Child and adolescent psychiatry training in Australia and New Zealand.

The specialty of Child and Adolescent Psychiatry was formally recognised in the 1930s. The Faculty of Child and Adolescent Psychiatry was established in 1964 in Australia, as a subspecialty in The Royal Australian and New Zealand College of Psychiatrists (RANZCP). The aim of the current article is first to provide a brief summary and overview of the current status of Child and Adolescent Psychiatry (CAP), followed by an outline of the requirements of the Training Program for CAP in Australia and New Zealand. The training required to become a fully qualified child and adolescent psychiatrist in Australia and New Zealand consists of different stages and takes the form of competency-based training. Information relating to assessment types, supervision and research requirements is also described. Accreditation procedures for the training program are stipulated by RANZCP to monitor standards and to ensure consistency within the programs delivered across Australia and New Zealand. Employment opportunities for trainees upon completion of the program are discussed. In summary, this article highlights the requirements of the training programs for CAP in Australia and New Zealand.

TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells.

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the α/ß-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

The effect of anaesthetic agents on induction, recovery and patient preferences in adult day case surgery: a 7-day follow-up randomized controlled trial.

BACKGROUND AND OBJECTIVE: To compare induction, pre- and post-discharge recovery characteristics and patient preferences between four anaesthetic regimens in adult day-surgery. METHODS: Randomized controlled trial. In all, 1158 adults assigned to: propofol induction and maintenance, propofol induction with isoflurane/N2O, or sevoflurane/N2O maintenance, or sevoflurane/N2O alone. We prospectively recorded induction and pre-discharge recovery characteristics, collected 7-day post-discharge recovery characteristics using patient diaries and patient preferences by telephone follow-up. RESULTS: Recruitment rate was 73%--of the 425 refusals, 226 were not willing to risk a volatile induction. During induction, excitatory movements and breath holding were more common with sevoflurane only (P < 0.01). Injection pain and hiccup were more common with propofol induction (P < 0.01). In the recovery room and the postoperative ward, both nausea and vomiting were more common with sevoflurane only (P < 0.01). This difference disappeared within 48 h. There was no difference between groups in the mental state on awakening, recovery time, time to discharge or overnight admissions; then was also no difference in pain between the four groups for each of the seven postoperative days (P < 0.01), nor any differences in concentration or forgetfulness. Patients took 6.5 days (95% CI: 6.0-7.0, n = 693) to resume normal activities. Patients who received sevoflurane only were more likely to recall an unpleasant induction and least likely to want the same induction method again (P < 0.01). CONCLUSION: Differences in outcome between the four regimens are transient; sevoflurane is not an ideal sole agent for adult day case anaesthesia and, in this setting, patients base their preferences for future anaesthetics on the method of induction.

Telomere maintenance is dependent on activities required for end repair of double-strand breaks.

Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.

Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.

Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.

Patients with the worst outcomes after paracetamol (acetaminophen)-induced liver failure have an early monocytopenia.

BACKGROUND: Acute liver failure (ALF) is associated with significant morbidity and mortality. Studies have implicated the immune response, especially monocyte/macrophages as being important in dictating outcome. AIM: To investigate changes in the circulating monocytes and other immune cells serially in patients with ALF, relate these with cytokine concentrations, monocyte gene expression and patient outcome. METHODS: In a prospective case-control study in the Scottish Liver Transplant Unit, Royal Infirmary Edinburgh, 35 consecutive patients admitted with paracetamol-induced liver failure (POD-ALF), 10 patients with non-paracetamol causes of ALF and 16 controls were recruited. The peripheral blood monocyte phenotype was analysed by flow cytometry, circulating cytokines quantified by protein array and monocyte gene expression array performed and related to outcome. RESULTS: On admission, patients with worst outcomes after POD-ALF had a significant monocytopenia, characterised by reduced classical and expanded intermediate monocyte population. This was associated with reduced circulating lymphocytes and natural killer cells, peripheral cytokine patterns suggestive of a 'cytokine storm' and increased concentrations of cytokines associated with monocyte egress from the bone marrow. Gene expression array did not differentiate patient outcome. At day 4, there was no significant difference in monocyte, lymphocyte or natural killer cells between survivors and the patients with adverse outcomes. CONCLUSIONS: Severe paracetamol liver failure is associated with profound changes in the peripheral blood compartment, particularly in monocytes, related with worse outcomes. This is not seen in patients with non-paracetamol-induced liver failure. Significant monocytopenia on admission may allow earlier clarification of prognosis, and it highlights a potential target for therapeutic intervention.

RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase.

Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.

Autologous anti-metatype immune response in rabbits.

Rabbits hyperimmunized with fluorescyl-conjugated KLH exhibited bound ligand associated with a high affinity circulating IgG anti-fluorescein population. After cessation of immunogen administration the liganded complexes were eventually spontaneously cleared from the circulation. Individual rabbits synthesized autologous anti-metatype antibodies specific for ligand-antibody complexes. Autologous anti-metatype antibodies reacted optimally with autologous liganded anti-fluorescein antibodies. However, cross reactivity was noted with allogenic rabbit liganded antibodies from three affinity-purified pools. An autologous anti-metatype response, reminiscent of autoanti-idiotype responses, has important implications concerning in vivo clearance of antigen-antibody complexes and may serve as a model to study immune complex diseases.

Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury.

Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury by profiling the circulating miRNome in patients with acetaminophen overdose. Plasma miRNAs were quantified in age- and sex-matched overdose patients with (N = 27) and without (N = 27) organ injury (APAP-TOX and APAP-no TOX, respectively). Classifier miRNAs were tested in a separate cohort (N = 81). miRNA specificity was determined in non-acetaminophen liver injury and murine models. Sensitivity was tested by stratification of patients at hospital presentation (N = 67). From 1809 miRNAs, 75 were 3-fold or more increased and 46 were 3-fold or more decreased with APAP-TOX. A 16 miRNA classifier model accurately diagnosed APAP-TOX in the test cohort. In humans, the miRNAs with the largest increase (miR-122-5p, miR-885-5p, miR-151a-3p) and the highest rank in the classifier model (miR-382-5p) accurately reported non-acetaminophen liver injury and were unaffected by kidney injury. miR-122-5p was more sensitive than ALT for reporting liver injury at hospital presentation, especially combined with miR-483-3p. A miRNA panel was associated with human kidney dysfunction. In mice, miR-122-5p, miR-151a-3p and miR-382-5p specifically reported APAP toxicity - being unaffected by drug-induced kidney injury. Profiling of acetaminophen toxicity identified multiple miRNAs that report acute liver injury and potential biomarkers of drug-induced kidney injury.

The primate cochlear nuclei: loss of lamination as a phylogenetic process.

In primates from prosimians to hominoids (lorisoid prosimians, marmoset and ceboid monkeys, cercopithecoid monkeys, and gibbon), there are differences in the location, depth, and extent of the granular layer of the cochlear nuclei. In the prosimians, the deep granular layer of the DCN is similar to that of other mammals, but there is, in addition, a superficial or subependymal layer of granule cells in the DCN. In ceboid and cercopithecoid monkeys, only a superficial or external granular layer is present in the DCN, and the granular layer over the VCN is much reduced. In the gibbon, there is no granular layer in either of the cochlear muclei. In conjunction with the progressive reduction of the cochlear granular layer in primates, fusiform cells lose their position as a radially oriented peripheral cell layer in the DCN and become located in the central region of the nucleus. These changes in primates are interpreted as resulting from failure of inward migration and increasing cell death in the ontogeny of the cochlear external granular layer, with concomitant changes occurring in the position and orientation of their target neurons, the fusiform cells.

Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat.

