Enhancing the immunogenicity of tumour lysate-loaded dendritic cell vaccines by conjugation to virus-like particles.

BACKGROUND: Tumour cell lysates are an excellent source of many defined and undefined tumour antigens and have been used clinically in immunotherapeutic regimes but with limited success. METHODS: We conjugated Mel888 melanoma lysates to rabbit haemorrhagic disease virus virus-like particles (VLP), which can act as vehicles to deliver multiple tumour epitopes to dendritic cells (DC) to effectively activate antitumour responses. RESULTS: Virus-like particles did not stimulate the phenotypic maturation of DC although, the conjugation of lysates to VLP (VLP-lysate) did overcome lysate-induced suppression of DC activation. Lysate-conjugated VLP enhanced delivery of antigenic proteins to DC, while the co-delivery of VLP-lysates with OK432 resulted in cross-priming of naïve T cells, with expansion of a MART1(+) population of CD8(+) T cells and generation of a specific cytotoxic response against Mel888 tumour cell targets. The responses generated with VLP-lysate and OK432 were superior to those stimulated by unconjugated lysate with OK432. CONCLUSION: Collectively, these results show that the combination of VLP-lysate with OK432 delivered to DC overcomes the suppressive effects of lysates, and enables priming of naïve T cells with superior ability to specifically kill their target tumour cells.

Could musculo-skeletal radiograph interpretation by radiographers be a source of support to Australian medical interns: A quantitative evaluation.

INTRODUCTION: Public expectations regarding access to the emergency department (ED) challenges providers and policy makers with finite resources being stretched beyond capacity. To enable education of a greater numbers of doctors the format of the medical internship in Australia has changed and assumes that sufficient supervision is provided to interns to enable image interpretation skills development. Furthermore this assumes that appropriate foundational skills are established during undergraduate education. METHODS: A mixed methods approach using a convenience, self selecting sample population of radiographers and final year medical students was adopted. The study measured the interpretive ability of final year medical students and radiographers in musculo-skeletal trauma (MSK) plain radiographic images. An image test bank based upon radiologist consensual agreement was corrected for prevalence and bias. Performance across a range of measurements was completed and compared for statistical significance using Mann-Whitney U. RESULTS: Results were divided to enable analysis across age ranges and types of skeletal presentation. Radiographer performance was better numerically and demonstrated statistically significant difference in several areas. CONCLUSION: Radiographers have the knowledge base to assist junior doctors to clinically interpret the musculo-skeletal radiographic image. To meet the requirements of AMC and the Medical Board of Australia (MBA), a tailored clinically based educational system could be developed and provided by an accredited radiographer. Australian radiographers could also be employed to provide a safety net to avoid misinterpretation, such as seen in the UK commenting system, operating as an interprofessional team.

Targeting early events in T cell activation to construct improved vaccines.

Live, attenuated vaccines currently offer the best protection against virulent pathogens. Recent advances in Immunology and Molecular Biology provide an opportunity to design vaccines that will be more effective and safer than existing ones. Immunologists are rapidly developing the capacity to identify and construct the minimal immunogenic units from pathogens. The molecular signals required to fully activate antigen presenting cells (APCs) and responder T cells are becoming apparent. Improved vaccine delivery systems are being designed which will mimic the actions of pathogens in vivo. These vaccines will incorporate protective epitopes fused to immunoregulatory cytokines in chimeric proteins. They will be encapsulated in formulations which allow for the slow release of these chimeric proteins thereby inducing the memory T cells required for long-lived immunity. These vaccine formulations will target receptors present on the most active APCs. Here we discuss how these advances will allow us to rationally construct "virtual pathogens" which will provide improved protection against new and old microbial foes.

Further observations on transfusion collapse curves in inbred rats.

Transfused allogeneic red blood cells (RBC) are very rapidly cleared from the host circulation in some strain combinations among inbred rats. The phenomenon, termed collapse curve clearance, an analogy with its human counterpart, is spleen-dependent. Allogeneic 51Cr-labeled red cells taken up by the spleen are retained for at least 6 weeks, and appear to represent a pool of intact cells that are destroyed no more quickly than syngeneic red cells. Sequential transfusions delivered at weekly intervals gradually abrogate rapid clearance, but a longer interval between injections allows the collapse curve pattern to persist. Increasing the dosage of donor red cells above the standard dose used in these experiments decreases the clearance rate, while reducing the dosage further accelerates clearance. Several attempts to implicate anti-RBC antibody, and to correlate the presence of such antibody with accelerated clearance, have failed. The grafting of an additional syngeneic spleen neither increases the proportion of allogeneic RBC removed from the circulation, nor reverses a temporary paralysis of the phenomenon induced by red cell overloading. These experiments imply that rapid clearance of allogeneic red cells is not mediated by a purely cellular mechanism, but that it requires a humoral substance that is readily exhausted, but fairly quickly replenished. The possibility that the rat equivalent of the H-2.7 mouse antigen might be responsible was considered, but appropriate experiments on mice have failed to support this idea. The findings in these experiments strengthen the analogy to the clinical event.

