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These two studies investigate the immune responses of lung and kidney transplant recipients to the influenza vaccine. The study involving kidney transplant recipients developed a novel flow cytometry assay to measure cell-mediated immunity in response to influenza vaccination. The activation of T-cells was assessed through the change in T-cell production of interferon gamma after vaccination. In lung transplant recipients, the study examined the formation of de novo anti-HLA antibodies following influenza vaccination. Anti-HLA antibodies were classified as donor-specific or not. The study in kidney transplant recipients found that the influenza vaccine is effective at stimulating the immune response and producing long-lived memory in these patients, as evidenced by high baseline T-cell activity. The study of lung transplant recipients found that receiving the influenza vaccine did not result in the production of anti-HLA antibodies. Both studies found vaccine to be safe for use in these populations. / Experimental Medicine

<p>Southern Africa is facing one of the most serious HIV epidemics. This project contributes to the HIVNET, Network for Prevention Trials cohort for vaccine development. HIV’s biology and rapid mutation rate have made vaccine design difficult. We examined HIV-1 subtype C diversity and how it relates to CTL epitope location along viral gag sequences. We found a negative correlation between codon sites under positive selection and epitope regions / suggesting epitope regions are evolutionarily conserved. It is possible that epitopes exist in non-conserved regions, yet fail to be detected due to the reference strain diverging from the circulating viral population. To test if CTL clustering is an artifact of the reference strain, we calculated differences between the gag codons and the reference strain. We found a weak negative correlation, suggesting epitopes in less conserved regions maybe evading detection. Locating conserved and optimal epitopes that can be recognized by CTLs is essential for the design of vaccine reagents.</p>

El efecto deletéreo de la presencia de anticuerpos anti-HLA donante-específicos (ADS) pre-trasplante renal en la supervivencia del injerto a corto plazo quedó patente en las primeras experiencias clínicas en los años 60. Sin embargo, resulta controvertido el papel que juegan los ADS anti-HLA que aparecen post-trasplante. La experiencia preliminar de nuestro grupo de trabajo y de otros sugiere que la aparición de ADS post-trasplante se relaciona con el desarrollo de un episodio de rechazo agudo de características diferenciadas. Diseñamos los estudios que componen esta tesis con objeto de definir adecuadamente el rechazo agudo humoral o rechazo agudo asociado a la aparición de ADS, así como determinar su incidencia, características clínicas, serológicas e histológicas y valorar la eficacia de una propuesta terapéutica alternativa en una población determinada. Estudiamos el grupo de enfermos que recibió un trasplante renal en el Massachusetts General Hospital de Boston entre julio de 1995 y julio de 1999 (n=232). Revisamos la evolución clínica de los 81 receptores que habían sufrido algún episodio de rechazo agudo durante los tres primeros meses post-trasplante; realizamos pruebas cruzadas donante-específicas con los sueros peri-rechazo; utilizamos técnicas de inmunofluorescencia para detectar depósitos de la fracción C4d del complemento en las biopsias correspondientes y examinamos las características histológicas de las mismas con las tinciones tradicionales. Las conclusiones fundamentales de nuestros estudios son las siguientes:1) El rechazo agudo humoral es una entidad clínica y patológica diferenciada que podemos diagnosticar con criterios: -CLÍNICOS: Disfunción renal severa precoz típicamente córtico-resistente y con frecuencia resistente al tratamiento anti-linfocitario convencional.-SEROLÓGICOS: Acompañada de la aparición de anticuerpos donante- específicos en el momento del rechazo, habitualmente IgG anti-HLA de clase I o de clase II (sin descartar un posible papel patogénico de IgM).-HISTOLÓGICOS: Con depósitos difusos de C4d en capilares peritubulares.2)La presencia de sólo dos de los tres criterios anteriores permitiría establecer el diagnóstico de "probable rechazo agudo humoral", en cuyo caso probablemente resulta acertado aplicar la misma pauta terapéutica.3) La incidencia de rechazo agudo humoral en la población estudiada es de 7.7%.4) Los factores de riesgo claramente identificados son: sensibilización (pre-trasplante o histórica) y retrasplante renal.5) El estudio histológico tradicional resulta con frecuencia insuficiente para diagnosticar el rechazo agudo humoral, aunque la presencia de neutrófilos en capilares peritubulares y/o glomerulares, la necrosis fibrinoide arterial o glomerular y la presencia de trombos de fibrina en glomérulos resultan datos sugestivos. En virtud de estos parámetros sugerimos distinguir entre rechazo agudo humoral tipo 1 (con afectación capilar) y rechazo agudo humoral tipo 2 (con afectación arterial) con severidad y pronóstico diferenciados.6) El rescate con plasmaféresis, tacrólimus y micofenolato muestra una eficacia elevada en los casos severos de rechazo agudo humoral (89% de éxito a corto plazo en nuestra serie). Parece aconsejable asociar gamaglobulina policlonal al tratamiento, como medida profiláctica e inmunomoduladora. Fármacos recientemente incorporados al mercado o en fase de estudio clínico podrían constituir alternativas eficaces en el control de las respuestas humorales post-trasplante en el futuro.

