD45RO+ T‐cell population. As previously shown by us, a time‐dependent induction of caspase activity, in the absence of apoptosis, can be observed in CD3‐stimulated human peripheral blood lymphocytes. We here show that a population of CD45RO+ cells, with activated caspase‐3 and with resistance to tributyltin‐induced apoptosis, develops after 3 days of stimulation. A concomitant expression of the anti‐apoptotic protein Bcl‐xL accompanied the caspase activity and the development of the apoptosis‐resistant phenotype. Finally, upon co‐culturing with dexamethasone (DEX), the CD3‐induced caspase‐3 activity was blocked. During this condition, the expression of the activation marker HLA‐DR as well as the cellular proliferative response was strongly suppressed. The development of memory cells with a CD45RO+ phenotype was also blocked. Our data support the hypothesis that caspase‐3 activity, observed in CD3‐stimulated cells, may be an important component in the proliferation process and, furthermore, might play a role for the development of memory T cells, and DEX inhibits this process.
Immunoglobulin G3 and Immunoglobulin M Isotype Plasma Levels are Influenced by Interleukin‐1α Genotype
Authors: Kilpinen S. , Laine S. , Hulkkonen J. , Hurme M.
Published: December 2004

The immunoglobulin (Ig) plasma levels are known to be, at least partially, genetically regulated, but all the genes involved are not known. Interleukin‐1 (IL‐1) is a potent proinflammatory cytokine able to serve as an adjuvant for immune responses. IL‐1α gene is polymorphic, and at least one of the polymorphisms has been identified in the 5′ regulatory region of the promoter, a biallelic base exchange (C→T) at position −889.

We set out to study whether the IL‐1α genotype might contribute to the genetic component seen in the steady‐state antibody levels of healthy individuals.

Four hundred healthy blood donors (218 males and 182 females) were genotyped, and the plasma levels of IgM, IgG as well as IgG subclasses were measured.

An association was found between IgG3 plasma levels and the IL‐1α genotype; the 1.1 homozygotes had increased IgG3 levels compared with the 1.2 heterozygotes (P < 0.001 in males and P = 0.04 in females, Mann–Whitney U‐test). A similar significant association was also found between IgM plasma levels and the IL‐1α genotype in males, but it was no longer present in females; the 1.1 homozygotes had higher IgM levels than the 2.2 homozygotes (P = 0.03, Mann–Whitney U‐test).

The data suggest that IL‐1α‐mediated signals are critical for IgG3 and IgM responses, which are induced by thymus‐independent antigens and are important in activating complement.
Brown Norway Rat Ovalbumin‐Specific Immunoglobulin E Antibodies Increase the Human Basophil Expression of CD63 Marker
Authors: Bellou A. , Saint‐Laudy J. , Knippels L. , Montémont C. , Vauthier E. , Gerard P. , Pellegrom H. , Koerkamp E. K. , Lesesve J. F. , Guéant J. L. , Lambert H. , Mallié J. P.
Published: December 2004

Anaphylactic shock is an immunoglobulin E (IgE)‐dependent hypersensitivity. Biological tests like leucocyte histamine release (LHR) and human basophil activation (HBA), frequently used in human allergy, reflect both the amount of IgE fixed on cells and the cellular reactivity. To assess whether serum‐specific IgE from Brown Norway (BN) rats prepared for ovalbumin (OVA)‐induced anaphylactic shocks can activate human basophils which has a potential interest in experimental allergy: such a test could rapidly assert an IgE sensitization in laboratory animals genetically T‐helper 2 (Th2)‐predisposed. Rats (n = 39) were immunized three times (day 0, day 5 and day 21) with OVA injected subcutaneously. One week after the third immunization, a shock was induced with an intravenous (i.v.) bolus of OVA. Sensitization was assessed by passive cutaneous anaphylaxis (PCA) test and dosages of serum IgE antibodies anti‐OVA by enzyme‐linked immunosorbent assay. Blood basophils were counted before and during the shock. Before the shock induction (at day 21), an LHR test was performed on rat blood, and human basophils were sensitized with rat sera. HBA was demonstrated by the increase in the percentage of cells expressing CD63 antigen membrane, measured by flow cytometry. Twenty‐one days after the first subcutaneous (s.c.) immunization, the rat serum induced a significant HBA. HBA was observed neither with the same serum previously heated nor with the serum from nonimmunized rats (NIRs). OVA‐specific IgEs were significantly increased in immunized rat (IR) serum. The PCA test was negative when the serum was previously heated (56 °C). We never observed any circulating basophils, and LHR test was negative. After OVA i.v. administration, all IRs died rapidly. HBA testing strongly suggests a mediation by specific IgE in the increase of CD63 in BN rats. Thus, HBA test seems useful in assessing whether an experimental allergy was induced in animals genetically predisposed to an immune response, Th2‐mediated, like BN rat. We also conclude that rat basophil activation does not participate in the histamine release during anaphylactic shock in sensitized BN rats.
Long‐Term CD4+ and CD8+ Memory T Cells Developed in Severe Combined Immunodeficiency Mice during Homoeostasis Exhibit Differences in Sensitivity to Antigen
Authors: Pettersson F. Ekholm , Grönvik K.‐O.
Published: December 2004

