bluuebunny/crossref_metadata_2025 huggingface dataset:
Psychometric testing of the Chinese version of nursing practice environment scale
Authors: Chiang Hui‐Ying , Lin Shu‐Yuan
Published: November 2008

Aims.  To test the reliability and validity of the Chinese version of the Nursing Practice Environment Scale (C‐NPES) translated from the Lake’s Practice Environment Scale of the Nursing Work Index and to explore nurses’ perceptions of the nursing work environments in Taiwan

Background.  Magnet hospitals are characterised by professional autonomy, control over nursing practice, adequacy of staffing, supportive management and effective interdisciplinary relationships, which are successful in attracting and retaining nurses. In Taiwan, this concept has gained importance since the SARS crisis of 2003. However, there have been limited Chinese instruments based on magnet hospital traits to explore Taiwan’s nursing work environment.

Design.  This study was designed as a cross‐sectional survey. Purposive convenience samples of 842 nurses were recruited from five acute hospitals in Taiwan. Internal consistency reliability, content validity using expert review, construct validity using factor analysis and criterion‐related validity were examined.

Results.  The Cronbach’s alphas were 0·90 for the total scale and 0·87–0·65 for the subscales. The validity was obtained using a content validity index and principal component analysis of five‐factor structure (variance explained 47·89%). The criterion‐related validity was supportive of the turnover rate (t = 7·84, p < 0·001). The participants disagreed on staffing and resource adequacy and participation in hospital affairs but agreed on professional development.

Conclusion.  The preliminary psychometric properties of C‐NPES have been established. Considering cultural appropriateness, the subscales of staffing and participation in hospital affairs need advanced modification.

Relevance to clinical practice.  The C‐NPES will provide hospital administrators with an overview of magnet hospital settings for nursing practice. It is beneficial for the stabilisation of the nursing workforce as well as for the optimisation of nursing work environments. Additionally, the use of professional development programs to enhance nurses’ knowledge of SARS prevention is favorable.
A multi‐centre comparison of nursing staff time required for the preparation and administration of liposomal amphotericin B and amphotericin B deoxycholate vs. voriconazole
Authors: Flynn Elizabeth , Marciniak Anne , Barbabietola Giuliana , Oppenheim Beryl A , Roberts Craig , Barker Kenneth
Published: November 2008

Aims and objectives.  To compare the nursing time and cost required for preparation and administration of liposomal amphotericin B, amphotericin B deoxycholate and voriconazole.

Design.  Cost comparison study.

Methods.  Nurse activities associated with the preparation and administration of the three study drugs were divided into 11 tasks and timed by observers at five hospitals. Target tasks were defined as those likely to be affected by the differences between drugs and excluded those tasks likely to differ owing to site‐specific factors. Mean times for administration of a single day of therapy for each study drug were compared. Costs of preparation and administration of a 14‐day regimen were estimated.

Results.  Sixty‐nine patients were observed receiving a total of 256 doses of study medications. Labour times were 20, 16, 14 and 3 minutes per day for liposomal amphotericin B, amphotericin B deoxycholate, intravenous voriconazole and oral voriconazole, respectively. Administration time was significantly lower for intravenous voriconazole compared with liposomal amphotericin B (p < 0·05), and for oral voriconazole compared with all intravenous regimens (p < 0·05). Preparation of medications took the longest time for intravenous formulations and was longer for liposomal amphotericin B than for the other drugs by 3–5 minutes. Average non‐drug costs associated with preparation and administration of a 14‐day regimen were greatest in the amphotericin B deoxycholate arm at US$ 335, followed by liposomal amphotericin B (US$ 310) and voriconazole (US$ 180).

Conclusion.  Intravenous voriconazole required less time to prepare and administer on a daily basis than liposomal amphotericin B, and was similar to amphotericin B deoxycholate. Measurements of intravenous vs. oral voriconazole administration suggest the opportunity to save 10–17 minutes per day with the oral formulation.

Relevance to clinical practice.  Oral voriconazole may provide significant savings in terms of nursing time compared with intravenous antifungal drugs.
‘I don’t know how we coped before’: a study of respite care for children in the home and hospice
Authors: Eaton Nicola
Published: November 2008

Aim and objective.  To describe the experiences of families, whose children have life‐limiting and life‐threatening conditions and who have complex healthcare needs, of receiving respite care at home or in a hospice.

Background.  Respite provision is an extremely important service in assisting families to cope with the extra stresses and problems of coping with children with complex healthcare needs. There are different issues when the venue is home or a hospice.

Design.  Semi‐structured interviews were carried out with families of children with complex healthcare needs, receiving respite care at home or in a hospice.

Methods.  A convenience sample of 11 families was interviewed using an interview schedule, exploring their experiences of the service and their views on the service.

Results.  The areas of concern identified as significant to all the families were referral to respite service, service organisation, communication, relinquishing control to respite carers and satisfaction with service.

Conclusion.  Within the provision of respite care, there needs to be more overt referral systems and criteria, negotiation of appropriate roles, continuity of care, regular assessment of need and acknowledgement of the difficulty, which parents have in relinquishing control to respite carers.

Relevance to clinical practice.  High‐quality respite care for families involves more than just organising a respite session. Healthcare professionals organising and providing care could manage a service more effectively, if taking the above issues into consideration.
Navigating the maze: parents’ views and influence on a paediatric haematology and oncology day care service
Authors: Lowe Rochelle , Bravery Karen , Gibson Faith
Published: November 2008

Aim. To examine with families their views on the service they receive in a Paediatric Haematology/Oncology Day Care Unit.