The distribution of GABA-producing neurons in the brainstem auditory nuclei of the rat was investigated immunohistochemically by using an antibody to glutamic acid decarboxylase (GAD). In the cochlear nuclei, GAD immunoreactive neurons are present only in the superficial granular and molecular layers, whereas terminals are found in all subdivisions of the nuclei and are particularly dense surrounding large spherical cells and one type of stellate cell. In the superior olivary complex, GAD immunoreactive neurons are located in the lateral olivary nucleus and throughout the periolivary region. Immunoreactive terminals are distributed along dendrites of principal cells of the medial and lateral olivary nuclei and are clustered around somata of globular neurons of the nucleus of the trapezoid body. An extremely dense band of immunoreactive somata and terminals is present along the ventral edge of the olivary complex. The ventral, intermediate, and dorsal nuclei of the lateral lemniscus contain small fusiform GAD-immunoreactive neurons and a moderately dense plexus of immunoreactive terminals. The inferior colliculus contains a large population of GAD-immunoreactive perikarya and an extremely dense accumulation of immunoreactive terminals in the central, dorsomedial, and external nuclei. These observations indicate that GABA systems are involved in function at all levels of the brainstem auditory pathway.

Nigrotectal projection to the inferior colliculus: horseradish peroxidase transport and tyrosine hydroxylase immunohistochemical studies in rats, cats, and bats.

The present study investigated descending projections from the substantia nigra to the auditory tectum. Small (0.02-0.05 microliters) injections of a 30-60% aqueous solution of horseradish peroxidase (HRP) were made unilaterally into the inferior colliculus in rats, cats, and bats (Eptesicus fuscus). Tissue blocks including the substantia nigra, superior colliculus, and inferior colliculus were removed, sectioned, and processed for visualization of HRP. Results show that the substantia nigra, pars lateralis, projects to the inferior colliculus ipsilaterally. In addition, retrogradely labeled cells are found dorsal to the pars lateralis, in a column within the lateral tegmental area of the midbrain. Analysis of injection sites suggests that the principal target of this nigral projection is the dorsal and rostral pericentral region of the inferior colliculus. Immunohistochemical studies with an antibody to tyrosine hydroxylase demonstrate catecholaminergic neurons within the pars lateralis and lateral tegmentum that are similar in location and morphology to one class of HRP retrogradely labeled cells within these structures. These immunohistochemical studies also demonstrate a plexus of fine, varicose tyrosine hydroxylase-positive axons in the rostral pericentral region of the colliculus. The presence of this nigrotectal projection to the inferior colliculus is discussed in relation to its possible role in the control of acousticomotor behavior.

Immunohistochemical organization of the ventral lateral geniculate nucleus in the ground squirrel.

The ventral lateral geniculate nucleus (vLGN) of the thirteen-lined ground squirrel (Citellus tridecemlineatus) is a highly differentiated nucleus that is divisible into five major subdivisions on the basis of retinal projections and cytoarchitecture. To pursue the likelihood that these subdivisions (the dorsal cap, intergeniculate leaflet, external magnocellular lamina, internal magnocellular lamina, and parvicellular segment) correlate with the functional diversity of this complex, the present study examined the neurochemical composition of the vLGN with regard to substances that have previously proved useful in distinguishing functionally distinct subregions within nuclei (i.e., neuropeptide Y (NPY), substance P (SP), leucine and methionine enkephalins, gamma-aminobutyric acid (GABA), cytochrome oxidase (CO), acetylcholinesterase (AChE), and NADPH-diaphorase). The results showed a clear differential neurochemical distribution within the nucleus. Neuropeptide Y immunoreactive perikarya were found predominantly in the intergeniculate leaflet and external magnocellular lamina, with only a few present in the internal magnocellular lamina and dorsal cap, and none observed in the parvicellular segment. NPY+ fibers, however, were present in all divisions except the parvicellular segment. The highest concentration of SP immunoreactive cells was observed in the internal magnocellular lamina, and substantial numbers also were scattered in the external magnocellular lamina and parvicellular segment. SP+ fibers were seen predominantly in the intergeniculate leaflet and the magnocellular laminae. The heaviest concentration of enkephalinergic fibers occurred in the internal magnocellular lamina and dorsal cap, but fibers were also observed in the external magnocellular lamina and intergeniculate leaflet. GABA reactivity was widespread throughout the vLGN, with the dorsal cap and external magnocellular lamina most heavily labeled, followed by the intergeniculate leaflet and the internal magnocellular lamina. Cytochrome oxidase, AChE, and NADPH-diaphorase histochemistry revealed rich reactivity within the dorsal cap, and external and internal magnocellular laminae and paler reactivity in the intergeniculate leaflet and parvicellular segment. The external magnocellular lamina was more reactive for CO and NADPH-diaphorase than AChE, while the internal magnocellular lamina showed the opposite pattern of reactivity. In addition, NADPH-diaphorase reactive cells were present in caudal intergeniculate leaflet and lateral external magnocellular lamina. These local differences in the neurochemical character of the vLGN support its parcellation into multiple subdivisions. Taken in conjunction with the differences in cytoarchitecture and retinal projections, these results suggest substantial functional diversity within the ventral lateral geniculate complex.