T cells transferred in small numbers to lymphopenic hosts proliferate spontaneously, and naïve T cells turn into memory cells without complete cellular reconstitution of the lymphoid compartment. In this study, neonatal severe combined immunodeficiency mice were treated with peripheral CD4+ or CD8+ T cells purified from the spleen of syngeneic C.B‐17 mice. At 2 weeks and more pronounced at 10 weeks post treatment, a majority of the residing donor T cells showed memory phenotype, with high expression of CD44 and an early onset of proliferation and cytokine production upon stimulation. These memory type of donor cells were sustained in numbers for at least 1.5 years post treatment in a homoeostatic fashion, recognized by normal CD4/CD8 ratio and no bias towards type 1 or type 2 immune response. Furthermore, amongst the memory type of cells, there was a striking difference in their response, where the CD8+ donor cells had higher threshold for stimulation than the CD4+ donor cells.
Nodular celestite in the Chihsia Formation (Middle Permian) of south China
Authors: Yan Jiaxin , Carlson Ernest H.
Published: December 2004

The middle Permian Chihsia Formation of south China accumulated on a shallow shelf, and consists mainly of black to dark grey micritic limestone rich in chert nodules and organic matter. A unique type of nodular crystal cluster is distributed widely in the carbonate succession. Most crystal clusters consist of calcite. Some, however, are composed of celestite, and geochemical, microscopic and crystal morphological data suggest that celestite was the precursor of the calcite. The celestite developed displacively within the sediments during early diagenesis, before compaction and before local dolomitization of the host rock. Similar strontium isotopic values were obtained from the celestite clusters, replacement calcite, vein calcite and host rock. The values are within the range of middle Permian sea water. The strontium in the celestite was furnished chiefly by either diagenetic alteration of strontium‐rich marine aragonite to strontium‐poor calcite, or aragonite dissolution induced by aerobic oxidation of organic matter, or both. The sulphur isotopic values of the celestite are about 6–11‰ heavier than the sulphur isotopic value of sulphate in coeval sea water. Based on geological context, this difference is attributed to microbial reduction of porewater sulphate in the Chihsia sediments.
Sub‐Milankovitch and Milankovitch forcing on a model Mesozoic carbonate platform – the Latemar (Middle Triassic, Italy)
Authors: Zühlke Rainer , Bechstädt Thilo , Mundil Roland
Published: December 2004

ABSTRACT Basic shallowing‐upward cycles (shu‐cycles) and five‐bundled megacycles in the Latemar platform have been widely regarded as a model example for precessional and eccentricity forcing in the Mesozoic. Based on bio‐/chronostratigraphic data, recent studies questioned this particular type of Milankovitch forcing on the Latemar cyclic series. We present an integrated model that incorporates (i) new cyclostratigraphic data, (ii) new and existing bio‐/chronostratigraphic data and (iii) new spectral analyses. The basic shu‐cycles in the Latemar reflect sub‐Milankovitch control. Cycle bundles of 1 : 4–5 (megacycles : shu‐cycles) indicate precessional forcing. They do not reflect eccentricity superimposed on precessional forcing. Spectral analyses reveal highly significant ratios in the large‐scale cycle bundlings. Stacking patterns of 1 : 9.9 and 1 : 24.0 shu‐cycles represent obliquity and short eccentricity forcing. Both sub‐Milankovitch and Milankovitch forcing potentially controlled shallow subtidal carbonate deposition in Mesozoic greenhouse times. Cyclostratigraphic models require an integrated approach including bio‐ and chronostratigraphic data.
Analysis of large DMA from soybean (Glycine max L. Merr.) by pulsed‐field gel electrophoresis
Authors: Honeycutt Rhonda J. , Sobral Bruno W.S. , McClelland Michael , Atherly Alan G.
Published: December 2004

The technique of pulsed‐field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DMA from embedded soybean protoplasts and the conditions for separating large DMA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.
Correlated clustering and virtual display of gene expression patterns in the wheat life cycle by large‐scale statistical analyses of expressed sequence tags
Authors: Ogihara Yasunari , Mochida Keiichi , Nemoto Yasue , Murai Koji , Yamazaki Yukiko , Shin‐I Tadasu , Kohara Yuji
Published: December 2004

Compared to rice, wheat exhibits characteristic growth habits and contains complex genome constituents. To assess global changes in gene expression patterns in the wheat life cycle, we conducted large‐scale analysis of expressed sequence tags (ESTs) in common wheat. Ten wheat tissues were used to construct cDNA libraries: crown and root from 14‐day‐old seedlings; spikelet from early and late flowering stages; spike at the booting stage, heading date and flowering date; pistil at the heading date; and seeds at 10 and 30 days post‐anthesis. Several thousand colonies were randomly selected from each of these 10 cDNA libraries and sequenced from both 5′ and 3′ ends. Consequently, a total of 116 232 sequences were accumulated and classified into 25 971 contigs based on sequence homology. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were identified. Furthermore, relationships of gene expression profiles among the 10 wheat tissues were inferred from global gene expression patterns. Genes with similar functions were grouped with one another by clustering gene expression profiles. This technique might enable estimation of the functions of anonymous genes. Multidimensional analysis of EST data that is analogous to the microarray experiments may offer new approaches to functional genomics of plants.
In vitro analysis of transcription initiation and termination from the Lhcb1 gene family in Nicotiana sylvestris: detection of transcription termination sites
Authors: Hasegawa Keiko , Yukawa Yasusi , Sugiura Masahiro
Published: December 2004