Background. National policy emphasises the importance of involving children, young people and families in the planning of services they are receiving. To be responsive to real, as opposed to perceived needs, health care professionals are actively seeking ways to capture the voice of the families they care for through a process of consultation and action.

Design. Focus groups.

Method. A series of focus groups were undertaken with 16 families who were either on treatment or within six months of completion of treatment for acute lymphoblastic leukaemia. Tape‐ and note‐based analysis was initially undertaken and the data sorted by means of an overview grid. Data were then analysed by the research team through comparison and agreement of final themes.

Results. The majority of families were satisfied with the care they received. Six themes are described that capture their combined thoughts on their overall experience; navigating the maze, communication, continuity of care, environment, waiting and organisation of care.

Conclusions. Focus groups are an effective means of obtaining data from service users. Group discussion allowed for the sharing and development of ideas to be incorporated into developments within the service.

Relevance to clinical practice. Increasingly, children, young people and their families are being cared for within Paediatric Haematology/Oncology Day Care units, it is, therefore, vital that this aspect of care is designed, co‐ordinated and delivered around the needs of the family. This can only be achieved by listening to the stories of those families who use our service to confirm what it is that works well and what areas of care may need to be enhanced.
An ethnography of a children’s renal unit: experiences of children and young people with long‐term renal illness
Authors: Lindsay Waters Anne
Published: November 2008

Aim.  This study explored the experience of long‐term renal illness, including issues concerning compliance with treatment, from the perspectives of children and young people.

Background.  Children and young people have difficulty complying with renal treatment; research in this area usually focuses on their compliance with a narrow range of behaviours: compliance with dialysis, tablet taking and dietary and fluid restrictions. Renal compliance studies are usually premised on the assumption that children will automatically comply with treatment and/or that blame for non‐compliance rests with the child and family.

Design.  The study, underpinned by a view of children as competent social agents, used ethnography to explore and describe the everyday experience of long‐term renal illness.

Method.  Ethnographic fieldwork – involving participant observation, informal interviews and the use of children’s drawings – took place over 16 months in a hospital renal unit comprising an inpatient ward, a haemodialysis unit and an outpatient clinic. The primary participants were 13 children; 14 carers and 36 staff members also participated. Data were interrogated through descriptive and thematic forms of analysis.

Results.  This study reveals participants’ perspectives on the physical and social impact of renal treatment. It also provides a conceptualisation of life with long‐term renal illness that highlights children’s management of ‘illness labour’, their inhabitation ‘renal geographical space’, ‘a renal body’ and a ‘renal social world’.

Conclusions.  These findings show how compliance with renal treatment involves particular embodied experiences that are hard for children to manage. Traditional views of compliance/non‐compliance render children’s embodied experience and their labour in managing dialysis, thirst and their difficulties with tablet taking irrelevant, whilst this study shows these as highly relevant to children.

Relevance to clinical practice.  The conceptualisation of children’s experience arising in this study provides a new way of considering the embodied experience of children with long‐term renal illness.
An analysis of job search behaviour of inactive nurses in Japan
Authors: Kawaguchi Akira , Yasukawa Fumiaki , Matsuda Yoichiro
Published: November 2008

Aim.  The aim of this paper is to examine inactive nurses’ job‐searching behaviours to return to healthcare workplace at where serious difficulty of meeting demand and supply of nurses exist.

Design and methods.  To clarify the factors that would make their attempts easy or difficult, successful or not, we employed econometric method to analyse the probability of job finding in terms of both the length of duration and the quality of consequence in job‐seeking behaviour by using microdata pooled on Japan Nursing Association Computer System (NCCS), in which inactive nurses’ individual attributes (gender, age, academic background, the types of qualifications, the number of preschool aged children, etc) are stated.

Results.  From the estimations, we found that (i) having preschool children is one of the factors of delaying job finding, and failing to obtain desired workplace and working style; (ii) nurses with higher educational backgrounds tend to spend a longer time in finding jobs and (iii) training programme for the returners may not work well in case of shorter career blank, while may support for those who have longer one.

Relevance to clinical practice.  To meet the desired demand for qualified nurses in healthcare facilities, we should (i) develop more supportive working environment for nurses with preschool children, such as more flexible working time and shifts; (ii) encourage the employment of higher educated nurses by reforming compensation scheme, as well as reconsidering of the standard of skill‐mix; and (iii) redevelop training programme for returners with more practical contents.
Extensive population expansion of Pedicularis longiflora (Orobanchaceae) on the Qinghai‐Tibetan Plateau and its correlation with the Quaternary climate change
Authors: YANG FU‐SHENG , LI YU‐FEI , DING XIN , WANG XIAO‐QUAN
Published: November 2008

