Exogenous administration of protease-resistant, non-matrix-binding IGFBP-2 inhibits tumour growth in a murine model of breast cancer.

BACKGROUND: Insulin-like growth factors (IGF-I and IGF-II) signal via the type 1 IGF receptor (IGF-1R) and IGF-II also activates the insulin receptor isoform A (IR-A). Signalling via both receptors promotes tumour growth, survival and metastasis. In some instances IGF-II action via the IR-A also promotes resistance to anti-IGF-1R inhibitors. This study assessed the efficacy of two novel modified IGF-binding protein-2 (IGFBP-2) proteins that were designed to sequester both IGFs. The two modified IGFBP-2 proteins were either protease resistant alone or also lacked the ability to bind extracellular matrix (ECM). METHODS: The modified IGFBP-2 proteins were tested in vitro for their abilities to inhibit cancer cell proliferation and in vivo to inhibit MCF-7 breast tumour xenograft growth. RESULTS: Both mutants retained low nanomolar affinity for IGF-I and IGF-II (0.8-2.1-fold lower than IGFBP-2) and inhibited cancer cell proliferation in vitro. However, the combined protease resistant, non-matrix-binding mutant was more effective in inhibiting MCF-7 tumour xenograft growth and led to inhibition of angiogenesis. CONCLUSIONS: By removing protease cleavage and matrix-binding sites, modified IGFBP-2 was effective in inhibiting tumour growth and reducing tumour angiogenesis.

Ex vivo restimulation of human PBMC expands a CD3+CD4-CD8- γδ+ T cell population that can confound the evaluation of CD4 and CD8 T cell responses to vaccination.

The measurement of vaccine-induced humoral and CD4(+) and CD8(+) cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3(+)CD4(-)CD8(-) γδ (+) T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3(+)CD4(-)CD8(-) γδ (+) T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4(+) and CD8(+) immune responses following vaccination, the CD3(+)CD4(-)CD8(-) γδ (+) T cells are either excluded or separately enumerated from the overall frequency determination.

The impact of COVID-19 on major trauma (ISS>15) in London, across its four Level 1 centres.

INTRODUCTION: The COVID-19 pandemic has led to reconfiguration of healthcare resources to manage increased demand for acute hospital beds and intensive care places. Concerns were raised regarding continuing provision of critical care for non-COVID patients during the pandemic. The aim of this study was to assess the impact of the COVID-19 pandemic on patients admitted with major trauma (Injury Severity Score >15) across the four Level 1 trauma centres in London. METHODS: Data were collected from all four major trauma centres (MTCs) in London using the Trauma Audit and Research Network database and from local databases at each centre. A 2-month period from 5 March to 5 May 2020 was selected and the same period during 2019 was used to compare changes due to the pandemic. RESULTS: There was a 31% decrease in overall number of patients presenting to the four MTCs during the COVID-19 period compared with 2019. There was no difference in patient demographics or mechanism of injury between the two periods. Sports-related injuries and proportion of self-presentation to hospital were reduced slightly during the pandemic, although the differences were not statistically significant. The mortality rate and association between mortality and injury severity were similar. Proportion of patients requiring intensive care unit facilities also did not change. CONCLUSION: Despite diversion of critical care resources to deal with COVID-related admissions, we did not observe a change in mortality rate or proportion of severely injured patients requiring critical care. Our results suggest London MTCs were able to provide their usual standard of care for critically injured major trauma (Injury Severity Score >15) patients during the pandemic.

A Prospective Study of Cosmetic Outcomes for Patients Treated with Breast Conservation and Radiation Therapy using Shorter Fractionation Schedules at Auckland Hospital, New Zealand.