Genes encoding chlorophyll a/b‐binding proteins of photosystem II (Lhcb) constitute a multigene family. Nine Lhcb1 genes have previously been isolated from the tobacco species, Nicotiana sylvestris, and the transcription initiation sites in vivo have been mapped. Reaction conditions from a previously developed in vitro transcription system from tobacco cultured cells were optimized for the Lhcb1 genes. Transcription initiation sites in vitro predominantly coincided with those found in vivo and were typically cytidines, a system unique to N. sylvestris Lhcb1 genes. CTC*A (C* for initiation site) was a consensus motif for the initiation region in vitro, as reported in vivo. Mutation analysis defined functionally that the TATA box is essential for transcription initiation and that the CTCA motif is a determinant of transcription initiation sites but not transcript levels. Polyadenylation sites were determined from in vivo transcripts, located 12–21 nt downstream from likely poly(A) signals. Four major 3′‐ends of in vitro transcripts from Lhcb1*6 were detected, 40–300 nt downstream of the poly(A) site, suggestive of multiple, discrete transcription termination sites. These 3′‐ends are mapped in or nearby T‐rich sequences.
Increased dehydrin promoter activity caused by HvSPY is independent of the ABA response pathway
Authors: Robertson Masumi
Published: December 2004

A barley SPINDLY protein, HvSPY, is a negative regulator of gibberellin (GA) action. It is also found to be a positive regulator of the promoter of a barley dehydrin (Dhn) gene which is abscisic acid (ABA) upregulated. To investigate whether HvSPY acts through the ABA signaling pathway to upregulate the Dhn promoter, functional characterization was carried out by co‐bombardment experiments. These experiments used Dhn promoter‐GUS reporter constructs and an effector construct to overexpress HvSPY protein in barley aleurone. ABA dose–response experiments with and without HvSPY overexpression showed that the induction by HvSPY occurred in addition to the ABA effect. Gibberellic acid (GA3) did not reduce the induction by ABA, but it had a small, although significant, effect on the ability of HvSPY to upregulate. The induction of promoter activity of Dhn by HvSPY required the intact protein, and a small deletion in the tetratricopeptide repeat (TPR) region reduced this ability significantly. When a promoter region containing an element for ABA responsiveness was mutagenized or deleted, the mutant promoters lost ABA responsiveness but remained responsive to HvSPY. In addition, HvSPY did not increase promoter activities of other ABA‐upregulated genes. Taken together, these results indicate that HvSPY and ABA both regulate promoter activity of Dhn, and that HvSPY acts independently of the ABA signaling pathway.
Flower development in carrot CMS plants: mitochondria affect the expression of MADS box genes homologous to GLOBOSA and DEFICIENS
Authors: Linke Bettina , Nothnagel Thomas , Börner Thomas
Published: December 2004

Maternally inherited defects in the formation of male flower organs leading to cytoplasmic male sterility (CMS) indicate an involvement of mitochondrial genes in the control of flower formation. In the ‘carpeloid’ CMS type of carrot, stamens are replaced by carpels. The florets thus resemble well‐investigated homeotic flower mutants of Arabidopsis and Antirrhinum, in which organ identity is impaired because of the mutation of specific nuclear MADS box genes. We have isolated five cDNAs encoding MADS box proteins (DcMADS1–5) from a flower‐specific library of carrot. Structural features deduced from their sequence and transcript patterns in unmodified carrot flowers determined by in situ hybridisation relate them to known MADS box transcription factors involved in specification of flower organs. In ‘carpeloid’ CMS flowers, we detected a distinctly reduced expression of DcMADS2 and DcMADS3, homologues of the Antirrhinum genes GLOBOSA and DEFICIENS. Our data strongly suggest that the ‘carpeloid’ CMS phenotype is caused by a cytoplasmic (mitochondrial) effect on the expression of two MADS box factors specifying organ development at whorls 2 and 3 of carrot flowers.
Automated whole mount localisation techniques for plant seedlings
Authors: Friml Jiří , Benková Eva , Mayer Ulrike , Palme Klaus , Muster Gerhard
Published: December 2004

Plant biology is currently experiencing a growing demand for easy and reliable mRNA and protein localisation techniques. Here, we present novel whole mount in situ hybridisation and immunolocalisation protocols, suitable to localise mRNAs and proteins in Arabidopsis seedlings. We demonstrate that these methods can be used in different organs of Arabidopsis seedlings as well as in other plant species. In order to achieve better reproducibility and higher throughput, we modified these protocols for automation to be performed by a liquid handling robot. In addition, we show that other procedures such as reporter enzyme assays and tissue clearing can be similarly automated. We present examples of application of our protocols including mRNA localisation and proteins and epitope tag (co)localisations which demonstrate that these methods provide reliable and versatile tools for expression, localisation and anatomical studies in plants.
The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis
Authors: Brady Siobhan Mary , Sarkar Sara F. , Bonetta Dario , McCourt Peter
Published: December 2004