The Qinghai‐Tibetan Plateau (QTP) is thought to be more strongly affected by the Quaternary glaciations than most other regions of the same latitude. It would be of great interest to investigate the population genetic structure of organisms distributed on the platform and its correlation with the Quaternary climatic oscillations. Here we used the chloroplast (cp)DNA trnT‐trnF sequence to study genetic variation and phylogeography of Pedicularis longiflora, an alpine herb with extensive distribution on the QTP. Based on a range‐wide sampling comprising 41 populations and 910 individuals, we detected 30 cpDNA haplotypes that were divided into five clades by phylogenetic and network analyses and a strong phylogeographical structure. All haplotypes but one in the three basal clades occur exclusively in the southeast QTP, whereas haplotypes in the young clade V occupy almost the whole species range. In particular, the young haplotype H18 occurs in 420 individuals, even at a frequency of 100% in some QTP platform populations and the Altai population. The haplotype distribution pattern, together with molecular clock estimation and mismatch distribution analysis, suggests that the southeast QTP was either a refuge for P. longiflora during the Quaternary climatic change or is the place of origin of the species. The present wide distribution of the species on the QTP platform has resulted from recent population expansions which could be dated back to 120 000–17 000 years ago, a period mostly before the last glacial maximum. The possible relationships among geographic genetic structure, climatic change and species diversification in Pedicularis are also discussed.
dhfr and dhps genotype and sulfadoxine‐pyrimethamine treatment failure in children with falciparum malaria in the Democratic Republic of Congo
Authors: Alker Alisa P. , Kazadi Walter M. , Kutelemeni Albert K. , Bloland Peter B. , Tshefu Antoinette K. , Meshnick Steven R.
Published: November 2008

Objective  To determine the relationship between mutations in dhfr and dhps and SP treatment failure in Plasmodium falciparum malaria in the Democratic Republic of the Congo (DRC).

Methods  Therapeutic efficacy trial was conducted in Rutshuru, Eastern DRC, between June and September 2002, comparing sulfadoxine‐pyrimethamine (SP), SP plus amodiaquine (AQSP) and artesunate plus SP (ASSP) regimens for treating malaria in children under 5 years old. We genotyped 212 samples for mutations associated with SP resistance and investigated their association with treatment failure.

Results  In the SP arm, 61% of the subjects experienced treatment failure after 14 days. The failure rate was lower in the combination arms (AQSP: 32%, ASSP: 21%). The dhfr‐108 and dhfr‐51 mutations were nearly universal while 89% of the samples had at least one additional mutation at dhfr‐59, dhps‐437 or dhps‐540. Dhps mutations had a bigger impact on treatment failure in children with high parasite density: for children with a parasite density <45 000 parasites/μl, the risk of treatment failure was 37% for mutations at dhps‐437 and dhps‐540 mutation and 21% for neither mutation [risk difference (RD) = 17%, 95% CI: −3%, 36%]. In children with a parasite density >45 000 parasites/μl, the treatment failure risk was 58% and 8% for children with both mutations or neither mutation, respectively (RD = 51%, 95% CI: 34%, 67%).

Conclusions  Dhps‐437 and dhps‐540 are strongly associated with SP treatment failure and should be evaluated further as a method for surveillance of SP‐based therapy in DRC.
Health economics of asthma: assessing the value of asthma interventions
Authors: Campbell J. D. , Spackman D. E. , Sullivan S. D.
Published: November 2008

The aim of this systematic review was to summarize and assess the quality of asthma intervention health economic studies from 2002 to 2007, compare the study findings with clinical management guidelines, and suggest avenues for future improvement of asthma health economic studies. Forty of the 177 studies met our inclusion criteria. We assessed the quality of studies using The Quality of Health Economic Studies validated instrument (total score range: 0–100). Six studies (15%) had quality category 2, 26 studies (65%) achieved quality category 3, and the remaining eight (20%) studies were scored as the highest quality level, category 4. Overall, the findings from this review are in line with the Global Initiative for Asthma clinical guidelines. Many asthma health economic studies lacked appropriate long term time horizons to match the chronic nature of the disease and suffered from using effectiveness measures that did not capture all disease related risks and benefits. We recommend that new asthma simulation models: be flexible to allow for long term time horizons, focus on using levels of asthma control in their structure, and estimate both long term asthma specific outcomes like well‐controlled time as well as generic outcomes such as quality adjusted survival.
Latent manganese deficiency increases transpiration in barley (Hordeum vulgare)
Authors: Hebbern Christopher A. , Laursen Kristian Holst , Ladegaard Anne H. , Schmidt Sidsel B. , Pedas Pai , Bruhn Dan , Schjoerring Jan K. , Wulfsohn Dvoralai , Husted Søren
Published: November 2008

To investigate if latent manganese (Mn) deficiency leads to increased transpiration, barley plants were grown for 10 weeks in hydroponics with daily additions of Mn in the low nM range. The Mn‐starved plants did not exhibit visual leaf symptoms of Mn deficiency, but Chl a fluorescence measurements revealed that the quantum yield efficiency of PSII (Fv/Fm) was reduced from 0.83 in Mn‐sufficient control plants to below 0.5 in Mn‐starved plants. Leaf Mn concentrations declined from 30 to 7 μg Mn g−1 dry weight in control and Mn‐starved plants, respectively. Mn‐starved plants had up to four‐fold higher transpiration than control plants. Stomatal closure and opening upon light/dark transitions took place at the same rate in both Mn treatments, but the nocturnal leaf conductance for water vapour was still twice as high in Mn‐starved plants compared with the control. The observed increase in transpiration was substantiated by 13C‐isotope discrimination analysis and gravimetric measurement of the water consumption, showing significantly lower water use efficiency in Mn‐starved plants. The extractable wax content of leaves of Mn‐starved plants was approximately 40% lower than that in control plants, and it is concluded that the increased leaf conductance and higher transpirational water loss are correlated with a reduction in the epicuticular wax layer under Mn deficiency.
Characterization of cDNA clones for porcine α(l,3)galactosyl transferase: The enzyme generating the Galα(l,3)Gal epitope
Authors: Sandrin Mauro S. , Dabkowski Paul L. , Henning Margaret M. , Mouhtouris Effie , McKenzie Ian F.C.
Published: November 2008