AIMS: Breast cosmesis is an important outcome for women after breast conservation. It has been shown to be correlated with better patient satisfaction, sexuality and self-esteem. This study reports the subjective and objective breast cosmesis outcomes for women treated at Auckland Hospital Radiation Oncology Department using hypofractionated radiation therapy. MATERIALS AND METHODS: Breast cosmesis was evaluated using a subjective questionnaire and standardised photographs before radiation, at 6 weeks, 1 year, 3 years and 5 years after radiation. Objective evaluation of the photographs and completion of questionnaires were undertaken by clinicians at the same time points. The questionnaire evaluated global cosmesis on a four-point scale - excellent (E), good (G), fair (F) and poor (P). Patient, tumour and treatment factors were evaluated to assess the impact on cosmesis. RESULTS: Three hundred and fifty-eight patients completed the baseline assessments and two hundred and two patients completed the 5-year assessments. Sixty-eight per cent of patients at baseline and 70% at 5 years scored their cosmesis as E/G compared with 52% and 51%, respectively, by clinician assessment. Age >50 years, separation ≤25 cm, non-diabetic, T1 tumours, node negative, quadrant of the scar, no boost and no adjuvant endocrine therapy were correlated with E/G cosmesis. On multivariate analysis at baseline, the individual breast factors that were significantly associated with a score of E/G cosmesis were breast shape (P = 0.028) and scar appearance (P = 0.001). At 5 years, breast shape (P = 0.003), nipple shape (P = 0.019) and scar appearance (P = 0.001) were found to be significant. CONCLUSION: This study shows that most women reported no significant change in their breast cosmesis after hypofractionated radiation treatment over 5 years and that a number of patient, tumour and treatment factors may impact on breast cosmesis.

Microbiological and functional outcomes after open extremity fractures sustained overseas: The experience of a UK level I trauma centre.

BACKGROUND: Open extremity fractures carry a high risk of limb loss and poor functional outcomes. Transfer of extremity trauma patients from developing countries and areas of conflict adds further layers of complexity due to challenges in the delivery of adequate care. The combination of extensive injuries, transfer delays and complex microbiology presents unique challenges. METHODS: A retrospective review was conducted to analyse the surgical and microbiological themes of patients with open extremity fractures transferred from overseas to our institution (Imperial College NHS Trust) between January 2011 and January 2016. RESULTS: Twenty civilian patients with 21 open extremity fractures were referred to our unit from 11 different countries. All patients had poly-microbial wound contamination on initial surveillance cultures. Five patients (25%) underwent amputation depending on the extent of osseous injury; positive surveillance cultures did not preclude limb reconstruction, with seven patients undergoing complex reconstruction and eight undergoing simple reconstruction to achievewound coverage. Hundred percent of patients demonstrated infection-free fracture union on discharge. CONCLUSION: Patients with open extremity fractures transferred from overseas present the unique challenge of poly-microbial infection in addition to extensive traumatic wounds. Favourable outcomes can be achieved despite positive microbiological findings on tissue culture with adequate antimicrobial therapy. The decision to salvage the limb and the complexity of reconstruction used should be based on the chance of achieving meaningful functional recovery, mainly determined by the extent of bony injury. The complexity of reconstruction was based on the predicted long-term functionality of the salvaged limb.

The treatment of segmental tibial fractures: does patient preference differ from surgeon choice?

INTRODUCTION: Segmental tibial fractures are complex injuries with a prolonged recovery time. Current definitive treatment options include intramedullary fixation or a circular external fixator. However, there is uncertainty as to which surgical option is preferable and there are no sufficiently rigorous multi-centre trials that have answered this question. The objective of this study was to determine whether patient and surgeon opinion was permissive for a randomised controlled trial (RCT) comparing intramedullary nailing to the application of a circular external fixator. MATERIALS AND METHODS: A convenience questionnaire survey of attending surgeons was conducted during the United Kingdom's Orthopaedic Trauma Society annual meeting 2017 to determine the treatment modalities used for a segmental tibial fracture (n=63). Patient opinion was obtained from clinical patients who had been treated for a segmental tibial fracture as part of a patient and public involvement focus group with questions covering the domains of surgical preference, treatment expectations, outcome, the consent process and follow-up regime (n=5). RESULTS: Based on the surgeon survey, 39% routinely use circular frame fixation following segmental tibial fracture compared to 61% who use nail fixation. Nail fixation was reported as the treatment of choice for a closed injury in a healthy patient in 81% of surgeons, and by 86% for a patient with a closed fracture who was obese. Twenty-one percent reported that they would use a nail for an open segmental tibia fracture in diabetics who smoked, whilst 57% would opt for a nail for a closed injury with compartment syndrome, and only 27% would use a nail for an open segmental injury in a young fit sports person. The patient and public preference exercise identified that sleep, early functional outcomes and psychosocial measures of outcomes are important. CONCLUSION: We concluded that a RCT comparing definitive fixation with an intramedullary nail and a circular external fixator is justified as there remains uncertainty on the optimal surgical management for segmental tibial fractures. Furthermore, psychosocial factors and early post-operative outcomes should be reported as core outcome measures as part of such a trial.

