Naive B Cell Output in HIV-Infected and HIV-Uninfected Children.

In this study, we aimed to quantify KREC (kappa-deleting recombination excision circle) levels and naive B cell output in healthy HIV-uninfected children, compared with HIV-infected South African children, before and after starting ART (antiretroviral therapy). Samples were acquired from a Child Wellness Clinic (n = 288 HIV-uninfected South African children, 2 weeks-12 years) and the Children with HIV Early Antiretroviral Therapy (CHER) trial (n = 153 HIV-infected South African children, 7 weeks-8 years). Naive B cell output was estimated using a mathematical model combining KREC levels to reflect B cell emigration into the circulation, flow cytometry measures of naive unswitched B cells to quantify total body naive B cells, and their rates of proliferation using the intracellular marker Ki67. Naive B cell output increases from birth to 1 year, followed by a decline and plateau into late childhood. HIV-infected children on or off ART had higher naive B cell outputs than their uninfected counterparts (p = .01 and p = .04). This is the first study to present reference ranges for measurements of KRECs and naive B cell output in healthy and HIV-infected children. Comparison between HIV-uninfected healthy children and HIV-infected children suggests that HIV may increase naive B cell output. Further work is required to fully understand the mechanisms involved and clinical value of measuring naive B cell output in children.

Clinical T Cell Receptor Repertoire Deep Sequencing and Analysis: An Application to Monitor Immune Reconstitution Following Cord Blood Transplantation.

Spectratyping assays are well recognized as the clinical gold standard for assessing the T cell receptor (TCR) repertoire in haematopoietic stem cell transplant (HSCT) recipients. These assays use length distributions of the hyper variable complementarity-determining region 3 (CDR3) to characterize a patient's T cell immune reconstitution post-transplant. However, whilst useful, TCR spectratyping is notably limited by its resolution, with the technique unable to provide data on the individual clonotypes present in a sample. High-resolution clonotype data are necessary to provide quantitative clinical TCR assessments and to better understand clonotype dynamics during clinically relevant events such as viral infections or GvHD. In this study we developed and applied a CDR3 Next Generation Sequencing (NGS) methodology to assess the TCR repertoire in cord blood transplant (CBT) recipients. Using this, we obtained comprehensive TCR data from 16 CBT patients and 5 control cord samples at Great Ormond Street Hospital (GOSH). These were analyzed to provide a quantitative measurement of the TCR repertoire and its constituents in patients post-CBT. We were able to both recreate and quantify inferences typically drawn from spectratyping data. Additionally, we demonstrate that an NGS approach to TCR assessment can provide novel insights into the recovery of the immune system in these patients. We show that NGS can be used to accurately quantify TCR repertoire diversity and to provide valuable inference on clonotypes detected in a sample. We serially assessed the progress of T cell immune reconstitution demonstrating that there is dramatic variation in TCR diversity immediately following transplantation and that the dynamics of T cell immune reconstitution is perturbed by the presence of GvHD. These findings provide a proof of concept for the adoption of NGS TCR sequencing in clinical practice.

Thymic Output and CD4 T-Cell Reconstitution in HIV-Infected Children on Early and Interrupted Antiretroviral Treatment: Evidence from the Children with HIV Early Antiretroviral Therapy Trial.

OBJECTIVES: Early treatment of HIV-infected children and adults is important for optimal immune reconstitution. Infants' immune systems are more plastic and dynamic than older children's or adults', and deserve particular attention. This study aimed to understand the response of the HIV-infected infant immune system to early antiretroviral therapy (ART) and planned ART interruption and restart. METHODS: Data from HIV-infected children enrolled the CHER trial, starting ART aged between 6 and 12 weeks, were used to explore the effect of ART on immune reconstitution. We used linear and non-linear regression and mixed-effects models to describe children's CD4 trajectories and to identify predictors of CD4 count during early and interrupted ART. RESULTS: Early treatment arrested the decline in CD4 count but did not fully restore it to the levels observed in HIV-uninfected children. Treatment interruption at 40 or 96 weeks resulted in a rapid decline in CD4 T-cells, which on retreatment returned to levels observed before interruption. Naïve CD4 T-cell count was an important determinant of overall CD4 levels. A strong correlation was observed between thymic output and the stable CD4 count both before and after treatment interruption. CONCLUSION: Early identification and treatment of HIV-infected infants is important to stabilize CD4 counts at the highest levels possible. Once stabilized, children's CD4 counts appear resilient, with good potential for recovery following treatment interruption. The naïve T-cell pool and thymic production of naive cells are key determinants of children's CD4 levels.