Abstract: Galoα(l,3)Gal is a terminal carbohydrate found on many glycosylated cell surface molecules of species other than humans and Old World monkeys, and is produced by the α(l,3)galactosyl transferase enzyme's adding galactose to a substrate. We have previously shown, by the transfection of COS cells with the cloned mouse α(l,3)galactosyl transferase, that most human anti‐pig antibodies react with Galα(l,3)Gal. Using cross‐species hybridization with the mouse α(l,3)galactosyl transferase cDNA, bacteriophage λ, gt11 and λgt10 pig cDNA libraries were screened and overlapping clones isolated which encode the pig α(l,3)galactosyl transferase. Sequencing of the clones demonstrated a single open reading frame coding for a protein with high homology to murine (75% identity) and bovine (82% identity) α(l,3)galactosyl transferases. Southern blot analysis shows the porcine α(l,3)galactosyl transferase gene to be a single copy gene, and northern analysis demonstrated an mRNA of 3.9 kb. After splicing the clones to produce a single full length clone, transfection of Galα(l,3)Gal‐ COS cells led to strong reactivity with human serum and with the IB4 lectin (which reacts only with Galα(l,3)Gal), indicating that the expression of the transferase led to the expression of Galα(l,3)Gal. The cloning of the cDNA gene for the pig α(l,3)galactosyl transferase is the first step in the production of a transgenic pig lacking the transferase and therefore the Galα(l,3)Gal epitope; such animals could serve as donors for human transplantation.
Limited specificity of xenoantibodies in diabetic patients transplanted with fetal porcine islet cell clusters. Main antibody reactivity against α‐linked galactose‐containing epitopes
Authors: Satake Masahiro , Kawagishi Naoki , Rydberg Lennart , Samuelsson Bo E. , Tibell Annika , Groth Carl‐Gustav , Möller Erna
Published: November 2008

Abstract:The immunological specificity of antibodies formed as a result of xenotransplantation of fetal porcine islet‐like cell clusters to diabetic patients was characterized. High titer increases were recorded against porcine cells, solubilized membrane fractions of porcine cells, and purified MHC class I antigens. However, titer increases were also noted against ssDNA and dsDNA and against pig thyroglobulin but not against actin, myoglobin, or haptenated BSA. Antibody titers against tetanus toxoid were unaffected. The reactivity against porcine RBC could be completely blocked by absorption with pig thyroglobulin. Since pig thyroglobulin contains the galαl‐3gal antigen, the reactivity against RBC was most probably mainly due to antibodies against this oligosaccharide epitope. The reactivity against porcine mononuclear cells was only partially absorbed by pig thyroglobulin, indicating a heterogeneity of the clonal response. These conclusions were substantiated by data showing that the antigenic determinants on pig thyroglobulin were completely destroyed by treatment with α‐ but not with β‐galactosidase. Further studies showed that immune reactivity against pig RBCs, platelets, islet‐cells, endothelial cells and pig MHC class I molecules, caused by xenoimmunization, was almost completely blocked by either absorption of antibodies on a column of Sepharose beads coated with galα1‐3gal or by pretreatment of the antigen fractions with α‐galactosidase. Western blot experiments revealed that both the natural and xenoimmune antibodies reacted with a large number of different glycoproteins. There was no difference in heterogeneity of the response when comparisons were made between pre‐and posttransplantation sera, nor was there any difference of patterns caused by IgM or IgG antibodies. Absorption studies revealed that this epitope was present in a large number of different glycoproteins, a conclusion verified by staining with the eluate from a thyroglobulin‐immunosorbent column or with the eluate from the galα1‐3gal‐coupled Sepharose column. Our findings demonstrate that the xenoimmune response is mainly directed against oligosaccharide residues and that there is a limited clonal heterogeneity of antibodies in xenografted patients. There was no direct evidence of reactivity against any porcine proteins nor of specific immunization against porcine MHC peptides. The data form a basis for a future understanding of means to cope with and prevent the rejection of xenogeneic islet cells in man. We also suggest, based on the specificity found in xenoimmunized patients sera, that a major part of naturally occurring antibodies might be specifically reactive with carbohydrate antigens.
Cytolytic effect of human anti‐Gal IgM and complement on porcine endothelial cells: A kinetic analysis
Authors: Borche Luis , Thibaudeau Karen , Navenot Jean‐Marc , Soulillou Jean‐Paul , Blanchard Dominique
Published: November 2008