Major histocompatibility complex-linked spleen-dependent transfusion "collapse curves" in inbred rats.

In most donor-host combinations among seven inbred rat strains, transfused allogeneic red blood cells were cleared from the circulation of unsensitized hosts at a rate no different from that of syngeneic red blood cells. In four strain combinations, however, allogeneic red blood cells were cleared very rapidly, and in six combination, clearance was effected at a rate intermediate between the rapid clearance rate and the syngeneic rate. In strain combinations exhibiting rapid clearance, females cleared allogeneic red blood cells significantly more rapidly than did males. Rapid clearance was linked to the major histocompatibility complex (MHC) of the donor strain but not to the MHC of the host strain. All donor-host combinations showing rapid clearance possessed identical RT2, RT3, and AgS red blood cell alloantigens. Red blood cells removed rapidly from the circulation by allogeneic hosts were sequestered in the spleen rather than lysed in the circulation. The phenomenon was mediated by splenic reticuloendothelial cells and could be completely circumvented by prior splenectomy. Rapid clearance was apparently not antibody mediated. The phenomenon of rapid clearance of allogeneic red blood cells in rats appears to be analogous to the "collapse curves" of human blood transfusion.

Evidence that changes in expression of major histocompatibility complex antigens may underlie the immunosuppressive effect of heat-treated cells in vivo and in vitro.

The prior transfusion of heat-treated (60 degrees C for 1 hr) allogeneic spleen cells is known to bring about specific prolongation of the survival of subsequent donor strain heart allografts. In this communication we show that some polymorphic and monomorphic class 1 determinants on spleen cells are heat denatured so that they no longer provoke antibody formation in naive allogeneic hosts. By contrast, the heated cells remain able to provoke the formation of anti-class 2 antibodies. When measured in a binding assay, the levels of anti-class 2 antibodies are similar irrespective of whether the immunizing inoculum consists of normal or heated cells. In a cytotoxic assay, the antibodies produced following exposure to normal cells are cytotoxic; this activity is substantially reduced when the cellular immunizing inoculum is heated. Cells heated to 60 degrees C for 1 hr are unable to stimulate in a mixed lymphocyte reaction, but reactivity can be partially restored by the addition of exogenous IL-2 to the culture. From previous evidence it seems unlikely that suppressor cells play a major role in the immunosuppression effected by cells heated to 60 degrees C. The results presented in this communication suggest a possible role for anticlass 2 antibodies and also imply the defective production of a costimulatory signal that normally follows the presentation of allogeneic MHC antigens.

Immunosuppressive activity of class I antigens in the absence of antigen-presenting cells that are MHC-compatible with the host.

Pretransplant transfusions of heat-treated spleen and lymph node cells were shown to prolong the survival of DA strain heart grafts in 3 allogeneic host strains: BS, HS, and AS2. To examine whether MHC incompatibility was necessary for immunosuppression mediated by heat-treated cells, AS strain skin-graft recipients were pretreated with fresh or heated inocula from either MHC compatible or incompatible congenic donor strains, AS2.1L(AS) and AS.1F(AS2) prior to transplanting donor strain skin. Prolonged survival was observed only in the MHC-incompatible strain combination, and in this MHC-incompatible strain combination, and in this instance heated cells were conspicuously more immunosuppressive than fresh cells. To determine the effect of intra-MHC differences between donor and host on graft survival, cells from a recombinant donor strain (r22), which shared class II antigens with the graft donor strain and class I antigens with the host, were transfused prior to heart transplantation. Neither fresh nor heated r22 cells prolonged graft survival. Our data accord with the suggestion that in the absence of MHC-compatible antigen-presenting cells, foreign class I antigen is immunosuppressive.

Prolonged survival of cardiac allografts in rats following the administration of heat-treated donor lymphocytes. A possible immunosuppressive role for class I major histocompatibility complex antigens.

Pretransplant transfusions of spleen and lymph node cells heated to 45 degrees C or 50 degrees C for 1 hr prolong the survival of subsequent donor-specific heart grafts in the fully allogeneic donor-host combination DA (RT1a)----AS (RT1l). The results are comparable to survival times recorded following pretransplant transfusions of purified donor specific red blood cells (RBC) in the same strain combination. Both class I and class II major histocompatibility complex (MHC) antigens are serologically detectable on heat-treated cells; by contrast only class I antigens are expressed on red blood cells. Although heat-treated cells stimulate alloantibody formation, they fail to provoke a proliferative response in an in vivo host-versus-graft assay. Both red blood cells and heat-treated inocula persist in the host for long periods, possibly an important consideration in relation to their capacity to prolong the survival of subsequent donor strain allografts. The experimental data support the contention that class I MHC antigens can be immunosuppressive in the context of allografting. The present results recall the experiments carried out early in the century, which used heat-treated tumor cells to prolong the survival of subsequent viable tumor allografts, and which are sometimes cited as the first example of active enhancement.