Modelling CD4 T Cell Recovery in Hepatitis C and HIV Co-infected Children Receiving Antiretroviral Therapy.

BACKGROUND: The effect of hepatitis C virus (HCV) coinfection on CD4 T cell recovery in treated HIV-infected children is poorly understood. OBJECTIVE: To compare CD4 T cell recovery in HIV/HCV coinfected children with recovery in HIV monoinfected children. METHOD: We studied 355 HIV monoinfected and 46 HIV/HCV coinfected children receiving antiretroviral therapy (ART) during a median follow-up period of 4.2 years (interquartile range: 2.7-5.3 years). Our dataset came from the Ukraine pediatric HIV Cohort and the HIV/HCV coinfection study within the European Pregnancy and Paediatric HIV Cohort Collaboration. We fitted an asymptotic nonlinear mixed-effects model of CD4 T cell reconstitution to age-standardized CD4 counts in all 401 children and investigated factors predicting the speed and extent of recovery. RESULTS: We found no significant impact of HCV coinfection on either pre-ART or long-term age-adjusted CD4 counts (z scores). However, the rate of increase in CD4 z score was slower in HIV/HCV coinfected children when compared with their monoinfected counterparts (P < 0.001). Both monoinfected and coinfected children starting ART at younger ages had higher pre-ART (P < 0.001) and long-term (P < 0.001) CD4 z scores than those who started when they were older. CONCLUSIONS: HIV/HCV coinfected children receiving ART had slower CD4 T cell recovery than HIV monoinfected children. HIV/HCV coinfection had no impact on pre-ART or long-term CD4 z scores. Early treatment of HIV/HCV coinfected children with ART should be encouraged.

Distinguishing the Signals of Gingivitis and Periodontitis in Supragingival Plaque: a Cross-Sectional Cohort Study in Malawi.

UNLABELLED: Periodontal disease ranges from gingival inflammation (gingivitis) to the inflammation and loss of tooth-supporting tissues (periodontitis). Previous research has focused mainly on subgingival plaque, but supragingival plaque composition is also known to be associated with disease. Quantitative modeling of bacterial abundances across the natural range of periodontal severities can distinguish which features of disease are associated with particular changes in composition. We assessed a cross-sectional cohort of 962 Malawian women for periodontal disease and used 16S rRNA gene amplicon sequencing (V5 to V7 region) to characterize the bacterial compositions of supragingival plaque samples. Associations between bacterial relative abundances and gingivitis/periodontitis were investigated by using negative binomial models, adjusting for epidemiological factors. We also examined bacterial cooccurrence networks to assess community structure. The main differences in supragingival plaque compositions were associated more with gingivitis than periodontitis, including higher bacterial diversity and a greater abundance of particular species. However, even after controlling for gingivitis, the presence of subgingival periodontitis was associated with an altered supragingival plaque. A small number of species were associated with periodontitis but not gingivitis, including members of Prevotella, Treponema, and Selenomonas, supporting a more complex disease model than a linear progression following gingivitis. Cooccurrence networks of periodontitis-associated taxa clustered according to periodontitis across all gingivitis severities. Species including Filifactor alocis and Fusobacterium nucleatum were central to this network, which supports their role in the coaggregation of periodontal biofilms during disease progression. Our findings confirm that periodontitis cannot be considered simply an advanced stage of gingivitis even when only considering supragingival plaque. IMPORTANCE: Periodontal disease is a major public health problem associated with oral bacteria. While earlier studies focused on a small number of periodontal pathogens, it is now accepted that the whole bacterial community may be important. However, previous high-throughput marker gene sequencing studies of supragingival plaque have largely focused on high-income populations with good oral hygiene without including a range of periodontal disease severities. Our study includes a large number of low-income participants with poor oral hygiene and a wide range of severities, and we were therefore able to quantitatively model bacterial abundances as functions of both gingivitis and periodontitis. A signal associated with periodontitis remains after controlling for gingivitis severity, which supports the concept that, even when only considering supragingival plaque, periodontitis is not simply an advanced stage of gingivitis. This suggests the future possibility of diagnosing periodontitis based on bacterial occurrences in supragingival plaque.