Abstract: The binding of human natural antibodies to porcine endothelial cells is the first step leading to activation of complement and lysis of the cells. The kinetic of incorporation of propidium iodide into porcine endothelial cell line SVPAEC/6A or peripheral blood lymphocytes was investigated to monitor the permeabilization of target cells by membrane attack complex. In less than 5 min, more than 90% of porcine cells were highly fluorescent upon incubation with human sera, showing they incorporate propidium iodide. The lysis occurred neither with adult sera pretreated with dithiothreitol nor with umbilical cord sera, suggesting that lytic antibodies belong to the IgM class. By using antibodies purified from human sera, the bulk of lytic activity was found to be associated with IgM specific for the Galal‐3Gal epitope, whereas IgG were not able to lyse the porcine cells. These antibodies could be readily absorbed on rabbit red blood cells known to express the main epitope target of xeno human antibodies. Finally, our results show that permeabilization of porcine endothelial cells is a very early phenomenon, probably directly associated with the biological process responsible for the hyperacute rejection.
Natural antibodies do not inhibit xenogeneic transplantation of human PBL in lethally irradiated mice
Authors: Kulova Lydia , Denes Laszlo , Lubin Ido , Marcus Hadar , David Magda , Fire Gil , Reisner Yair
Published: November 2008

Abstract: Attempts to transfer human peripheral blood lymphocytes (hu‐PBL) into lethally irradiated mice resulted in limited engraftment in recipients lacking natural antibodies (nAb) and could not be achieved in immunologically normal mice. It has been proposed that nAb with antihuman specificity play a major role in the rejection of the hu‐PBL graft. In the present study we demonstrate that, following intensification of the conditioning protocol (thymectomy, supralethal dose of TBI, and radioprotection with bone marrow for donors with severe combined immune deficiency (SCID), transplants of 50 to 70 × 106 hu‐PBL were successfully engrafted in BALB/c, CBA/J and C3H/HeJ mice—regardless of the initial high levels of nAb. The percentage of human CD45+ cells in peritoneal lavage was not statistically different from that obtained in congenitally immune‐deficient corresponding strains (SCID and CBA/N) lacking natural antibodies. Significant differences in engraftment of hu‐PBL, between different human donors, were related neither to the nAb content (r = 0.29) nor to the ABO(H) blood group. The transfer of serum with high level of nAb into SCID and CBA/N mice or incubation of hu‐PBL in such a serum prior to implantation, did not impede the engraftment and did not decrease the production of human immunoglobulins. These data demonstrate that the presence of nAb in supralethally irradiated normal mice does not inhibit the engraftment of hu‐PBL, emphasizing the role of cellular mediated mechanisms in xenograft rejection.
Microbiological characterization of porcine fetal islet‐like cell clusters for intended clinical xenografting
Authors: Bjöersdorff A. , Korsgren O. , Feinstein R. , Andersson A. , Tollemar J. , Malmborg A.‐S. , Ehrnst A. , Groth C.G.
Published: November 2008

Abstract:A major concern in animal‐to‐man transplantation is the risk of transferring microorganisms from an animal to a patient. For this reason, before transplanting the porcine fetal pancreas to diabetic patients, the pregnant sows and the tissue to be transplanted—i.e., fetal islet‐like cell clusters (ICC) prepared by collagenase digestion and cell culture—were subjected to a comprehensive microbiological screening program. Serological testing of the pregnant sows revealed antibodies toLeptospira interrogans(2/59 sows, 3%) andAspergillus fumigatus(23/41, 56%). However, there was no evidence of colonization of the animals with these organisms. Serological testing for a multitude of other microorganisms was negative. Growth of bacteria was found in 4% of the specimens recovered from the endometrium and in 31% of those from the amniotic fluid. However, none of these bacterial species was recovered in the culture dishes containing ICC. A few samples taken from the culture dishes (2/53, 4%) revealed growth of bacteria, but no viruses, fungi, or mycoplasmas were detected. In nude mice that had carried ICC under the kidney capsule for 9 to 23 months, various lesions were observed, but none of them was due to the implanted porcine tissue. Microscopic examination of the mouse brains revealed no prion‐related alterations. If porcine tissue is to be transplanted into humans, a stringent microbiological screening program must be followed. The program used by us makes possible the weeding out of seropositive animals, as required, and the exclusion of contaminated tissue.
Method for mass separation and morphologic and functional qualification of adult porcine pancreatic islets
Authors: Basta Giuseppe , Osticioli Luca , Tortoioli Cristina , Rossodivita Maria Elena , Brunetti Paolo , Calafiore Riccardo
Published: November 2008

Abstract: We upgraded and implemented our original procedure for mass retrieval, morphologic, and in vitro/in vivo functional characterization and maintenance of adult porcine pancreatic islets (PPI). We addressed, in particular, donor pig pancreas selection and perfected the organ digestion technique to obtain considerably higher yield in large‐sized, intact, viable, and functional PPI. We attempted to define criteria for PPI qualification, in terms of better analysis of their in vitro and preliminary in vivo physiologic characteristics, as well as to study culture maintenance conditions in order to understand potential impact on future employment of this islet type.
Transplantation of organ cultured fetal pig pancreas in non‐obese diabetic (NOD) mice and primates (Macaca fascicularis)
Authors: Mandel TE , Koulmanda Maria , Kovarik J. , Georgiou H.M. , Francis D.M.A. , Dawson P. , Stainsby G.
Published: November 2008