Management of severe open tibial fractures: the need for combined orthopaedic and plastic surgical treatment in specialist centres.

Although it is widely accepted that grade IIIB open tibial fractures require combined specialised orthopaedic and plastic surgery, the majority of patients in the UK initially present to local hospitals without access to specialised trauma facilities. The aim of this study was to compare the outcome of patients presenting directly to a specialist centre (primary group) with that of patients initially managed at local centres (tertiary group). We reviewed 73 consecutive grade IIIB open tibial shaft fractures with a mean follow-up of 14 months (8 to 48). There were 26 fractures in the primary and 47 in the tertiary group. The initial skeletal fixation required revision in 22 (47%) of the tertiary patients. Although there was no statistically-significant relationship between flap timing and flap failure, all the failures (6 of 63; 9.5%) occurred in the tertiary group. The overall mean time to union of 28 weeks was not influenced by the type of skeletal fixation. Deep infection occurred in 8.5% of patients, but there were no persistently infected fractures. The infection rate was not increased in those patients debrided more than six hours after injury. The limb salvage rate was 93%. The mean limb functional score was 74% of that of the normal limb. At review, 67% of patients had returned to employment, with a further 10% considering a return after rehabilitation. The times to union, infection rates and Enneking limb reconstruction scores were not statistically different between the primary and tertiary groups. The increased complications and revision surgery encountered in the tertiary group suggest that severe open tibial fractures should be referred directly to specialist centres for simultaneous combined management by orthopaedic and plastic surgeons.

Improving the care of patients with severe open fractures of the tibia: the effect of the introduction of Major Trauma Networks and national guidelines.

AIMS: The management of open lower limb fractures in the United Kingdom has evolved over the last ten years with the introduction of major trauma networks (MTNs), the publication of standards of care and the wide acceptance of a combined orthopaedic and plastic surgical approach to management. The aims of this study were to report recent changes in outcome of open tibial fractures following the implementation of these changes. PATIENTS AND METHODS: Data on all patients with an open tibial fracture presenting to a major trauma centre between 2011 and 2012 were collected prospectively. The treatment and outcomes of the 65 Gustilo Anderson Grade III B tibial fractures were compared with historical data from the same unit. RESULTS: The volume of cases, the proportion of patients directly admitted and undergoing first debridement in a major trauma centre all increased. The rate of limb salvage was maintained at 94% and a successful limb reconstruction rate of 98.5% was achieved. The rate of deep bone infection improved to 1.6% (one patient) in the follow-up period. CONCLUSION: The reasons for these improvements are multifactorial, but the major trauma network facilitating early presentation to the major trauma centre, senior orthopaedic and plastic surgical involvement at every stage and proactive microbiological management, may be important factors. TAKE HOME MESSAGE: This study demonstrates that a systemised trauma network combined with evidence based practice can lead to improvements in patient care.

Anti-xenograft immune responses in alpha 1,3-galactosyltransferase knock-out mice.

