Neuroimaging for non-accidental head injury in childhood: a proposed protocol.

Non-accidental head injury (NAHI) is a major cause of neurological disability and death during infancy. Radiological imaging plays a crucial role in evaluating craniospinal injury, both for guiding medical management and the forensic aspects of abusive trauma. The damage sustained is varied, complex and may be accompanied by an evolving pattern of brain injury secondary to a cascade of metabolic and physiological derangements. Regrettably, many cases are poorly or incompletely evaluated leading to diagnostic errors and difficulties in executing subsequent child care or criminal proceedings. It is evident, from cases referred to the authors, that imaging protocols for NAHI are lacking (or only loosely adhered to, if present) in many centres throughout the U.K. Future research in this field will also be hampered if there is a lack of consistent and reliable radiological data. There is no nationally agreed protocol for imaging NAHI. We propose such a protocol, based upon a wide experience in the medical management of child abuse and extensive involvement in the medicolegal aspects of NAHI.

Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells.

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.

Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength.

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.

Differential requirement for SLP-76 domains in T cell development and function.

The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.

Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals.

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4(+)CD8(+) thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4(+)CD8(+) thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.

Paediatric brain tumours: an embryological perspective.

Primitive neuroectodermal tumours are amongst the most common paediatric tumours of the central nervous system. These tumours are composed of undifferentiated cells and a variable component of more differentiated cell types. Most analysis of these tumours has focused on molecules normally found in the differentiated cells or those found in all primitive neuronal precursors. In this article we describe recent advances in understanding of the molecular processes involved in normal neurogenesis. We discuss the relevance of these data to the biology of neuronal tumours and describe strategies we and others have adopted to investigate the usefulness of molecules found in undifferentiated neuronal tissues in understanding the events which underlie oncogenesis in this tumour type.

Third ventricular cysts and membranes unsuspected on conventional CT and MRI.

Three cases are presented of symptomatic cysts or membranes within the third ventricle interfering with CSF flow and presenting as non-communicating triventricular hydrocephalus. None was visible on conventional CT or MRI, two being discovered at neuroendoscopy and one only with a specific MRI sequence designed to show CSF partitioning.

Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.

During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death. Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner. We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets. Here we show that cells of the T lineage also express A1 in a developmentally regulated manner. Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes. It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes. Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes. Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.

Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4(+)CD8(+) thymocytes.

Thymocyte maturation is governed by antigen-T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen-TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.

Positive selection as a developmental progression initiated by alpha beta TCR signals that fix TCR specificity prior to lineage commitment.

During positive selection, immature thymocytes commit to either the CD4+ or CD8+ T cell lineage ("commitment") and convert from short-lived thymocytes into long-lived T cells ("rescue"). By formal precursor-progeny analysis, we now identify what is likely to be the initial positive selection step signaled by alpha beta TCR, which we have termed "induction". During induction, RAG mRNA expression is downregulated, but lineage commitment does not occur. Rather, lineage commitment (which depends upon the MHC class specificity of the alpha beta TCR) only occurs after downregulation of RAG expression and the consequent fixation of alpha beta TCR specificity. We propose that positive selection can be viewed as a sequence of increasingly selective developmental steps (induction-->commitment-->rescue) that are signaled by alpha beta TCR engagements of intrathymic ligands.

Altered functional responsiveness of thymocyte subsets from CD3delta-deficient mice to TCR-CD3 engagement.

CD3delta-deficient (delta degrees) mice are defective in alphabeta T cell development. Here we explore the capacity of TCR-CD3 signaling complexes expressed on delta degrees thymocytes to mediate the following functional outcomes in response to antibody cross-linking: (i) the transition from the CD4-CD8- to CD4+CD8+ stage, (ii) the transition from the CD4+CD8+ to CD4+CD8- or CD4-CD8+ stages and (iii) the induction of apoptosis. We provide evidence that CD3deltaepsilon complexes are dispensable for mediating the anti-CD3-mediated CD4-CD8- to CD4+CD8+ transition. On the other hand, CD3delta is critical at the CD4+CD8+ stage. We demonstrate that CD4+CD8+ thymocytes from delta degrees mice, unlike delta degrees CD4-CD8- thymocytes and wild-type CD4+CD8+ thymocytes, require prolonged or consecutive stimuli to elicit functional responses. Depending on the nature of the secondary stimulus, delta degrees thymocytes can be induced to undergo apoptosis or preferential maturation to the CD4-CD8+ stage. Taken together these results indicate that the signaling capacity of the TCR-CD3 complex is noticeably altered in the absence of CD3delta. The essential role of CD3delta at the CD4+CD8+ stage of development correlates with the onset of TCRalpha rearrangement, consistent with a critical structural and/or functional relationship between CD3delta and TCRalpha.