Abstract: Xenografts of organ cultured fetal pig pancreas in prediabetic NOD mice can survive for prolonged periods (>20 weeks) in recipients treated with anti‐T cell monoclonal antibodies (MAb) directed against host CD4 and CD3 cell surface molecules. Anti‐CD4 MAb treatment alone is only partly effective and xenograft rejection occurs over a period of many weeks. In diabetic recipients, by contrast, recurrence of autoimmune disease in isografts is rapid (<28 days) despite similar depletion of CD4+ve T cells. In spontaneously diabetic NOD mice immunosuppressed with anti‐CD3 and anti‐CD4 MAbs xenograft function occurs and the recipient's blood glucose levels fall into the pig range. Organ cultured fetal pig pancreas transplanted into cynomolgus monkeys is rapidly but not hyperacutely rejected when azathioprine‐cyclosporin A‐prednisolone immunosuppression is used. Anti‐T‐cell MAb treatment is now being studied in this primate model.
Xenotransplantation of pig kidneys to nonhuman primates: I. Development of the model
Authors: Sablinski Tomasz , Latinne Dominique , Gianello Pierre , Ballln Michael , Bergen Kate , Colvin Robert B. , Foleys Alicia , Hong Han‐Zhou , Lorf Thomas , Meehan Shane , Monroy Rod , Powelson John A. , Sykess Megan , Tanaka Mayumi , Cosimi A. Benedict , Sachs David H.
Published: November 2008

Abstract:Long‐term survival of discordant xenografts will require control of both humoral and cellular aspects of the immune response. Because cellular responses to xenografts appear to be more potent than those encountered by allografts, recipients of xenogeneic tissues are likely to require more immunosuppression, with potential for unacceptably high complication rates. We have therefore directed our attention toward the induction of tolerance to some or all of the xenogeneic antigens recognized in the primate anti‐pig cellular immune response, utilizing mixed lymphohematopoietic chimerism as the means for tolerance induction. We report here our initial series of 16 animals in which the conditions for application of this treatment regimen were established. In 14 cynomolgus monkey recipients, induction therapy consisted of low dose whole body irradiation, thymic irradiation, and ATG, followed by infusion of pig bone marrow and a pig kidney transplant. Other aspects of the regimen included splenectomy and removal of monkey anti‐porcine natural antibodies by extracorporeal perfusion of the recipient's blood through a pig liver. Two control animals received either no treatment or extracorporeal perfusion but no additional induction therapy. Five of the experimental animals were treated posttransplant with an anti‐IgM monoclonal antibody, five with Cyclosporin A, and two with a combination of both immunosuppressants. Both IgM and IgG natural antibodies were removed effectively by liver perfusion in all but one monkey, as determined by flow cytometry. Antibody liters remained low for 5–7 days, but increased progressively thereafter. The longest kidney survival in this series was 13 days, in an animal which maintained excellent kidney function for the first 11 days posttransplant. Peripheral chimerism was detected only transiently on day 10 in the peripheral blood of this recipient. We conclude that extracorporeal perfusion by this technique removes natural antibodies and prevents hyperacute rejection, permitting maintenance of excellent renal xenograft function for at least 11 days. Additional manipulations appear to be required to achieve mixed chimerism and tolerance of the cellular immune system in this model.
Immunoselection of human lymphocytes producing xenoreactive natural antibodies against Galocl,3Gal terminal residues
Authors: Pitre Joël , Möller Erna , Satake Masahiro
Published: November 2008

Abstract:In the pig‐to‐human xenograft combination, human xenoreactive natural antibodies (XNA) bind to carbohydrate moieties, especially those with Galα1,3Gal terminal residues, expressed on the surface of most cells. The aim of this study was to select the cells that produce XNA by functionally characterizing a subset of cells for future studies on immunosuppression of XNA formation. We especially addressed the question whether XNA could be produced by CD5+ B lymphocytes, a subset of cells producing antibodies of high connectivity and polyreactivity, possibly involved in autoimmune processes. For that purpose we used porcine thyroglobulin (PTg), which expresses several Galα,3Gal terminal residues, for the immunoselection of XNA‐producing cells. On FACS analysis, biotinylated PTg appeared to react with 5% of the B lymphocytes but the proportion of CD5+ B lymphocytes was not enriched in the PTg‐reactive population. Similarly, magnetic beads coated with PTg were used to sort lymphocytes with Ig receptors recognizing PTg. Two and one‐half percent of cells reacted, mainly B lymphocytes (89%), but they were not enriched for CD5+ B lymphocytes. When PTg‐reactive lymphocytes were cultivated in presence of irradiated T cells and stimulated with PWM, the synthesis of Galα1,3Gal reactive antibodies, mainly of the IgG class, was demonstrated. The results of this study suggest that a high percentage of B lymphocytes react with Galα1,3Gal terminal residues. Such XNA‐producing cells are not particularly common in the CD5+ subset.
Evidence that double transfection to xenoendothelial cells using GPI‐anchoring complement regulatory factor (DAF and HRF20) genes is useful for the inhibition of human complement‐mediated cytolysis
Authors: Hayashi Shuji , Emi Nobuhiko , Isobe Ken‐Ichi , Okada Hidechika , Yokoyama Itsuo , Takagi Hiroshi
Published: November 2008

Abstract: It has been known that xenogeneic cells transfected with regulator of complement activation (RCA) molecules such as DAF (CD55), MCP (CD46) and HRF20 (CD59) resist the lysis by the human complement. However, it has not been demonstrated that the induction of double RCA molecules is more effective than that of single RCA molecule to inhibit the human complement attack. In this study, the authors compared the effect on the protection from complement‐mediated lysis between single transfection and double transfection of DAF and HRF20 cDNA to bovine aortic endothelial cells (BAEC) using retro viral vector.