How many TCR clonotypes does a body maintain?

We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude.

Reactivity of routine HIV antibody tests in children who initiated antiretroviral therapy in early infancy as part of the Children with HIV Early Antiretroviral Therapy (CHER) trial: a retrospective analysis.

BACKGROUND: Early antiretroviral therapy (ART) and virological suppression can affect evolving antibody responses to HIV infection. We aimed to assess frequency and predictors of seronegativity in infants starting early ART. METHODS: We compared HIV antibody results between two of three treatment groups of the Children with HIV Early Antiretroviral Therapy (CHER) trial, done from July, 2005, until July, 2011, in which infants with HIV infection aged 5·7-12·0 weeks with a percentage of CD4-positive T lymphocytes of at least 25% were randomly assigned to immediate ART for 96 weeks (ART-96W) or deferred ART until clinical or immunological progression (ART-Def). We measured antibody from all available stored samples for ART-96W and ART-Def at trial week 84 using three assays: fourth-generation enzyme immunoassay HIV antigen-antibody combination, HIV-1 and HIV-2 rapid antibody test, and quantitative anti-gp120 IgG ELISA. We also assessed odds of seropositivity with respect to age of ART initiation and cumulative viral load. The CHER trial was registered with ClinicalTrials.gov, number NCT00102960. FINDINGS: The median age of the infants from when samples were taken (184 samples from 268 infants) was 92 weeks (IQR 90·6-93·4). More specimens from the ART-96W group were seronegative than from the ART-Def group by enzyme immunoassay (ART-96W 49 [46%] of 107 vs ART-Def eight [11%] of 75; p<0·0001) and rapid antibody test (54 [53%] of 101 vs eight [11%] of 74; p<0·0001). Median anti-gp120 IgG concentration was lower in the ART-96W group (230 µg/µL [IQR 133-13 129]) than in the ART-Def group (6870 µg/µL [1706-53 645]; p<0·0001). If ART was started between 12 and 24 weeks of age, odds of seropositivity were increased 13·7 times (95% CI 3·1-60·2; p=0·001) compared with starting it between 0 and 12 weeks. All children starting ART aged older than 24 weeks were seropositive. Cumulative viral load to week 84 correlated with anti-gp120 IgG concentrations (coefficient 0·54; p<0·0001) and increased odds of seropositivity (odds ratio 1·59 [95% CI 1·1-2·3]) adjusted for ART initiation age. INTERPRETATION: About half of children starting ART before 12 weeks of age were HIV seronegative by almost 2 years of age. HIV antibody tests cannot be used to reconfirm HIV diagnosis in children starting early ART. Long-term effects of seronegativity need further study. Clear guidelines are needed for retesting alongside improved diagnostic tests. FUNDING: Wellcome Trust, Medical Research Council, and National Institutes of Health.

Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods.

BACKGROUND: Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers. RESULTS: Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5' Rapid amplification of cDNA ends (5'RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA. CONCLUSIONS: Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.

Neuroblastoma killing properties of Vδ2 and Vδ2-negative γδT cells following expansion by artificial antigen-presenting cells.