Genetic screens have identified a number of genes that regulate abscisic acid (ABA) responsiveness in Arabidopsis. Using a combination of suppressor screens and double mutant analysis, we have determined a genetic relationship for a number of these ABA response loci. Based on germination in the presence of exogenous ABA, the ABI1 and ABI2 phosphatases act at or upstream of the ERA1 farnesyl transferase and the ABI3 and ABI5 transcription factors act at or downstream of ERA1. In contrast with ABI3 and ABI5, the ABI4 transcription factor appears to act at or upstream of ERA1. Based on reporter gene constructs, the upstream regulation of ABI3 by ERA1 occurs at least partially at the level of transcription, suggesting that this lipid modification is required to attenuate ABI3 expression. Similar experiments also indicate that ABI3 is auxin inducible in lateral root primordia. Related to this, loss‐of‐function abi3 alleles show reduced lateral root responsiveness in the presence of auxin and an auxin transport inhibitor, and era1 mutants have increased numbers of lateral roots. These results suggest the possibility that genes identified through ABA responsive germination screens such as ERA1 and ABI3 have functions in auxin action in Arabidopsis.
High‐Cot sequence analysis of the maize genome
Authors: Yuan Yinan , SanMiguel Phillip J. , Bennetzen Jeffrey L.
Published: December 2004

Higher eukaryotic genomes, including those from plants, contain large amounts of repetitive DNA that complicate genome analysis. We have developed a technique based on DNA renaturation which normalizes repetitive DNA, and thereby allows a more efficient outcome for full genome shotgun sequencing. The data indicate that sequencing the unrenatured outcome of a Cot experiment, otherwise known as High‐Cot DNA, enriches genic sequences by more than fourfold in maize, from 5% for a random library to more than 20% for a High‐Cot library. Using this approach, we predict that gene discovery would be greater than 95% and that the number of sequencing runs required to sequence the full gene space in maize would be at least fourfold lower than that required for full‐genome shotgun sequencing.
Transfusion for anaemia in the district hospital setting
Authors: Ridley J. , Revell P. , Ainsworth M. , Pryce A.
Published: December 2004

summary. We examined the practice of transfusion for anaemia in our hospitals using an audit method, with standards derived from guidelines published by the British Committee for Standards in Haematology.

Documentation was generally good, but simple tests were not performed prior to transfusion in many cases when the diagnosis was not known.
Early primary immune response against erythrocytes: a case report
Authors: Albiero A. L. , Novaretti M. C. Z. , Llacer P. E. D. , Chamone D. A. F.
Published: December 2004

summary. Background Primary immune response against red blood cell (RBC) antigens often takes weeks or months to be detected. In previous reports, for children receiving multiple units of blood components, ranging from five to 81 units, the elapsed time between the first RBC transfusion and antibody detection ranged from 18 to 78 days. Cytomegalovirus (CMV) is sometimes associated with immunohaematologic findings and may modulate immune response.

Case report A 24‐week‐old male infant with interstitial pneumonia and hepatitis because of CMV developed an RBC auto antibody and two RBC alloantibodies: anti‐Jka, detected in tube 11 days after a single RBC transfusion, and anti‐K, detected only in papain gel test 18 days later.

Conclusion As anti‐Jka is not a naturally occurring antibody, this is the most rapid primary immune response against an RBC antigen after a single RBC transfusion ever described, in the youngest child ever described.
Therapeutic efficacy of clindamycin in combination with quinine for treating uncomplicated malaria in a village dispensary in Gabon
Authors: Vaillant Michel , Millet Pascal , Luty Adrian , Tshopamba Paul , Lekoulou Faustin , Mayombo Justice , Georges Alain J. , Deloron Philippe
Published: December 2004

The efficacy of a 3‐day clindamycin‐quinine regimen to treat clinical malaria attacks was investigated in 256 children from western Gabon. Treatment was well tolerated by all of the children and its efficacy was higher than 97% by day 20. Thus this 3‐day clindamycin‐quinine regimen might constitute a potential alternative to chloroquine for treating clinical malarial attacks in children from Gabon.
Factors influencing the care‐seeking behaviour of chest symptomatics: a community‐based study involving rural and urban population in Tamil Nadu, South India
Authors: Sudha G. , Nirupa C. , Rajasakthivel M. , Sivasusbramanian S. , Sundaram V. , Bhatt S. , Subramaniam K. , Thiruvalluvan E. , Mathew R. , Renu G. , Santha T.
Published: December 2004

Our aim was to identify the factors that influence the care‐seeking behaviour of chest symptomatics in urban and rural areas in South India. We conducted in‐depth interviews with 649 participants: 80% of 310 urban residents and 63% of 339 rural people had sought care (P < 0.01), 93% within 1 month of onset of symptoms. Private health care facilities were the first and preferred point of contact for 57% of urban and 48% of rural participants; the major reasons were proximity to residence and their perception that good‐quality care would be available there. Symptomatics who did not seek care attributed their inaction to insufficient severity of symptoms (51%), unaffordability (46%) and lack of time due to work pressures (25%). Socio‐economic factors such as literacy and family income significantly influenced care‐seeking behaviour. Our results indicate that most chest symptomatics seek care promptly; their initial response is to go to the nearest private health care facility, shifting to another if they are dissatisfied. Fifty per cent of the participants who did not seek care felt that their symptoms were not severe.
Pharmacodynamics of clemastine in healthy horses
Authors: Bergvall K. , Ingvast Larsson C. , Broström H. , Törneke K.
Published: December 2004

The aim of this study was to evaluate the efficacy of the H1‐receptor inhibitor clemastine (a compound belonging to the first generation of antihistamines) in equine skin after a single intravenous and oral dose. Histamine binds to H1‐receptors involved in pruritus, increased vascular permeability, release of inflammatory mediators and recruitment of inflammatory cells. To counteract these effects antihistamines have classically been used for treatment of allergic hypersensitivity disorders and urticaria in several species including dogs, cats and horses. Six horses received clemastine orally, 200 µg kg−1, by stomach tube and 50 mg kg−1 as an intravenous infusion in a crossover study with crossover design. The ability to reduce wheal formation after intradermal injection of histamine was evaluated. Intradermal injections of 0.07 mL histamine dihydrochloride (0.1 mg mL−1) were given before and at seven time periods ranging from 0.5 to 24 h after clemastine administration. Wheal area was measured 20 min after the injection. Sterile saline (0.07 mL) served as a negative control. There was a marked reduction in wheal size for approximately 6 h after the intravenous administration, indicating that clemastine is distributed to, and effective in inhibiting H1‐receptors in, equine skin. After oral administration the effect was almost negligible, indicating low oral bioavailability of clemastine in the horse.