Although originally generated to test the effect of eliminating the alpha-Gal epitope on HAR, it is becoming increasingly clear that GalT KO mice offer a convenient and inexpensive model to investigate many aspects of the anti-xenorgraft immune response. Clearly, not all aspects of anti-xenograft rejection responses are identical in mice and primates, which should be kept in mind when interpreting results of GalT KO mouse studies. However, with this and other mouse models it is possible to test a large number of variables, which is impractical for both logistical and financial reasons with primates. Furthermore the short gestation time and large litter size of mice means that genetic strategies targeting different aspects of the anti-xenograft immune response can be combined and subsequently tested to identify the optimal combination of genetic and therapeutic approaches to achieve long term xenograft survival. In this regard the GalT KO mouse has been and will continue to be a valuable small animal model for the study of all facets of xenograft rejection involving anti-Gal antibodies.

Xenotransplantation: an update.

Over the past 20 years, the mortality and morbidity associated with cardiac allotransplantation has fallen significantly, providing a viable treatment for patients with terminal cardiac failure. Unfortunately, the increase in the number of patients who could benefit from cardiac transplantation has not been matched with an increase in the number of organs available for transplantation. Thus, many patients with cardiac failure die waiting for a suitable organ, unlike patients with renal failure, who can be maintained on dialysis while waiting for a kidney. Although the development of artificial hearts may provide a life-sustaining bridging therapy until a donor organ becomes available, the quality of life associated with cardiac prostheses is currently less than that following successful cardiac allotransplantation.

Identification of genes downregulated in the thymus by cyclosporin-A: preliminary characterization of clone CSA-19.

Using a novel subtractive hybridization approach, we have identified a set of cDNA clones whose expression is downregulated in the thymus by cyclosporin-A (CsA). A number of regulated genes were identified, but the major focus of this report is the clone termed CsA-19. In the adult mouse, CsA-19 is expressed at high levels in lymphoid organs, particularly in secondary lymphoid organs such as lymph node and spleen. CsA-19 expression was also found to be regulated in the brain and liver during mouse development. The full length sequences of the murine and human CsA-19 cDNAs have been determined. Both are 700 nucleotides in length and encode a predicted product of 217 amino acids. Comparison of the full length cDNA sequences of murine and human CsA-19 reveal that they are greater than 91% conserved. At the predicted amino acid level the homology is even greater, with only two amino acid differences in 217 residues (99% identity). The high degree of homology between murine and human CsA-19 indicates that has there been strong evolutionary pressure to conserve the amino acid sequence of CsA-19, and suggests that CsA-19 may play a critical role both during embryogenesis and in mature lymphoid cells.

Inhibition of NF-kappaB activation by a dominant-negative mutant of IkappaBalpha.

The activity of the transcription factor NF-kappaB is tightly regulated by the inhibitory molecule IkappaBalpha. Upon stimulation, IkappaBalpha is rapidly degraded and NF-kappaB translocates to the nucleus to induce gene expression. The IkappaBalpha degradation is preceded by phosphorylation, suggesting that this event plays a role in the activation of NF-kappaB. In this study, we have mutated three potential phosphorylation sites in porcine IkappaBalpha and found that expression of the Ser32 mutant of IkappaBalpha (IS32A), but not Tyr42 or Ser262 mutants or wild-type IkappaBalpha, blocked the activation of NF-kappaB by TNF-alpha. These results suggest that the Ser32 residue, a potential casein kinase II phosphorylation site, is critical for NF-kappaB activation.

Involvement of both the classical and alternate pathways of complement in an ex vivo model of xenograft rejection.

BACKGROUND: It is now generally accepted that complement activation is critical for the hyperacute rejection of xenografts. Activation of the classical pathway as the result of the interaction of xenoreactive IgM xenoantibodies with the vascular endothelium has been observed in all species combinations examined to date. A number of studies using a variety of species combinations have also implicated alternate pathway involvement; however, these studies do not enable a conclusion to be drawn as to whether the alternate pathway can be activated in the complete absence of classical pathway activation. METHODS: In this study, human plasma was depleted of both Clq and factor D and then reconstituted with purified Clq or factor D to restore the classical and alternate complement pathways, respectively. The ability of these modified plasmas to prosecute hyperacute rejection was then examined using an ex vivo isolated mouse heart perfusion model based on the Langendorff system. RESULTS AND CONCLUSIONS: In the mouse to human species combination, both the classical and alternate pathways of complement are independently capable of initiating complement activation and mediating xenograft rejection.