PURPOSE: The majority of circulating human γδT lymphocytes are of the Vγ9Vδ2 lineage, and have T-cell receptor (TCR) specificity for nonpeptide phosphoantigens. Previous attempts to stimulate and expand these cells have therefore focused on stimulation using ligands of the Vγ9Vδ2 receptor, whereas relatively little is known about variant blood γδT subsets and their potential role in cancer immunotherapy. EXPERIMENTAL DESIGN: To expand the full repertoire of γδT without bias toward specific TCRs, we made use of artificial antigen-presenting cells loaded with an anti γδTCR antibody that promoted unbiased expansion of the γδT repertoire. Expanded cells from adult blood donors were sorted into 3 populations expressing respectively Vδ2 TCR chains (Vδ2(+)), Vδ1 chains (Vδ1(+)), and TCR of other δ chain subtypes (Vδ1(neg)Vδ2(neg)). RESULTS: Both freshly isolated and expanded cells showed heterogeneity of differentiation markers, with a less differentiated phenotype in the Vδ1 and Vδ1(neg)Vδ2(neg) populations. Expanded cells were largely of an effector memory phenotype, although there were higher numbers of less differentiated cells in the Vδ1(+) and Vδ1(neg)Vδ2(neg) populations. Using neuroblastoma tumor cells and the anti-GD2 therapeutic mAb ch14.18 as a model system, all three populations showed clinically relevant cytotoxicity. Although killing by expanded Vδ2 cells was predominantly antibody dependent and proportionate to upregulated CD16, Vδ1 cells killed by antibody-independent mechanisms. CONCLUSIONS: In conclusion, we have demonstrated that polyclonal-expanded populations of γδT cells are capable of both antibody-dependent and -independent effector functions in neuroblastoma.

Revealing individual signatures of human T cell CDR3 sequence repertoires with Kidera Factors.

The recent development of High Throughput Sequencing technologies has enabled an individual's TCR repertoire to be efficiently analysed at the nucleotide level. However, with unique clonotypes ranging in the tens of millions per individual, this approach gives a surfeit of information that is difficult to analyse and interpret in a biological context and gives little information about TCR structural diversity. Using publicly available TCR CDR3 sequence data, we analysed TCR repertoires by converting the encoded CDR3 amino acid sequences into Kidera Factors, a set of orthogonal physico-chemical properties that reflect protein structure. This approach enabled the TCR repertoire from different individuals to be distinguished and demonstrated the close similarity of the repertoire in different samples from the same individual.

Antiretroviral therapy increases thymic output in children with HIV.

OBJECTIVE: Disease progression and response to antiretroviral therapy (ART) in HIV-infected children is different to that of adults. Immune reconstitution in adults is mainly from memory T cells, whereas in children it occurs predominantly from the naive T-cell pool. It is unclear however what proportion of reconstituted CD4 T cells comes from thymic export and homeostatic proliferation in the periphery. Thymic output is often estimated by measuring T-cell receptor excision circles and markers such as CD31 expressed on recent thymic emigrants but these are confounded by peripheral T-cell division and cannot in themselves be used as quantitative estimates of thymic output. DESIGN: To compare thymic output in HIV-infected children on ART, HIV-infected children not on ART and uninfected children of different ages. METHOD: Combined T-cell receptor excision circle (TREC) and proliferation data are used with a recently described mathematical model to give explicit measures of thymic output. RESULTS: We found that age-adjusted thymic output is reduced in untreated children with HIV, which increases significantly with length of time on ART. CONCLUSION: Our results suggest that a highly active thymus in early childhood may contribute to better immune reconstitution if ART is initiated early in life.

A mechanistic model for naive CD4 T cell homeostasis in healthy adults and children.