gamma-Aminobutyric acid and glycine in the baboon cochlear nuclei: an immunocytochemical colocalization study with reference to interspecies differences in inhibitory systems.

Previous studies of the cochlear nuclei in cat, rat, and guinea pig have demonstrated neural structures that are enriched in the inhibitory neurotransmitter amino acids gamma-aminobutyric acid (GABA) and glycine. In these mammals, inhibitory terminals are widely distributed throughout the nuclear complex, but somata of inhibitory neurons are concentrated in the dorsal cochlear nucleus, in granule cell regions, and in the cap area. Because these are the subdivisions that undergo the most pronounced phylogenetic changes in primates, we wanted to see whether the inhibitory systems are influenced by changes in cytoarchitecture. Therefore, we applied light microscopic postembedding immunostaining and optical densitometry to the cochlear nuclei of an anthropoid primate, the Senegalese baboon (Papio anubis). Our results demonstrate that, in baboon 1) glycinergic neurons and axons in the ventral cochlear nucleus seem to form a commissural system similar to that of other mammals; 2) the tuberculoventral system appears to be unchanged in morphology but exhibits a higher level of colocalization of GABA with glycine; 3) there is a reduction of the granule/cartwheel cell system, which is reflected in lesser numbers of inhibitory cartwheel, Golgi, and molecular layer stellate cells; 4) the cap area is larger than in rodents and carnivores and contains many neurons that colocalize GABA and glycine; and 5) throughout the nuclear complex, a higher proportion of the inhibitory terminals colocalize GABA and glycine. We conclude that modulation of the ascending auditory pathway in baboon is likely to differ from that in rodents and cat.

Durable donor-specific T and B cell tolerance in rhesus macaques induced with peritransplantation anti-CD3 immunotoxin and deoxyspergualin: absence of chronic allograft nephropathy.

Tolerance induction can prevent acute kidney allograft rejection without chronic immunosuppression. It is uncertain whether specific tolerance can prevent chronic allograft nephropathy (CAN), which involves both nonimmune and immune injury. This report provides evidence that immunologically tolerant macaques, induced with immunotoxin and deoxyspergualin, developed neither acute rejection nor CAN. Long survivors, bearing MHC-mismatched grafts without chronic immunosuppression for 0.8 to 3.4 years, exhibited general immune competence with donor-specific T and B cell tolerance and no functional or histological evidence of CAN. Stringent criteria for tolerance were satisfied by specific prolongation of donor skin grafts with rapid rejection of third-party skin, followed by indefinite acceptance of a second donor kidney graft and establishment of microchimerism. Primate tolerance with documented absence of CAN may give impetus to the clinical application of tolerance.

Phenotypic and functional analysis of T-cell recovery after anti-CD3 immunotoxin treatment for tolerance induction in rhesus macaques.