Developmental levels in family-centered medical care.

This paper describes a developmental sequence which many physicians seem to experience as they mature in their competence in family-centered medical care. The flow of the developmental levels is from information to affect to social systems. The paper concludes with implications for family systems curricula in family medicine residencies.

Risks and benefits of a family systems approach to medical care.

The "art of medicine" is struggling for survival in medical education, practice, and research. The authors embrace the biopsychosocial model as an ideal new paradigm for medicine and propose that the family systems approach provides the best available vehicle for applying the biopsychosocial model to everyday medical practice and research. Such an approach would require major changes in the way health care is delivered and offers both benefits and risks for family physicians to consider. One important change would involve the sharing of physicians' power in a health care system that emphasizes collaboration among individuals, families, and a variety of professionals. Such revolutionary changes should be approached with caution.

Levels of physician involvement with psychosocial concerns of individual patients: a developmental model.

BACKGROUND: Physician involvement in patients' psychosocial concerns is seen as desirable by practicing physicians and family medicine educators. Although the effectiveness of several approaches to psychosocial problems has been demonstrated, the skills required of the physician vary widely. We present a five-level developmental model of physician skills in addressing the psychosocial concerns of individual patients. METHODS: To validate the model, 171 outpatient office visits in a residency program were videotaped and rated according to the levels. The inter-rater agreement was 88%. RESULTS: Interviews with lower levels of psychosocial involvement occurred much more frequently than interviews rated at higher levels (48%, 34%, 16%, 2%, 0%, respectively). Involvement at each higher level added approximately two minutes to the length of the visit. The development of higher levels of physician involvement between the first and third year of residency training was not found in this sample. CONCLUSIONS: These results support the validity of the five-level sequence regarding the depth of physician involvement. Because the hierarchy can be used to reliably assess the degree of physician involvement with the psychosocial concerns of individual patients, the model offers potential applications for resident education and further research on the physician-patient relationship.

Prevention in college health: counseling perspectives.

Such problems as sexually transmitted diseases, alcohol and other drug use, and acquaintance rape require college health professionals to function in primary and secondary preventive roles. In this article, the authors draw upon counseling literature and college health practice to identify the central elements of preventive programs, highlight specific intervention formats used in preventive work, and describe how interventions are assembled into coherent programs of prevention. To illustrate the structure and process of long-range, institutionalized preventive efforts, the authors describe an initiative addressing the primary, secondary, and tertiary prevention of substance use at a health sciences campus.

Morbidity of therapeutic abortion in Auckland.

Twenty-six women were admitted to National Women's Hospital with complications following therapeutic abortion at the Auckland Medical Aid Centre, during its first year of operation. This represents 1.4 percent of the total number of cases at the Centre that year. The principal early complicatons were pelvic sepsis, suspected uterine perforation and incomplete abortion. The complication rate appears to be low and compares favourably with reports from other centres. During the same period, 17 women (8.4 percent) whose pregnancies were terminated at National Women's Hospital had significant complications. All 43 complicated cases are analysed.

Levels of physician involvement with patients and their families. A model for teaching and research.

BACKGROUND: We present an educational model that describes physician skills for addressing psychosocial concerns of patients, ranging from basic medical questions to in-depth psychotherapy. This model improves upon previously published models by integrating into one hierarchy levels of physician involvement with individual patients and levels of involvement with families. METHODS: Ten faculty family physicians were videotaped during 200 office visits. Interviews were categorized according to the model, with a 79% interrater agreement. RESULTS: Most visits involved the lower three levels of physician involvement (41%, level 1; 35.5%, level 2; and 23%, level 3). Discussion of family context occurred in a majority (58.5%) of visits, primarily when another family member was in the room and during preventive care visits. Higher levels were associated with longer visits--about 3 minutes more for each additional level. CONCLUSIONS: This investigation suggests that the levels of physician involvement model can be reliably measured. This model may be a useful tool for education and research, particularly the study of physician interview skills appropriate to family medicine.

A monomeric red fluorescent protein with low cytotoxicity.

Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.

p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors.

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which is important for the prevention of B-cell lymphomas. We created a mouse model (mDeltapro) that lacked residues 58-88 of the proline-rich domain of p53. mDeltapro is defective for apoptosis, but is able to arrest cell-cycle progression in hematopoietic tissues. mDeltapro develops late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-null mice. Interestingly, mDeltapro lymphomas comprised incorrectly differentiated B cells. B-cell irregularities were also detected in mDeltapro before tumor onset, in which aged mice showed an increased population of inappropriately differentiated B cells in the bone marrow and spleen. We predict that by keeping B-cell populations in check, p53-dependent apoptosis prevents irregular B cells from eventuating in lymphomas.