The size and composition of the T lymphocyte compartment is subject to strict homeostatic regulation and is remarkably stable throughout life in spite of variable dynamics in cell production and death during T cell development and immune responses. Homeostasis is achieved by careful orchestration of lymphocyte survival and cell division. New T cells are generated from the thymus and the number of peripheral T cells is regulated by controlling survival and proliferation. How these processes combine is however very complex. Thymic output increases in the first year of life and then decreases but is crucial for establishing repertoire diversity. Proliferation of new naive T cells plays a crucial role for maintaining numbers but at a potential cost to TCR repertoire diversity. A mechanistic two-compartment model of T cell homeostasis is described here that includes specific terms for thymic output, cell proliferation, and cell death of both resting and dividing cells. The model successfully predicts the homeostatic set point for T cells in adults and identifies variables that determine the total number of T cells. It also accurately predicts T cell numbers in children in early life despite rapid changes in thymic output and growth over this period.

Predicting patterns of long-term CD4 reconstitution in HIV-infected children starting antiretroviral therapy in sub-Saharan Africa: a cohort-based modelling study.

BACKGROUND: Long-term immune reconstitution on antiretroviral therapy (ART) has important implications for HIV-infected children, who increasingly survive into adulthood. Children's response to ART differs from adults', and better descriptive and predictive models of reconstitution are needed to guide policy and direct research. We present statistical models characterising, qualitatively and quantitatively, patterns of long-term CD4 recovery. METHODS AND FINDINGS: CD4 counts every 12 wk over a median (interquartile range) of 4.0 (3.7, 4.4) y in 1,206 HIV-infected children, aged 0.4-17.6 y, starting ART in the Antiretroviral Research for Watoto trial (ISRCTN 24791884) were analysed in an exploratory analysis supplementary to the trial's pre-specified outcomes. Most (n = 914; 76%) children's CD4 counts rose quickly on ART to a constant age-corrected level. Using nonlinear mixed-effects models, higher long-term CD4 counts were predicted for children starting ART younger, and with higher CD4 counts (p<0.001). These results suggest that current World Health Organization-recommended CD4 thresholds for starting ART in children ≥5 y will result in lower CD4 counts in older children when they become adults, such that vertically infected children who remain ART-naïve beyond 10 y of age are unlikely ever to normalise CD4 count, regardless of CD4 count at ART initiation. CD4 profiles with four qualitatively distinct reconstitution patterns were seen in the remaining 292 (24%) children. Study limitations included incomplete viral load data, and that the uncertainty in allocating children to distinct reconstitution groups was not modelled. CONCLUSIONS: Although younger ART-naïve children are at high risk of disease progression, they have good potential for achieving high CD4 counts on ART in later life provided ART is initiated following current World Health Organization (WHO), Paediatric European Network for Treatment of AIDS, or US Centers for Disease Control and Prevention guidelines. In contrast, to maximise CD4 reconstitution in treatment-naïve children >10 y, ART should ideally be considered even if there is a low risk of immediate disease progression. Further exploration of the immunological mechanisms for these CD4 recovery profiles should help guide management of paediatric HIV infection and optimise children's immunological development. Please see later in the article for the Editors' Summary.

Heterogeneity in thymic emigrants: implications for thymectomy and immunosenescence.

The development of mature, antigen-inexperienced (naive) T cells begins in the thymus and continues after export into the periphery. Post-thymic maturation of naive T cells, in humans, coincides with the progressive loss of markers such as protein tyrosine kinase 7 (PTK7) and platelet endothelial cell adhesion molecule-1 (CD31). As a consequence, subpopulations of naive T cells can be recognised raising questions about the processes that give rise to the loss of these markers and their exact relationship to recent thymic emigrants (RTE). Here, we combine a mathematical survival analysis approach and data from healthy and thymectomised humans to understand the apparent persistence of populations of 'veteran' PTK7 (+) T cells in thymectomised individuals. We show that a model of heterogeneity in rates of maturation, possibly linked to natural variation in TCR signalling thresholds or affinity for self-antigens, can explain the data. This model of maturation predicts that the average post-thymic age of PTK7 (+) T cells will increase linearly with the age of the host suggesting that, despite the immature phenotype, PTK7 (+) cells do not necessarily represent a population of RTE. Further, the model predicts an accelerated increase in the average post-thymic age of residual PTK7 (+) T cells following thymectomy and may also explain in part the prematurely aged phenotype of the naive T cell pool in individuals thymectomised early in life.