BAEC transfected with DAF or HRF20 cDNA resisted the lysis by human serum in parallel with the intensity of antigen expression, whereas BAEC transfected with both DAF and HRF20 cDNA resisted the lysis by human serum with anti‐BAEC antibody, not human serum alone, more effectively than those with DAF cDNA alone. We conclude that the xenogeneic cells doubly transfected with both DAF and HRF20 cDNA are protected from complement mediated lysis more effectively than those singly transfected with DAF cDNA alone.
Antibodies to human adhesion molecules and their ligands: Cross‐species reactivity and potential application in xenotransplantation
Authors: Carter Anthony S. , Welsh Kenneth I. , Morris Peter J. , Fuggle Susan V.
Published: November 2008

Abstract: Given the renewed interest in the possibility of using animals as an alternative source of organs for transplantation, it would be useful to have tools to study and manipulate the inflammatory response to a xenograft. With this in mind, we have screened cynomolgus monkey and porcine tissue with 37 antibodies specific for human leukocyte differentiation antigens and adhesion molecules. The cynomolgus monkey, an old world primate, is a concordant species with respect to humans, in contrast to the pig, which is a discordant species.

The cross‐species reactivities of the antibodies tested fall into three groups: i) no crossreactivity; ii) crossreactivity with a tissue distribution similar to that in the human; iii) crossreactivity, but with a distribution different from that in the human. Those antibodies directed against E‐ and P‐selectin, ICAM‐1 (intercellular adhesion molecule‐1) and VCAM‐1 (vascular cell adhesion molecule‐1), which were reactive with monkey tissue, had a similar distribution to that seen in human tissue, but were unreactive with the porcine tissue tested. While an anti‐CD31 antibody detected a conserved epitope on endothelium, species differences were apparent in leukocyte reactivity. One of the antibodies directed against CD 18 reacted with leukocytes in all three species, whereas the other antibody detected an epitope present on porcine muscle/connective tissue. While antibodies to VLA‐4 (Very Late Antigen‐4) detected a small number of leukocytes in the kidney, they also reacted with the Bowman's capsule in the kidney and matrix protein/connective tissue in the lymph node.

This study indicates that when antibodies react across species, some epitopes recognized in the old world nonhuman primates may have a distribution similar to those detected in human tissue, whereas in more distant species such as the pig, in many instances the epitope is present on entirely different structures. Nevertheless, the absence of crossreactivity of human reagents with porcine tissue may allow targeting of molecules in a species‐specific manner, allowing their use for diagnostic or therapeutic purposes.
A line of transgenic pigs in which the expression of human decay‐accelerating factor by endothelial cells is increased in the presence of inflammatory stimuli
Authors: Carrington Christine A. , Richards Andrew C. , Tucker Alexander W. , Peters Anna L. , White David J.G.
Published: November 2008

Abstract: Aortic endothelial cell cultures (PAE) from four lines of pigs transgenic for human decay‐accelerating factor (hDAF) have been used to study the response to the inflammatory stimuli bacterial lipopolysaccharide (LPS) and recombinant human TNF‐α. Human umbilical vein endothelial cells (HUVEC) and PAE from normal, non‐transgenic pigs were used as controls. The expression of hDAF and E‐selectin on the cell surface was determined by flow cytometry. After overnight incubation, HUVEC, normal and transgenic PAE increased the relative expression of E‐selectin 2–5‐fold in response to LPS (25 μg/ml), and 5–40‐fold in response to TNF‐α(10 ng/ml). In both normal and transgenic PAE the increase in expression of E‐selectin in response to TNF‐α was maximal at 4 hr and significantly decreased after 20 hr. There was no significant increase in DAF expression by HUVECs in response to LPS or TNF‐α, and three of the four lines of transgenic pigs studied did not increase expression of hDAF in response to either stimulus. However, endothelial cells from the transgenic line A74 exhibited a dose‐dependent increase in expression of hDAF in response to LPS and TNF‐α. A study of the time course of up‐regulation triggered by incubation with TNF‐α showed that, in contrast to the up‐regulation of E‐selectin, hDAF expression continued to increase for at least 3 days. This response may afford additional protection to organs from this line of transgenic pigs.
Manipulation of the anti‐αGal antibody‐αGal epitope system in experimental discordant xenotransplantation
Authors: Cooper D.K.C. , Koren E. , Oriol B.
Published: November 2008