Dendritic cells (DC) are potent antigen presenting cells involved in the initiation of the alloimmune response and organ transplant rejection. This thesis, has investigated pharmacological and genetic approaches to manipulate DC in order to generate tolerogenic DC which elicit poor allostimulatory activity as potential cell therapy agents to treat allograft rejection. In the first aspect of this study, human monocyte-derived DC were used to study the influence of Rapamycin (RAPA) on DC phenotype and function. This study showed that RAPA when added to monocytes prior to DC differentiation or after DC maturation generated tolerogenic DC as evidenced by the ability of these cells to induce T cell hyporesponsiveness. However, T cell hyporesponsiveness was associated with downregulation of costimulatory molecules only when added prior to differentiation and surprisingly was not influenced by the induction of CD4 ⁺FoxP3 ⁺ T cells. To assess the effects of RAPA on DC function in the transplant setting an in vivo chimeric model of ovine vascularised skin allograft transplantation was established in immunocompromised NOD/SCID mice as a host. This model was established as a preliminary model to acquire in vivo data prior to testing the effect of pharmacologically modified DC in the preclinical ovine model of renal allograft transplantation, also established in the host laboratory. Firstly, comparison of ovine DC obtained from cannulation of the prefemoral lymphatic vessels in sheep demonstrated that RAPA-modified ovine DC acted as poor stimulators of allogeneic ovine T cells similar to human DC treated with RAPA. Secondly, in NOD/SCID mice engrafted with ovine skin, the infusion of allogeneic ovine T cells together with RAPA-modified ovine DC reduced histological rejection in comparison to control DC. In the second aspect of this study, the effects of genetic manipulation of DC were investigated. In order to investigate the effects of genetic modification of DC, two isoforms of the human HLA-G molecule, HLA-G1 (membrane bound) and HLA-G5 (soluble isoform) were used to generate adenoviral vectors. Unexpectedly, both HLA-G isoforms expressed by human DC transfectants were unable to induce allogeneic T cell hyporesponsiveness in the mixed lymphocyte reaction (MLR). Surprisingly, in the MLR the allogeneic T cells acquired HLA-G1, but not HLA-G5, indicating that direct cell contact and membrane transfer from DC to T cells occurred (Trogocytosis). In addition to HLA-G1, costimulatory molecules (CD40, CD80, CD86 and MHC Class II) were also cotransferred from DC to allogeneic T cells. Accordingly, in secondary proliferation assays T cells immunoselected after co-culture with allogeneic untransfected DC (TUT) demonstrated potent antigen presenting activity when used as stimulators of autologous T cells (analogous to the indirect pathway of antigen presentation). In contrast to TUT, immunoselected T cells that acquired HLA-G1 (THLA-G1) upon co-culture with DCtransfectants showed poor stimulatory capacity. Thus the data reported in this thesis supports the proposed novel concept that HLA-G acquired by T cells through genetically modified DC, functions to autoregulate T cells via T-T cell interaction through the HLA-G receptor ILT2 (negative signalling receptor) expressed on T cells. In conclusion, this thesis has firstly provided supportive evidence that the pharmacological modification of human and ovine DC with RAPA has potential therapeutic effects on allograft rejection. Secondly, the genetic modification of DC to induce expression of HLA-G has specifically allowed the transfer of this molecule to T cells by trogocytosis and the inhibition of alloreactive T cell expansion. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Identification of the complement C4d fragment in peritubular capillaries as a specific marker for antibody mediated rejection in renal transplantation revealed the critical role of antibodies in graft survival. In this thesis, I document the design and findings of studies performed to investigate the clinical impact of anti-HLA antibodies present before and/or after transplantation. Over time, the detection techniques for anti-HLA antibodies has evolved from the less sensitive complement-dependent lymphocytotoxicity (CDC) crossmatching (XM) to more sensitive solid phase assays such as Luminex®. Studies have been conducted to compare the predictive value of different antibody detection techniques. The first result chapter presents antibody specificity in positive CDC B-cell crossmatch (BXM), analysed with highly specific Luminex® assays. The study also investigates the predictive value of BXM in the general transplant population. I found that donor-specific anti-HLA antibodies (DSA) are only present in one third of positive BXM and are associated with poor outcomes. The novel finding is that >80% of the DSA detected by BXM are complement-fixing IgG₁ and IgG₃ subclasses. Transplant glomerulopathy (TG) is type of chronic renal graft rejection. The pathogenesis of TG is unclear. In the second result chapter, I report risk factors and involvement of anti-HLA antibodies in the development of TG. This study shows that glomerular rejection, delayed graft function, HLA presensitization and DSA have a univariate effect on TG development. Multivariate analysis revealed that DSA are an independent predictor of TG, after adjustment for other risk factors. I have further investigated the role of BXM in a unique, well-matched, highly sensitized patient group transplanted under the national renal exchange programme. I compared Luminex® antibody analysis with BXM in predicting transplant outcomes. In highly sensitized patients, DSA are found in two thirds of positive BXM. In univariate analyses, BXM is associated with humoral rejection whereas DSA defined by Luminex® are associated with total and all rejection types. The major finding is that, by multivariate analysis, DSA defined by Luminex® are an independent predictor of total and humoral rejection, but BXM are not. These interesting findings are reported in the third result chapter. Studies reported in this thesis define the clinical significance of anti-HLA antibodies in renal transplant outcomes. Method comparison studies provide useful information on antibody specificity and their impact on graft survival. Collectively, a better understanding of alloantibodies associated with graft rejection and limitation of antibody detection methods may facilitate donor selection and choice of immunosuppressants, and consequently improve transplant outcomes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1379925 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2010

In examining the role of the �legal rule� in Indonesian law, and in particular environmental law related to water quality management, this thesis questions the often expressed view that laws in Indonesia are sound, they merely fail to be implemented. It proposes that this appraisal of the situation does not take a sufficiently deep assessment and that a cause for non-implementation lies within the drafting of the laws themselves. It is argued that the ineffective system for environmental protection in Indonesia can be related to a failure to recognise the role of the �legal rule� in environmental law. A proposition presented in this thesis is that the arrangements for environmental law making in Indonesia lacks a strong rule foundation and, for this reason, it is not capable of producing shared understandings by lawmakers about producing and reproducing environmental law as legal sub-system. Another central proposition is that Indonesian environmental law has a form and style, which negates the role of the legal rule in environmental management and control. Despite the changes brought by reformasi, the central position of the legal rule in environmental law and, indeed, the necessary rule foundation to the development of the legal system, has yet to achieve full recognition. If this situation is related to the system of water quality management and pollution control in Indonesia, it can be seen that environmental improvement will not be achieved until underlying issues concerning the structure, form and style of environmental law making are addressed.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.