Clonally diverse T cell homeostasis is maintained by a common program of cell-cycle control.

Lymphopenia induces T cells to undergo cell divisions as part of a homeostatic response mechanism. The clonal response to lymphopenia is extremely diverse, and it is unknown whether this heterogeneity represents distinct mechanisms of cell-cycle control or whether a common mechanism can account for the diversity. We addressed this question by combining in vivo and mathematical modeling of lymphopenia-induced proliferation (LIP) of two distinct T cell clonotypes. OT-I T cells undergo rapid LIP accompanied by differentiation that superficially resembles Ag-induced proliferation, whereas F5 T cells divide slowly and remain naive. Both F5 and OT-I LIP responses were most accurately described by a single stochastic division model where the rate of cell division was exponentially decreased with increasing cell numbers. The model successfully identified key biological parameters of the response and accurately predicted the homeostatic set point of each clone. Significantly, the model was successful in predicting interclonal competition between OT-I and F5 T cells, consistent with competition for the same resource(s) required for homeostatic proliferation. Our results show that diverse and heterogeneous clonal T cell responses can be accounted for by a single common model of homeostasis.

The identification of a new role for LEKTI in the skin: The use of protein 'bait' arrays to detect defective trafficking of dermcidin in the skin of patients with Netherton syndrome.

Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI) has been demonstrated to be an inhibitor of various kallikreins and is thought to play a role in the regulation of skin desquamation. In order to identify and investigate the potential of LEKTI to interact with other proteins, a method was developed using immobilised proteins onto arrays and nanoUPLC/MALDI-TOF MS. Using various domains of LEKTI, we demonstrated that these domains bound a number of kallikreins (5, 13 and 14) to varied extents on the array surface. Inhibitory assays confirmed that binding on the protein array surface corresponded directly to levels of inhibition. The method was then tested using skin epidermal extracts. All forms of rLEKTI with the exception of rLEKTI 12-15, demonstrated the binding of several potential candidate proteins. Surprisingly, the major binding partners of LEKTI were found to be the antimicrobial peptide dermcidin and the serine protease cathepsin G and no kallikreins. Using confocal microscopy and Netherton syndrome skin sections, we confirmed the co-localisation of LEKTI with dermcidin and demonstrated altered trafficking of dermcidin in these patients. This potential new role for LEKTI as a multifunctional protein in the protection and transport of proteins in the epidermis and its role in disease are discussed.

Age and CD4 count at initiation of antiretroviral therapy in HIV-infected children: effects on long-term T-cell reconstitution.

BACKGROUND: Effective therapies and reduced AIDS-related morbidity and mortality have shifted the focus in pediatric human immunodeficiency virus (HIV) from minimizing short-term disease progression to maintaining optimal long-term health. We describe the effects of children's age and pre-antiretroviral therapy (ART) CD4 count on long-term CD4 T-cell reconstitution. METHODS: CD4 counts in perinatally HIV-infected, therapy-naive children in the Paediatric European Network for the Treatment of AIDS 5 trial were monitored following initiation of ART for a median 5.7 years. In a substudy, naive and memory CD4 counts were recorded. Age-standardized measurements were analyzed using monophasic, asymptotic nonlinear mixed-effects models. RESULTS: One hundred twenty-seven children were studied. Older children had lower age-adjusted CD4 counts in the long term and at treatment initiation (P < .001). At all ages, lower counts before treatment were associated with impaired recovery (P < .001). Age-adjusted naive CD4 counts increased on a timescale comparable to overall CD4 T-cell reconstitution, whereas age-adjusted memory CD4 counts increased less, albeit on a faster timescale. CONCLUSIONS: It appears the immature immune system can recover well from HIV infection via the naive pool. However, this potential is progressively damaged with age and/or duration of infection. Current guidelines may therefore not optimize long-term immunological health.