Analysis and modelling of immune cell behaviour in lymph nodes based on multi-photon imaging

This thesis presents quantitative studies of T cell and dendritic cell (DC) behaviour in mouse lymph nodes (LNs) in the naive state and following immunisation. These processes are of importance and interest in basic immunology, and better understanding could improve both diagnostic capacity and therapeutic manipulations, potentially helping in producing more effective vaccines or developing treatments for autoimmune diseases. The problem is also interesting conceptually as it is relevant to other fields where 3D movement of objects is tracked with a discrete scanning interval. A general immunology introduction is presented in chapter 1. In chapter 2, I apply quantitative methods to multi-photon imaging data to measure how T cells and DCs are spatially arranged in LNs. This has been previously studied to describe differences between the naive and immunised state and as an indicator of the magnitude of the immune response in LNs, but previous analyses have been generally descriptive. The quantitative analysis shows that some of the previous conclusions may have been premature. In chapter 3, I use Bayesian state-space models to test some hypotheses about the mode of T cell search for DCs. A two-state mode of movement where T cells can be classified as either interacting to a DC or freely migrating is supported over a model where T cells would home in on DCs at distance through for example the action of chemokines. In chapter 4, I study whether T cell migration is linked to the geometric structure of the fibroblast reticular network (FRC). I find support for the hypothesis that the movement is constrained to the fibroblast reticular cell (FRC) network over an alternative 'random walk with persistence time' model where cells would move randomly, with a short-term persistence driven by a hypothetical T cell intrinsic 'clock'. I also present unexpected results on the FRC network geometry. Finally, a quantitative method is presented for addressing some measurement biases inherent to multi-photon imaging. In all three chapters, novel findings are made, and the methods developed have the potential for further use to address important problems in the field. In chapter 5, I present a summary and synthesis of results from chapters 3-4 and a more speculative discussion of these results and potential future directions.

Quantification of variable palmar ligaments around the triquetrum-hamate joint determined by lunate type

The ligaments of the wrist are highly variable and poorly described, which is more obvious on the ulnar side of the wrist. Previous studies highlighted the potential differences within the ligaments of the wrist but no consensus has been reached. Poor tissue description and inconsistent use of terminology hindered the reproducibility of the results. Improved understanding of the morphological variations between carpal bones may facilitate improved understanding of the ligamentous structure within the wrist. This study aims to identify the potential variations between carpal bones that could be used to separate palmar ligamentous patterns around the triquetrum-hamate joint into subgroups within the sample population. Investigations were performed following a detailed nomenclature and a clear definition of ligamentous structures to facilitate detailed description and reproducible results. Quantitative analyses were conducted using 3D modelling technique. Histological sections were then analysed to identify the structure of each ligamentous attachment. Variable patterns of ligamentous attachments were identified. Differences were not only obvious between samples but also between the right and left hands of the same person. These identifications suggested that the palmar ligamentous patterns around the triquetrum-hamate joint are best described as a spectrum with a higher affinity of the triquetrum-hamate-capitate ligament and the lunate-triquetrum ligament to be associated with type I lunate wrists on one extreme and type II lunate wrists with the palmar triquetrum-hamate ligament, triquetrum-hamate-capitate ligament and palmar radius-lunate-triquetrum ligament attachments at the other extreme. Histological analyses confirmed pervious established work regarding the mechanical role of ligaments in wrist joint biomechanics. Also, there were no significant differences between the quantitative data obtained from the Genelyn-embalmed and unembalmed specimens (p>0.05). The current study demonstrated variable ligamentous patterns that suggest different bone restraints and two different patterns of motion. These findings support previous suggestions regarding separating the midcarpal joint into two distinct functional types. Type I wrists were identified with ligamentous attachments that are suggestive of rotating/translating hamate whilst type II wrists identified with ligamentous attachments that are suggestive of flexing/extending hamate motion based upon the patterns of the ligamentous attachments in relation to the morphological features of the underlying lunate type of the wrist. This opens the horizon for particular consideration and/or modification of surgical procedures, which may enhance the clinical management of wrist dysfunction.