 The study was funded by Linnea and Axel Ericsson’s Grant fund at the Veterinary Faculty, Swedish University of Agricultural Sciences.
Concurrent hyperadrenocorticism in a minature schnauzer with severe Trichophyton mentagrophytes infection
Authors: Chen C. , Su B.
Published: December 2004

A 4‐year‐old intact female schnauzer dog was presented with a history greater than 1 year of alopecia, thick crusting and secondary seborrhea with mild pruritus. The dog was diagnosed pituitary‐dependant hyperadrenocorticism (PDH) concurrent with severe Trichophyton mentagrophytes infection. The dog showed generalized alopecia, enlarged pendulous abdomen, mild polydipsia, polyuria and polyphagia. Liver enzymes alanine aminotransferase (ALT) and alkaline phosphatase (ALKP) were normal. ACTH stimulation and low‐dose dexamethasone suppression tests were performed prior to therapy. The concentrations of serum cortisol were determined by radioimmunoassay (RIA) and direct enzyme immunoassay (EIA) methods. The results of both assay systems indicated PDH. Fungal cultures showed T. mentagrophytes infection in 4 days. Histological examination of a skin biopsy specimen revealed severe irregular acanthosis accompanied by compact hyperkeratosis, and numerous dermatophytic spores and hyphae heavily colonized in the keratin layer. After 3 months of oral terbinafine (25 mg kg−1, once daily) and mitotane therapy, the hair regrew and the result of an ACTH stimulation test were normal. Three sets of biweekly fungal cultures were negative after 6 months. In this case, terbinafine was given concurrently with mitotane without detectable adverse effects (no abnormal clinical signs were observed and liver enzymes remained within normal ranges). Thus, the combination of terbinafine and mitotane appears to be a safe therapeutic protocol in treatment of PDH with dermatophytosis.

 This study was supported by The Association of Veterinary Dermatology, Taipei.
Systemic candidiasis due to Candida albicans in a dog
Authors: Chen C. , Su C. H. , Lee L. W.
Published: December 2004

In this case resport, we describe pyogranulomatous lymphadenitis due to Candida albicans that progressed to a systemic infection in a 10‐kg, 1‐year‐old spayed female mixed‐breed dog. The dog had bilaterally enlarged submandibular lymph nodes with peripheral oedema. Fine‐needle aspirate of fluid from a lymph node and associated area was collected for fungal culture; a skin biopsy was also performed at the same time. Growth of budding yeast was observed on mycobiotic agar subcultured via inoculation in human serum. The characteristic germ tube of C. albicans was observed. Histopathology revealed chronic pyogranulomatous lymphadenitis with fungal elements. Lymphadenitis due to C. albicans was diagnosed tentatively. The dog was treated initially with itracononazole (30 mg kg−1 orally once daily) without an adequate response; treatment was changed to fluconazole (150 mg every other day). Leucocytosis, lymphocytosis, elevated alanine aminotransferase (ALT) and alkaline phosphataes (ALKP) were observed to wane and wax during 4 months of therapy. The skin lesions did not respond to fluconazole therapy and progressed to involve the subcutaneous scapular and ocular regions, and the dog developed generalized lymphadenopathy. Finally, the dog appeared to seizure and the owner requested euthanasia. At necropsy, gross lesions showed disseminated white spots on the cerebrum, pancreas, liver, kidney, lung, heart and all the lymph nodes, especially the abdominal lymph system. Candida albicans was also cultured from the spinal fluid. No obvious invasion site was noted and the dog did not have a previous history of long‐term usage of corticosteroids and/or antibiotics.

 This study was supported by The Association of Veterinary Dermatology, Taipei.
Prevention of Microsporum canis infection in a cat challenge model
Authors: Moriello K. , Deboer D. J. , Volk L. , Blum J.
Published: December 2004

Oral lufenuron is reportedly an effective treatment for some cats with existing dermatophytosis. We sought to determine if lufenuron, when used as a pre‐treatment prior to challenge exposure and infection, would prospectively protect against development of infection by direct application of Microsporum canis spores. Three groups (n = 6 per group) of juvenile cats were treated with either monthly oral lufenuron (30 or 133 mg kg−1) or placebo. After 2 months of treatment, kittens were challenged using 105Microsporum canis spores applied to the skin under occlusion. Cats were observed weekly, including: Wood’s lamp examination; scoring for scale/crust, erythema, induration; lesion size; and development of satellite lesions. Fungal cultures were performed biweekly. All cats became infected; the infections progressed, and then regressed, in a similar fashion in all groups. There were no consistent statistically significant differences in weekly infection scores throughout the study. Treated cats did not recover faster than untreated cats. We concluded that oral lufenuron at the dosing schedule and conditions used in this study did not prevent dermatophyte infection by direct topical challenge. Further studies are warranted to evaluate alternate dosing schedules, less severe challenge exposures, varying strains of M. canis, and other factors that may be present under field conditions.

