Pathological and Clinical Analysis of Vascular Catheterization Models in Rats, with Exploration of Interventions to Improve Clinical Tolerance.

Permanent vascular catheterization for intravascular access is one of the most commonly applied techniques used on rodents in pharmacology studies. However, use of the intravascular catheters is complicated by nontolerance due to thromboembolic disease and sepsis. We have undertaken an extensive pathologic and clinical analysis of an intravascular catheterization model in Wistar Han and Sprague-Dawley rats, with a particular focus on carotid artery catheterization with or without jugular vein catheterization, in order to define the pathologic mechanisms behind nontolerance and define clinical end points to ensure maximal animal welfare. Further, we have explored various potential solutions to increase the tolerance of the procedure. In these studies, indwelling catheters were found to cause a high degree of thromboembolic disease with infarction in the brain, cecal tip, and kidneys being the primary causes of nontolerance. Loss of greater than 10% body weight was determined to be the most sensitive indicator of nontolerance and was closely correlated with degree of renal parenchymal loss. Sepsis was noted as a very rare complication, indicating that routine aseptic surgical techniques are adequate for preventing this complication.

Loss of T cell tolerance in the skin following immunopathology is linked to failed restoration of the dermal niche by recruited macrophages.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1ß and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

A wave of monocytes is recruited to replenish the long-term Langerhans cell network after immune injury.

A dense population of embryo-derived Langerhans cells (eLCs) is maintained within the sealed epidermis without contribution from circulating cells. When this network is perturbed by transient exposure to ultraviolet light, short-term LCs are temporarily reconstituted from an initial wave of monocytes but thought to be superseded by more permanent repopulation with undefined LC precursors. However, the extent to which this process is relevant to immunopathological processes that damage LC population integrity is not known. Using a model of allogeneic hematopoietic stem cell transplantation, where alloreactive T cells directly target eLCs, we have asked whether and how the original LC network is ultimately restored. We find that donor monocytes, but not dendritic cells, are the precursors of long-term LCs in this context. Destruction of eLCs leads to recruitment of a wave of monocytes that engraft in the epidermis and undergo a sequential pathway of differentiation via transcriptionally distinct EpCAM+ precursors. Monocyte-derived LCs acquire the capacity of self-renewal, and proliferation in the epidermis matched that of steady-state eLCs. However, we identified a bottleneck in the differentiation and survival of epidermal monocytes, which, together with the slow rate of renewal of mature LCs, limits repair of the network. Furthermore, replenishment of the LC network leads to constitutive entry of cells into the epidermal compartment. Thus, immune injury triggers functional adaptation of mechanisms used to maintain tissue-resident macrophages at other sites, but this process is highly inefficient in the skin.

Peripheral tissues reprogram CD8+ T cells for pathogenicity during graft-versus-host disease.

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic stem cell transplantation induced by the influx of donor-derived effector T cells (TE) into peripheral tissues. Current treatment strategies rely on targeting systemic T cells; however, the precise location and nature of instructions that program TE to become pathogenic and trigger injury are unknown. We therefore used weighted gene coexpression network analysis to construct an unbiased spatial map of TE differentiation during the evolution of GVHD and identified wide variation in effector programs in mice and humans according to location. Idiosyncrasy of effector programming in affected organs did not result from variation in T cell receptor repertoire or the selection of optimally activated TE. Instead, TE were reprogrammed by tissue-autonomous mechanisms in target organs for site-specific proinflammatory functions that were highly divergent from those primed in lymph nodes. In the skin, we combined the correlation-based network with a module-based differential expression analysis and showed that Langerhans cells provided in situ instructions for a Notch-dependent T cell gene cluster critical for triggering local injury. Thus, the principal determinant of TE pathogenicity in GVHD is the final destination, highlighting the need for target organ-specific approaches to block immunopathology while avoiding global immune suppression.

Redefining the Role of Langerhans Cells As Immune Regulators within the Skin.

Langerhans cells (LC) are a unique population of tissue-resident macrophages that form a network of cells across the epidermis of the skin, but which have the ability to migrate from the epidermis to draining lymph nodes (LN). Their location at the skin barrier suggests a key role as immune sentinels. However, despite decades of research, the role of LC in skin immunity is unclear; ablation of LC results in neither fatal susceptibility to skin infection nor overt autoimmunity due to lack of immune regulation. Our understanding of immune processes has traditionally been centered on secondary lymphoid organs as sites of lymphocyte priming and differentiation, which is exemplified by LC, initially defined as a paradigm for tissue dendritic cells that migrate to draining LN on maturation. But, more recently, an awareness of the importance of the tissue environment in shaping effector immunity has emerged. In this mini-review, we discuss whether our lack of understanding of LC function stems from our lymph node-centric view of these cells, and question whether a focus on LC as immune regulators in situ in the skin may reveal clearer answers about their function in cutaneous immunology.

Rational development of a protective P. vivax vaccine evaluated with transgenic rodent parasite challenge models.