T-cell reduction utilizing specific antibody has been widely used in human transplantation, and is a cornerstone of several tolerance induction strategies in nonhuman primates. We have established a population of long-term tolerant rhesus macaques induced with an anti-CD3epsilon immunotoxin (IT). This treatment effects transient, specific and profound ablation of T cells in blood and lymphoid tissues. In most instances the IT was used in combination with the NF-kappaB inhibitor, 15-Deoxyspergualin. This 2-week long protocol produces a "window of opportunity" for tolerization in which the animal exhibits an enduring quiescent state of unresponsiveness to the allograft, all accomplished without maintenance immunosuppressive drugs. During this induction period, the treated immune system bears some resemblance to that of the neonate, in that T cell numbers are abnormally low and antigen presentation by dendritic cells is precluded by an arrest in their NF-kappaB dependant maturation. In addition, IL-4 production is prominent during and after the tolerance induction interval. For this study we focused on measuring the monkey's ability to repopulate T cells with particular emphasis on the memory T-cell phenotype. Three "memory" phenotypes were utilized; CD3(+)CD45RO(+), CD3(+)CRTH2(+), and CD3(+)CD4(+)CD8(+). All three phenotypes exhibited different patterns of recovery, all of which included transient bursts in their numbers during repopulation. We also estimated thymic activity after T-cell ablation with the use of a newly-described RTE or recent thymic émigré phenotype (a naïve CD8(+)CD103(+) T cell). This marker revealed production of RTE cells including supranormal levels at approximately 6 months post-transplant, implicating thymic function in the repopulation of T-cells. Finally, we measured antibody responses to a panel of antigens (vaccines, environmental antigen, and foreign proteins) that indicated there was no apparent loss of immunologic function during or after the tolerance induction period. Results of studies of T-cell receptor repertoire expression suggest preservation of the pretreatment repertoire, which is consistent with rapid recovery of immune competence to the test antigens. Taken together, these results suggest that while aggressive, this tolerance induction protocol does not appear to incur a prolonged immunologically-compromised state, if at all.

MAP2 expression in developing dendrites of human brainstem auditory neurons.

Immunostaining of cytoskeletal elements has proved to be a useful technique for tracing ontogenetic development in the human central auditory system. In the present study, dendritic development in brainstem auditory nuclei (dorsal and ventral cochlear nuclei, medial and lateral superior olivary nuclei, and inferior colliculus) was studied using an antibody to a microtubule-associated protein, MAP2, a molecule which stabilizes dendritic processes by promoting assembly of microtubules. At 21-22 weeks of gestation, cells within the auditory nuclei first demonstrate cytoplasmic MAP2 immunoreactivity, but no dendritic structures have formed. Filamentous background staining at this stage may represent immunoreactivity in astrocytic processes. By the 24th fetal week, somata of auditory neurons are strongly immunostained and have developed short dendritic processes. During the perinatal period, dendrites extend up to 100-120 microm in length but are still sparsely branched and lack terminal formations. By the sixth postnatal month, neurons in all auditory nuclei have acquired dendritic arbors with a mature appearance. Thus MAP2 immunohistochemistry demonstrates that dendrogenesis in human brainstem auditory nuclei begins 16 weeks prior to term birth but does not reach the stage of mature dendritic morphology until several months into the postnatal period. This extended course of development implies a significant period of time during which neuronal activity could influence dendritic structure and function.

Cytoarchitectural and axonal maturation in human auditory cortex.

This study followed the maturation of human auditory cortex from the beginning of the second trimester of gestation to young adulthood. Histological and immunohistochemical techniques were used to trace the development of a laminar cytoarchitecture and an adult pattern of axonal neurofilament expression. From the 16th fetal week to the 4th postnatal month, the cortex progresses from a marginal layer and an undifferentiated cortical plate to incipient lamination. Between the 22nd fetal week and the 4th postnatal month, a two-tiered band of neurofilament-immunoreactive axons develops in layer I, but subsequent to the 4th month, the number of immunopositive axons in this layer is greatly reduced. Between the middle of the first year of life and age 3 years, the laminar pattern of cytoarchitecture becomes fully mature and a network of immunostained axons develops in layers VI, V, IV, and IlIc. This axonal plexus in the deep cortical layers continues to increase in density until age 5. Beginning at 5 years of age, a network of neurofilament-positive axons develops in the superficial layers IIIb, IIIa, and II, and by 11-12 years of age, overall axonal density is equivalent to that seen in young adulthood. This extended time span of axonal maturation has implications for the emergence of auditory cortical function.