 This study was funded by Novartis Animal Health.
Putrescentiae and Lepidoglyphus destructor in normal dogs and dogs with atopic dermatitis
Authors: Mueller R. S. , Fieseler K. V. , Rosychuk R. A. W. , Greenwalt T.
Published: December 2004

The purpose of this study was to evaluate the threshold concentration for the storage mites Tyrophagus putrescentiae and Lepidoglyphus destructor in intradermal testing of healthy and atopic dogs, and to compare the incidence of positive reactions in the two groups. Twenty‐one healthy dogs and twenty‐six atopic dogs were tested intradermally with T. putrescentiae and L. destructor extracts at 1000, 500, 250, 125, 63, 32 and 16 PNU mL−1. Reactions were evaluated objectively and subjectively. A Mann–Whitney U‐test was used to determine differences in grade of reaction to storage mites between healthy dogs and dogs with atopic dermatitis. Positive reactions to storage mite extract were most common at 1000 PNU mL−1, with approximately one‐third of normal and atopic dogs showing a positive reaction to T. putrescentiae and an even higher number to L. destructor. No positive reactions were seen in normal dogs at a concentration of 63 PNU mL−1 of L. destructor extract and 32 PNU mL−1 of T. putrescentiae extract. There was no significant difference in the incidence of positive reactions between normal versus atopic dogs for any of the Tyrophagus or Lepidoglyphus extract concentrations. Based on the findings in this study, the preliminary recommended concentrations for intradermal testing with T. putrescentiae and L. destructor extract are 16 and 32 PNU mL−1, respectively.

 Storage mite extracts were provided by Heska Corporation, Fort Collins, CO.
Effect of recent cropping history and herbicide use on the degradation rates of isoproturon in soils
Authors: Walker A , Austin C R
Published: December 2004

Five soil samples were taken from each of five fields with different crop management histories. Three of the fields were in an arable rotation, the fourth field was temporary grassland, and the final field was under permanent grass. Of the three arable fields, two had been cropped with winter wheat in three of the preceding 6 years, and the third had last been cropped with winter wheat once only, 6 years previously. With one exception, the winter wheat had been sprayed with the herbicide isoproturon. The rate of isoproturon degradation in laboratory incubations was strongly related to the previous management practices. In the five soils from the field that had been treated most regularly with isoproturon in recent years, <2.5% of the initial dose remained after 14 days, indicating considerable enhancement of degradation. In the soils from the field with two applications of the herbicide in the past 6 years, residues after 27 days varied from 5% to 37% of the amount applied. In soils from the other three sites, residue levels were less variable, and were inversely related to microbial biomass. In studies with selected soils from the field that had received three applications of isoproturon in the previous 6 years, kinetics of degradation were not first‐order but were indicative of microbial adaptation, and the average time to 50% loss of the herbicide (DT50) was 7.5 days. In selected soils from the field that had received just one application of isoproturon, degradation followed first‐order kinetics, indicative of cometabolism. Pre‐incubation of isoproturon in soil from the five fields led to significant enhancement of degradation only in the samples from the two fields that had a recent history of isoproturon application.
Evaluation of a New HTLV‐I/II Polymerase Chain Reaction
Authors: Vrielink H. , Zaaijer H. L. , Cuypers H. T. M. , van der Poel C. L. , Woerdeman M. , Lelie P. N. , Winkel C. , Reesink H. W.
Published: December 2004

Aim: Evaluation of a qualitative HTLV‐I/II DNA polymerase chain reaction (PCR) test for the detection of HTLV‐I/II DNA (Roche Diagnostic Systems, Branchburg, N.J., USA) in various panels. Methods: The panels consisted of fresh EDTA blood samples from blood donors who were anti‐HTLV‐I/II ELISA repeatably reactive: 53 were Western blot (WB) positive, 228 were WB indeterminate and 15 were WB negative. Elevent ELISA‐negative blood donors were used as negative controls. Furthermore, specimens from 1 HTLV‐II‐infected intravenous drug user and from 1 HTLV‐II‐infected blood donor were included in the panel. Peripheral blood lymphocytes were prepared by red blood cell lysis with the Roche washing solution and stored at < –23 °C until processing. Amplification products were analyzed with the HTLV‐I/II detection kit. Results: All 53 anti‐HTLV‐I/II ELISA‐ and WB‐positive samples and both HTLV‐II‐positive samples tested positively by PCR. All 228 anti‐HTLV‐I/II ELISA‐positive and WB‐indeterminate, all 15 ELISA‐positive and WB‐negative and all 11 ELISA‐negative control samples tested negative by PCR. Conclusion: The Roche Amplicor HTLV‐I/II test is a simple test, suitable for the confirmation of HTLV‐I and ‐II infection in individuals with indeterminate or positive WB patterns.
Identification of Novel Factor VIII Inhibitor Epitopes using Synthetic Peptide Arrays
Authors: Palmer Douglas S. , Dudani Anil K. , Drouin Jeanne , Ganz Peter R.
Published: December 2004