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement.

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.

Changes in cell surface glycosylation in alpha1,3-galactosyltransferase knockout and alpha1,2-fucosyltransferase transgenic mice.

BACKGROUND: Inactivation of the alpha1,3-galactosyltransferase (GalT) gene by homologous recombination (knockout [KO] mice) and competition for the enzyme's N-acetyllactosamine substrate by transgenically expressed alpha1,2-fucosyltransferase (H-transferase) are two genetic approaches to elimination of the Gal alpha1,3Gal (alphaGal) epitope, which is the major xenoantigen in pigs against which humans have preformed antibodies. Such genetic manipulations often have unpredictable results. METHODS: A panel of 19 selected lectins was used to characterize the changes in cell surface glycosylation in GalT KO and H-transferase transgenic mice, compared with nontransgenic littermate controls. RESULTS: GalT KO mice showed complete elimination of the alphaGal epitope, as reported previously. Surprisingly, however, this was associated with only a modest increase in N-acetyllactosamine residues and had little other effect on the pattern of lectin binding. In contrast, the pattern of lectin binding to H-transferase transgenic mouse cells was more profoundly disturbed and indicated, in addition to the expected expression of H substance and suppression of the alphaGal epitope, that there was a marked reduction in alpha2,3-sialylation and exposure of the normally cryptic antigens, sialylated Tn and Forssman antigens. Similar changes in lectin reactivity with porcine aortic endothelial cells were induced by neuraminidase treatment. CONCLUSIONS: Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

Combination of decay-accelerating factor expression and alpha1,3-galactosyltransferase knockout affords added protection from human complement-mediated injury.

BACKGROUND: Hyperacute rejection (HAR) currently prevents the use of pigs as organ donors for humans. It is now generally accepted that the key instigators of HAR are naturally occurring xenoantibodies against the terminal disaccharide galactose alpha1,3-galactose (Gal), and the species incompatibility between human complement and porcine complement regulatory molecules. Using two in vitro models and an ex vivo mouse heart perfusion model, we have shown previously that cells and tissues from Gal knockout (Gal KO) and transgenic mice expressing the human cell surface complement regulator decay-accelerating factor (DAF/CD55) are partially, but not completely, protected from human complement-mediated injury. METHODS: In the present study, Gal KO mice were crossed with DAF transgenic mice and bred to homozygosity (DAF/Gal KO). Isolated splenocytes were incubated with human serum, and the protective effect of DAF and Gal KO was assessed by measuring complement deposition and cell lysis. Hearts perfused ex vivo with human plasma were examined for human antibody and complement deposition, and assessed functionally by measuring work performed by the heart. RESULTS: Splenocytes from DAF/Gal KO mice were found to be more resistant to complement-mediated injury than cells from either DAF transgenic or Gal KO mice. In addition, hearts from DAF/Gal KO mice, when perfused with human plasma, displayed prolonged survival compared with hearts from Gal KO mice. This was associated with a reduction in the extent of endothelial deposition of IgG, IgM, and complement C3b. CONCLUSIONS: These findings demonstrate that expression of human DAF in association with elimination of the Gal epitope provides added protection from complement-mediated injury in these models of HAR.

High-level endothelial expression of human CD59 prolongs heart function in an ex vivo model of xenograft rejection.