Abstract:In 1991, evidence was provided to indicate that hyperacute rejection of pig organs by humans and baboons was initiated by the binding of anti‐agalactosyl (αGal) antibodies to the vascular endothelium. A search for suitable donor species that do not express aGal epitopes has demonstrated their absence in birds, including ratites (e.g., ostrich, emu) and a reptile (alligator), and very weak expression in a large rodent (the capybara). Studies on ratites would suggest that they are unlikely to be suitable organ donors for humans. In 1991, it was also proposed that anti‐aGal antibody‐antigen binding could be prevented either by depletion of antibody using an immunoaffinity column of the specific aGal oligosaccharide, or by the continuous intravenous infusion of the oligosaccharide, leading to binding (and thus inactivation) of the antibody to the aGal hapten. Synthetic aGal oligosaccharides have recently become available in sufficient quantities to allow experience to be gained with immunoaffinity columns, which have demonstrated complete, but temporary, elimination of baboon serum cytotoxicity to pig PK15 cells in vivo. A search for a natural and inexpensive source of aGal has revealed a subfraction of porcine stomach mucin that demonstrates high potency in inhibiting anti‐aGal antibodies both in vitro and in vivo. As an alternative therapeutic option, anti‐idiotypic antibodies have been produced in mice that are specific for human and baboon anti‐aGal antibodies. Their intravenous administration to baboons results in immediate reduction in serum cytotoxicity for 24 hr. Their use in delivering a toxin, such as ricin, to the B lymphocytes that produce the antibody (and express the same idiotype) is suggested. Studies on complement depletion by the administration of purified cobra venom factor (CVF) to baboons have demonstrated that, even in the absence of measurable levels of CH50, both Clq and properdin are deposited on the vascular endothelium, and that IgM, IgG, and IgA bind to the endothelium of a transplanted pig heart. The prior identification of anti‐αGal IgM, IgG, and IgA by our group suggests that these antibodies might be playing a role in both classical (by IgM and IgG) and alternative (by IgA) complement pathway activation in this experimental model and in pig‐to‐human xenotransplantation. CVF contains a terminal aGal structure, and its administration to an otherwise unmodified baboon leads to a massive increase in anti‐αGal level, suggesting that anti‐CVF antibodies may, at least in part, comprise anti‐aGal. The important role of anti‐aGal antibodies in xenograft rejection has now been clearly established, and techniques for preventing expression of aGal epitopes in donor animals, either by genetic engineering or by gene therapy, are discussed.
The potential role of xenogeneic antigen‐presenting cells in T‐cell co‐stimulation
Authors: Restifo Anthony C. , Ivis‐Woodward Maria A. , Tran Hanh M. , Nickerson Peter W. , Lehnert Anne M. , Strom Terry B. , Chapman Jeremy R. , O'Connell Philip J.
Published: November 2008

Abstract: CTLA4‐Fc is a chimeric murine construct consisting of the CD28 homologue CTLA4 and the constant portion of the heavy chain of mouse IgG2a, with a potential to suppress cellular xeno‐immune responses. The aim of this study was to determine the degree of binding of CTLA4 to B7 ligands on cells of different species and to use CTLA4‐Fc as a tool for the study of cross‐species CD28‐B7 interactions. As assessed by flow cytometry, CTLA4‐Fc bound to mouse L‐cells and human Epstein Barr virus (EBV) transformed lymphoblastoid cells and concanavalin A (Con A) or LPS‐stimulated peripheral blood mononuclear cells (PBMC) or splenocytes from rat, dog, and pig. CTLA4‐Fc inhibited the proliferation of Con A‐stimulated PBMC or splenocytes from mouse, rat, dog, and pig, in a dose‐dependent fashion with approximately 80% inhibition at a concentration of 10 μg/ml. It did not inhibit the proliferation of Con A‐stimulated human PBMC, although it did inhibit the human versus human, and human versus pig primed mixed lymphocyte culture (MLC) in a dose‐dependent fashion. At submitogenic concentrations, purified human T‐cells did not proliferate after incubation with Con A alone. However, proliferation occurred with the addition of B7 positive L‐cells or pig PBMC, but not B7‐negative OKT4 cells. Furthermore, CTLA4‐Fc inhibited proliferation in a dose‐dependent fashion. CTLA4‐Fc bound to all species tested and resulted in inhibition of Con A‐stimulated proliferation in these species, except for humans. Human T‐cells proliferated in response to co‐stimulation with xenogeneic B7, and this could be inhibited by CTLA4‐Fc, suggesting that xenogeneic B7 was capable of providing a functionally significant co‐stimulatory signal necessary for human T cell activation in vitro.
The effect of plasmapheresis and deoxyspergualin or cyclophosphamide treatment on anti‐porcine Gal‐α(1,3)‐Gal antibody levels in humans
Authors: Gannedahl G. , Tufveson G. , Sundberg B. , Groth C.G.
Published: November 2008

Abstract: The levels of human natural anti‐porcine antibodies were followed in six HLA‐immunized patients treated with plasmapheresis (PF) in combination with either 15‐deoxyspergualin (DSG) or cyclophosphamide (CYP). PF was carried out three times a week for 4 weeks, and DSG was administered in a dose of 5 mg/kg following every third PF. CYP was given in a daily dose of 1–1.5 mg/kg. Four patients were randomized to DSG treatment and two were given CYP. Following the last (4th) DSG infusion, all patients continued on CYP treatment. Levels of antibodies toward porcine thyroglobulin were measured in an ELISA assay; thyroglobulin carries the major target for human anti‐porcine natural antibodies, Gal‐α(1,3)‐Gal determinant.

In all patients there was a drop in antibody levels. However, the antibodies never disappeared. The drop was more pronounced and more rapid in the DSG‐treated group, but following the end of the PF series, antibody levels in both treatment groups rapidly returned to baseline or above baseline. In two patients in the DSG group, treatment had to be stopped because of side effects. No adverse reactions were noted in the CYP group.

It is concluded that plasmapheresis in conjunction with DSG seems more effective than CYP treatment in reducing the levels of anti‐porcine Gal‐α(1,3)‐Gal antibody. However, the dose of DSG should be reduced to lower the risk of side effects.