Objectives: Mapping the antibody‐binding sites on the factor VIII (FVIII) protein opens the prospect of studying the development of FVIII inhibitors and the alteration of inhibitor specificities over time. This paper describes a novel approach to the mapping of FVIII antibody‐binding sites. Methods: Immobilized synthetic peptide arrays covering 80% of the complete 2351 amino acid sequence of factor VIII (FVIII) were used to determine epitope specificity of 6 alloantibodies and 3 autoantibodies inhibitory to FVIII activity. This detailed assessment was carried out using a modified enzyme‐linked immunosorbent assay with plasma from normal persons or hemophilia A patients without inhibitors as negative controls. Results: Antibody‐combining sites could be differentiated in both a qualitative and quantitative manner and were patient‐specific. Highly reactive peptides were restricted to specific sites in the Al‐A3 and Cl‐C2 domains and were not proximal to known proteolytic cleavage sites. Free peptides incubated in vitro with the plasmas of 3 patients significantly reduced residual inhibitor titers in a dose‐dependent manner. Conclusion: This technique permits the study of the development and specificity of FVIII inhibitors, can detect and differentiate between inhibitory and noninhibitory antibodies using immobilized or free peptides respectively, permits correlation of antibody‐combining sites with inhibition of FVIII activity and provides a basis for the development of inhibitor adsorption or neutralization technology.
Flow‐Cytometric Quantitation of Anti‐D Antibodies
Authors: Hildén Jan‐Olof , Backteman Karin , Nilsson Joacim , Ernerudh Jan
Published: December 2004

Objectives: Quantitation of Rh antibodies is important clinically in predicting the risk of hemolytic disease of the newborn. We describe a flow cytometry method for the quantitation of anti‐D antibodies that we developed in parallel to a recently described method. Methods: As a secondary antibody we used whole IgG instead of Fab molecules. The advantages, besides lower cost, include a stronge fluorescence signal with no need for amplification, and the possibility of diluting samples to minimize the risk of agglutination by IgM antibodies. We did extensive studies on reproducibility. Results: Reproducibility was superior to the autoanalyzer method. The two methods were roughly in agreement in estimating low, medium, or high levels of anti‐D with a correlation coefficient of 0.89. The autoanalyzer measures the in vitro agglutination of all anti‐D antibodies whereas flow cytometry measures the amount of IgG anti‐D bound to red cells, which is more like the in vivo situation. Conclusion: Further studies in a clinical setting will show whether flow‐cytometric quantitation may improve the diagnostic value of anti‐D concentration measurement.
Sensitive Determination of the Rh D Genotype in Mixed Samples Using Fluorescence‐Based Polymerase Chain Reaction
Authors: Tonn T. , Westrup D. , Seidl C. , Kirchmaier C. M. , Seifried E.
Published: December 2004

Objectives: Prenatal determination of the fetal RhD status in pregnancies of Rh‐negative (Rh‐neg) women by polymerase chain reaction (PCR) on DNA has become of increasing importance. Since determination of the RhD genotype in these cases is usually performed in samples containing both maternal Rh‐neg and fetal Rh‐neg or Rh‐positive (Rh‐pos) DNA, the sensitivity and specificity of PCR‐based DNA detection are of crucial importance in the diagnosis of the fetal RhD status. Methods: We developed a method for RhD typing using the PCR and a fluorescence‐based detection method that allows us to determine RhD‐pos DNA even if it is mixed with large amounts of Rh‐neg DNA. Results: Using this approach, Rh‐pos DNA could be detected in dilutions with Rh‐neg DNA of as high as 1 in 10,000 (Rh‐pos/Rh‐neg). Furthermore PCR amplification could also be carried out on DNA samples from persons with weak (Dweak) or partial (Dcat) RhD antigens. Conclusion: This method will be valuable in prenatal RhD typing after amniocentesis or after the isolation of fetal cells from maternal peripheral blood.
Report of EPFA/NIBSC Workshop ‘Nucleic Acid Amplification Tests (NAT) for the Detection of Blood‐Borne Viruses’ Held on 31 October 1996 in Amsterdam, The Netherlands
Authors: Rogers P. M. , Saldanha J. , Allain J. P.
Published: December 2004

The 3rd annual European Plasma Fractionators Association/National Institut of Biological Standard and Control (EPFA/NIBSC) meeting provided a forum for regulators, blood product and test kit manufacturers and organisations developing standards to present and discuss their latest data. The main conclusions were as follows. There has been substantial progress during the last year in the development of NAT technology specifically for improving the safety of blood products though there is an urgent need for the development of international reference materials. The technology is not yet sufficiently developed to be used as a routine screening test though testing of plasma pools for hepatitis C virus may be achieved within a year. Introduction of testing should not result in the creation of dual standards for plasma derived and cellular products. Once the technology is fully developed it could significantly improve the safety of all blood products, particularly those derived from starting materials with a high incidence of viral markers.
Gel Test Application for IgG Subclass Detection in Auto‐Immune Haemolytic Anaemia
Authors: Fabijańska‐Mitek J. , Θopieńska H. , ¿upańska B.
Published: December 2004