Thymic function in young/old chimeras: substantial thymic T cell regenerative capacity despite irreversible age-associated thymic involution.

Age-associated thymic involution results in a diminished capacity to regenerate T cell populations, although the magnitude of this effect is unknown. In this report, thymic function was studied in aged vs. young adult mice after lethal irradiation and administration of T cell-depleted bone marrow (BM) from young mice. Abnormalities observed in aged thymi (reduced thymocyte numbers, histologic abnormalities) were not reversed by administration of young BM via bone marrow transplantation (BMT), but aged thymi displayed a normal thymocyte subset distribution and appropriately deleted MIs-reactive T cells after BMT. Aged BMT recipients regenerated significantly reduced numbers of splenic T cells compared to young recipients and showed increased peripheral expansion of thymic emigrants since a higher proportion of BM-derived T cells expressed a memory phenotype in aged vs. young BMT recipients. Because peripheral expansion of thymic emigrants could substantially increase the number of thymic progeny present in the spleen, we sought to measure thymic T cell regenerative capacity after BMT in a setting devoid of peripheral expansion. To do this, TCR-transgenic (Tg+) T cell-depleted BM was administered to aged and young recipients lacking antigen specific for the Tg+ TCR. Aged recipients regenerated approximately 50 % of the TCR Tg+ cells regenerated in young BMT recipients, providing evidence that even very aged thymi retain the capacity to regenerate significant numbers of mature T cell progeny. Therefore, thymic function is reduced with aged but it is not lost, suggesting that therapeutic approaches to enhance thymic function may be successful even in very aged hosts.

Developmentally regulated expression of peanut agglutinin (PNA)-specific glycans on murine thymocytes.

Intrathymic maturation of T lymphocytes is characterized by variable expression of O-linked Gal beta 1,3GalNAc glycans reactive with peanut agglutinin (PNA) lectin. Recent studies on human thymocytes show that conversion from PNA+ to PNA- phenotype is correlated with increased expression of alpha 2,3 O-linked sialyltransferase (ST), which sialylates Gal beta 1,3GalNAc glycans, masking their binding sites for PNA. Interestingly, alpha 2,3 O-linked ST expression is highest within the regions of the thymus containing the most immature and most mature thymocyte subsets, suggesting that PNA-specific glycans are intermittently masked by sialylation during thymic selection processes. Here, we studied expression of PNA receptors on developing thymocytes in the murine system using thymocytes from both normal mice and transgenic mice that are genetically arrested at the early phases of T cell development. Our results confirm and extend recent findings in the human system by showing that murine T cells sequentially progress from PNAlo-->PNAhi-->PNAlo stages during their differentiation within the thymus. In addition, our data demonstrate that a similar set of polypeptides is variably masked by sialylation throughout T cell development.

Surface molecules that drive T cell development in vitro in the absence of thymic epithelium and in the absence of lineage-specific signals.

Differentiation of immature double positive (DP) CD4+ CD8+ thymocytes into single positive (SP) CD4+ and CD8+ T cells is referred to as positive selection and requires physical contact with thymic cortical epithelium. We now have identified "coinducer" molecules on DP thymocytes that, together with TCR, signal DP thymocytes to differentiate into SP T cells in vitro in the absence of thymic epithelium. A remarkable number of different molecules on DP thymocytes possessed "coinducing" activity, including CD2, CD5, CD24, CD28, CD49d, CD81, and TSA-1. Interestingly, in vitro differentiation occurred in the absence of lineage-specific signals, yet resulted in the selective generation of CD4+CD8- T cells. Thus, the present study has identified surface molecules that can signal DP thymocytes to differentiate into SP T cells in the absence of thymic epithelium and has characterized a default pathway for CD4+ T cell differentiation.

T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus.

Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.