BACKGROUND: Hyperacute rejection of discordant xenografts is dependent on activation of the complement system of the recipient. Transgenic expression of recipient complement regulatory factors in donor tissue has proved to be a promising approach to dealing with hyperacute rejection, although the relationship between the level of complement regulatory factor expression and the degree of protection is not well established. Here, we examine this relationship using CD59 transgenic mouse hearts in an ex vivo model of xenograft rejection. METHODS: The level of expression of CD59 in two lines of transgenic mice, in which CD59 is expressed under the control of either the murine H2Kb (MHC class I) promoter (line CA-17) or the endothelium-specific human intercellular adhesion molecule-2 promoter (line 237-7), was compared by immunohistochemistry and flow cytometry. Hearts from both groups and wild-type controls were perfused ex vivo with human plasma, and mean heart work for each group was compared over a 60-min period. RESULTS: CD59 expression on cardiac endothelial cells isolated from homozygous CA-17 mice was 25- to 30-fold lower than that on cardiac endothelial cells from heterozygous 237-7 mice. CA-17 hearts perfused with 6% human plasma exhibited a reduction in deposition of the membrane attack complex, but not a prolongation of function, compared with nontransgenic mouse hearts. In contrast, 237-7 hearts showed significantly prolonged function during perfusion with 20% plasma. CONCLUSIONS: High-level endothelial-specific expression of CD59 was effective in prolonging the function of mouse hearts perfused with 20% human plasma, whereas low-level, broader expression did not provide protection from 6% plasma.

Transgenic expression of human alpha1,2-fucosyltransferase (H-transferase) prolongs mouse heart survival in an ex vivo model of xenograft rejection.

BACKGROUND: The expression of human alpha1,2-fucosyltransferase (H-transferase, HT) has been proposed as an alternative strategy to alpha1,3-galactosyltransferase (GT) gene knockout, which is not currently feasible in pigs, to reduce the galactose-alpha1,3-galactose (Gal) epitope expression. HT expression has recently been shown in transgenic mice and pigs to significantly reduce Gal expression on a variety of cells; however, its ability to do so on endothelial cells and its effectiveness at prolonging xenograft survival are yet to be determined. METHODS: HT-transgenic, Gal knockout (Gal KO) mice, and mice containing both genetic modifications (HT-transgenic/Gal KO) were tested for H-substance and Gal expression on splenocytes and endothelial cells by flow cytometric analysis. In addition, the hearts of these mice were perfused ex vivo with 6% human plasma, and the effect on cardiac function was determined. RESULTS AND CONCLUSION: H-substance expression was detected on both splenocytes and endothelial cells of HT-transgenic mice. The level of H-substance expression was not affected by the presence or absence of GT in the cells, consistent with HT being dominant over GT. The ability of HT expression to reduce Gal expression was highly variable depending on the cell type. Gal expression on splenocytes was almost completely eliminated, whereas on endothelial cells, substantial Gal remained despite a 70% reduction. When perfused ex vivo with human plasma, hearts from HT-transgenic, Gal KO, and HT-transgenic/Gal KO mice demonstrated a similar prolongation in survival, compared with wild-type controls. Therefore, as far as hyperacute rejection is concerned, HT expression may be as effective as Gal KO in protecting against xenoantibody and complement mediated injury. However, the effect of residual Gal on non-hyperacute rejection responses remains to be determined.

Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhesion molecule 2 promoter.

Genetic engineering of donor animals in xenotransplantation research has been directed largely toward obtaining expression of various immunoregulatory molecules on vascular endothelium, the initial target of recipient antibody and complement. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. Using the promoter of the gene for intercellular adhesion molecule 2 (ICAM-2), which is constitutively expressed on all vascular endothelium, we have developed a system for endothelial cell gene targeting in vivo. A 334-basepair fragment from the 5' flanking region of the human ICAM-2 gene was used to drive the expression of human CD59 in transgenic mice. Strong and uniform expression of CD59 was observed on the endothelial cells of all blood vessels in the heart, kidney, lung, liver, and pancreas in the three lines of mice examined. Little or no expression was seen in other cell types, with the exception of neutrophils and monocytes. These results suggest that this small promoter region contains most of the signals necessary to endow it with endothelial cell specificity, making it a potentially valuable tool in areas ranging from xenotransplantation to gene therapy.