Background and objectives: Commercially available gel test microtubes are not available with antisera for the determination of IgG subclasses. The aim of this study was to adapt the gel technique for this purpose and apply it to the investigation of patients with autoimmune haemolytic anaemia (AIHA). Materials and methods: We studied 66 red cell samples from 49 patients with AIHA of the warm‐active IgG type. Standard serologic and haematologic methods were used. We adapted the DiaMed gel test by using IgG‐subclass antisera. Results: We found the adapted test useful in determining the subclass of autoantibodies in eluates. We could identify the IgG subclass in all the AIHA patients, even those that were ‘Coombs‐negative’, with less than 200 IgG molecules bound in vivo per red cell. Comparison of the gel test with the standard spin tube test and the microtiter plate test showed the superiority of the gel test, i.e., detection of IgG subclasses was much better than with the tube test and the results were more clear‐cut than those of the microplate test. The gel test is also the simplest and least time‐consuming and permits a later reading of results. Application of the test in our 66 samples confirmed that IgG1 was the most frequent (96%). In 59% of the cases it was accompanied by IgG of other subclasses. Multiple subclasses were most common in the cases with stronger in vivo IgG red cell sensitization and severe haemolysis. Accompanying IgG3 was detected only in patients with obvious haemolysis. Conclusion: The gel test is more sensitive than other procedures for the determination of IgG subclass and has the advantages of simplicity, rapidity, low cost, and stability of the agglutinates.
Increased in vitro Immunosuppressive Action of Anti‐CMV and Anti‐HBs Intravenous Immunoglobulins due to Higher Amounts of Interferon‐Gamma Specific Neutralizing Antibodies
Authors: Denys C. , Toungouz M. , Dupont E.
Published: December 2004

Background and objectives: We previously found that interferon‐γ (IFN‐γ) antibodies in intravenous immunoglobulins (IVIG) can block not only IFN‐γ production and tumor necrosis factor‐α secretion, but also T‐cell proliferation. Since the presence of IFN‐γ antibodies has been attributed to previous viral infection, we hypothesized that the viral status of the plasma donors used for IVIG pools might be a decisive factor in controlling the immunosuppressive capacity of IVIG. Materials and methods: We tested three different pooled, human IVIG preparations for the presence of IFN‐γ antibodies by ELISA. Results: Comparison of the immunomodulatory activity of polyvalent IVIG with that of specific CMV and HBs IVIG showed that the latter had higher levels of IFN‐γ antibodies and an increased capacity to block mixed lymphocyte reaction and cytokine production. Conclusion: We propose that these in vitro assays constitute a basis for the selection of plasma intended for manufacturing IVIG aimed at immunosuppression in the transplant setting.
Prestorage Leukocyte Reduction with In‐Line Filtration of Whole Blood: Evaluation of Red Cells and Plasma Storage
Authors: Rapaille André , Moore Glenn , Siquet Jean , Flament Jocelyne , Sondag‐Thull Danièle
Published: December 2004

Objectives: Prestorage filtration of blood components appears to be an effective method to reduce leukocyte‐induced adverse reactions and other complications. To determine whether it is better to filter whole blood before component separation, we compared the efficiency of in‐line filtration of whole blood with that of postseparation filtration. Methods: Blood was collected from normal, healthy donors into either regular triple‐bag containers or into whole‐blood integral‐filter container systems. We then compared the in vitro storage values of leukocyte‐depleted red blood cell concentrates (RBCC) kept at 4 °C, and plasma frozen for 1 year with nonfiltered blood components as control. Results: All counts of white blood cells after filtration were < 1 × 106 per unit. For almost all storage parameters no significant differences were found between leukocyte‐reduced RBCC and control units. The plasma fibrinopeptide A values below 30 ng/ml prior to freezing indicate that filtration does not activate the coagulation factors. Furthermore, the filtration did not influence either the biological values or the coagulation factors of plasma units. Conclusions: Whole blood filtration prior to component preparation seems to offer a useful alternative technique for obtaining leukocyte‐reduced RBCC and plasma.
Transfusion‐Related Acute Lung Injury following Random Donor Platelet Transfusion: A Report of Two Cases
Authors: Ramanathan Ramesh K. , Triulzi Darrell J. , Logan Theodore F.
Published: December 2004

Objectives: Transfusion‐related acute lung injury (TRALI) following random donor platelet (RDP) transfusion is a rare complication of transfusion without any well‐documented case reported in the English language literature. We describe 2 patients in whom TRALI occurred following RDP transfusion. Methods: Conventional clinical and laboratory methods. Results: Both patients developed acute shortness of breath 30–60 min after completion of RDP transfusion and required mechanical ventilatory support. Chest X‐ray (CXR) in both cases revealed bilateral pulmonary infiltrates. Patient 1 required vasopressors for hypotension. Right heart catheterization ruled out fluid overload. Patient 2 remained hemodynamically stable. Both patients improved rapidly with continued respiratory support and were extubated within 48 h. CXR at this time showed clearing of infiltrates. In both cases a granulocyte antibody was identified in the plasma of a platelet donor supporting the diagnosis of TRALI. Conclusions: In suspected cases of TRALI, HLA and granulocyte antibody testing is indicated for the recipients and for donors of implicated components. Implicated donors need not be excluded from the donor pool, but can be used for fractionated plasma and plasma‐free components.
Consensus Statement from the Consensus Conference on Unrelated‐Donor Bone Marrow Transplants – 29 and 30 October 1996
Authors: Franklin I. M.
Published: December 2004

Bone marrow transplants from unrelated donors for leukaemias are increasing greatly in number and also in proportion to matched sibling‐donor transplants. The panel has considered unrelated donor transplant (UD‐BMT) on the basis of efficacy, toxicity and indications in leukaemias. The conclusions and statements are based largely but not exclusively on information provided at the Consensus Conference.