Development of a protective and broadly-acting vaccine against the most widely distributed human malaria parasite, Plasmodium vivax, will be a major step towards malaria elimination. However, a P. vivax vaccine has remained elusive by the scarcity of pre-clinical models to test protective efficacy and support further clinical trials. In this study, we report the development of a highly protective CSP-based P. vivax vaccine, a virus-like particle (VLP) known as Rv21, able to provide 100% sterile protection against a stringent sporozoite challenge in rodent models to malaria, where IgG2a antibodies were associated with protection in absence of detectable PvCSP-specific T cell responses. Additionally, we generated two novel transgenic rodent P. berghei parasite lines, where the P. berghei csp gene coding sequence has been replaced with either full-length P. vivax VK210 or the allelic VK247 csp that additionally express GFP-Luciferase. Efficacy of Rv21 surpassed viral-vectored vaccination using ChAd63 and MVA. We show for the first time that a chimeric VK210/247 antigen can elicit high level cross-protection against parasites expressing either CSP allele, which provide accessible and affordable models suitable to support the development of P. vivax vaccines candidates. Rv21 is progressing to GMP production and has entered a path towards clinical evaluation.

Expressions of nonabandonment during the intensive care unit family conference.

PURPOSE: Palliative care consultants play an increasing role in assisting critical care clinicians with end-of-life communication in the intensive care unit (ICU). One of the ethical principles these consultants may apply to such communication is nonabandonment of the patient. Limited data exist concerning expressions of nonabandonment in the ICU family conference. This analysis examines expressions of nonabandonment during ICU family conferences. Our goal was to categorize these expressions and develop a conceptual model for understanding this issue as it arises in the ICU setting. METHODS: We identified family conferences in the ICUs of four hospitals. Conferences were eligible if the attending physician believed that discussion about withholding or withdrawing life support or the delivery of bad news was likely to occur. Fifty-one conferences were audiotaped, transcribed, and analyzed using grounded theory. RESULTS: We identified categories capturing expressions of nonabandonment in the ICU family conference. Clinicians expressed nonabandonment of the patient or family in three ways: alleviating suffering/ensuring comfort, allowing family members to be present at the bedside for the death, and being accessible to patients and families. Families expressed their own nonabandonment of the patient or concern about abandonment of the patient by the health care team in five ways: ensuring the patient's suffering is eased, being present at the bedside, ensuring the patient's end-of-life preferences are respected, ensuring that everything possible be done to cure the patient, and "letting go." These categories were placed into a conceptual model that differentiates explicit and implicit statements of nonabandonment. CONCLUSIONS: This paper describes categories and a conceptual model for understanding expressions of nonabandonment that may allow palliative care consultants to help critical care clinicians express nonabandonment and respond to families' expressions of nonabandonment in the ICU family conference. Future studies could use this model to develop a communication intervention for the ICU family conference.

Abstracting Sequences: Reasoning That Is a Key to Academic Achievement.

The ability to understand sequences of items may be an important cognitive ability. To test this proposition, 8 first-grade children from each of 36 classes were randomly assigned to four conditions. Some were taught sequences that represented increasing or decreasing values, or were symmetrical, or were rotations of an object through 6 or 8 positions. Control children received equal numbers of sessions on mathematics, reading, or social studies. Instruction was conducted three times weekly in 15-min sessions for seven months. In May, the children taught sequences applied their understanding to novel sequences, and scored as well or better on three standardized reading tests as the control children. They outscored all children on tests of mathematics concepts, and scored better than control children on some mathematics scales. These findings indicate that developing an understanding of sequences is a form of abstraction, probably involving fluid reasoning, that provides a foundation for academic achievement in early education.

Reconsidering the team concept: educational implications for patient-centered cancer care.

Patient-centered cancer care has become a priority in the oncology field. Increasing efforts to train oncologists in communication skills have led to a growing literature on patient-centered cancer education. In addition, systems approaches have led to an increased emphasis on the concept of teams as an organizing framework for cancer care. In this essay, we examine issues involved in educating teams to provide patient-centered cancer care. In the process, we question the applicability of a tightly coordinated 'team' concept, and suggest the concept of a 'care community' as a more achievable ideal for the way that cancer care is commonly delivered. We discuss the implications that this has for cancer communication education, and propose three principles to guide the development of educational interventions aimed at increasing patient-centeredness in cancer care delivery systems.

Determining the Safety of Maltogenic Amylase Produced by rDNA Technology.

A maltogenic amylase produced by a genetically engineered Bacillus subtilis was studied to evaluate its safety in the food industry. First, the safety of the component parts used in the cloning process, i.e. the host organism ( B. subtilis ), the donor organism ( Bacillus stearothermophilus ) and the construction process, were evaluated. This evaluation indicated that the final construct should be regarded as a safe source for maltogenic amylase when manufactured according to current Good Manufacturing Practices. Additional experimental safety testing was carried out to confirm this conclusion. In a 13-week oral toxicity study rats tolerated the maltogenic amylase at dietary levels of 5% without toxicologically significant adverse reaction. Lack of mutagenic potential was confirmed in bacterial mutagenic as-says with Salmonella typhimurium and in an in vivo cytogenetic study in rat bone marrow cells. In an acute inhalation study with 4 h of exposure to rats, no death occurred at the highest dose level, i.e, 1.59 mg/L. The test material was non-irritating to skin and did not product eye injury in rabbits. A skin sensitization study in guinea pigs was negative. Antibiotic activity tests indicated that the microorganism did not produce antibiotics. Results indicated that maltogenic amylase should be generally recognized as safe for use in production of maltose syrups, and confirmed the conclusion drawn from the safety evaluation of the component parts used in the cloning process.