Decreased level of TREM like Transcript 1 (TLT-1) is associated with prematurity and promotes the in-utero inflammatory response to maternal lipopolysaccharide (LPS) exposure.

PROBLEM: The occurrence of preterm birth is associated with multiple factors including bleeding, infection and inflammation. Platelets are mediators of hemostasis and can modulate inflammation through interactions with leukocytes. TREM like Transcript 1 (TLT-1) is a type 1 single Ig domain receptor on activated platelets. In adults, it plays a protective role by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. TLT-1 is expressed in human placenta and found in cord blood. We thus hypothesized that TLT-1 deficiency is associated with prematurity and fetal inflammation. METHOD OF STUDY: To test this hypothesis, we examined cord blood levels of soluble TLT-1 (sTLT) in premature and term infants and compared the inflammatory response in C57BL/6 (WT) and TLT-1-/- (treml1-/- , KO) mice given intraperitoneal LPS mid-gestation RESULTS: The preterm infant cord blood level of sTLT was significantly lower than that found at term. On exposure to LPS, histology of KO (as compared to WT) placenta and decidua showed increased hemorrhage, and KO decidual RNA expression of IL-10 was significantly lower. KO fetal interface tissues (placenta, membranes, amniotic fluid) over time showed increased expression of inflammatory cytokines such as IL-6, IFN-γ, and TNF, but not MCP-1. However, fetal organs showed similar levels. CONCLUSION: There is a potential association between insufficient TLT-1 expression and increased fetal inflammatory responses in the setting of prematurity. The data support further study of TLT-1 in the mechanistic link between bleeding, inflammation and preterm birth, and perhaps as a biomarker in human pregnancy.

Development and Initial Validation of a Frontline Health Worker mHealth Assessment Platform (MEDSINC®) for Children 2-60 Months of Age.

Approximately 3 million children younger than 5 years living in low- and middle-income countries (LMICs) die each year from treatable clinical conditions such as pneumonia, dehydration secondary to diarrhea, and malaria. A majority of these deaths could be prevented with early clinical assessments and appropriate therapeutic intervention. In this study, we describe the development and initial validation testing of a mobile health (mHealth) platform, MEDSINC®, designed for frontline health workers (FLWs) to perform clinical risk assessments of children aged 2-60 months. MEDSINC is a web browser-based clinical severity assessment, triage, treatment, and follow-up recommendation platform developed with physician-based Bayesian pattern recognition logic. Initial validation, usability, and acceptability testing were performed on 861 children aged between 2 and 60 months by 49 FLWs in Burkina Faso, Ecuador, and Bangladesh. MEDSINC-based clinical assessments by FLWs were independently and blindly correlated with clinical assessments by 22 local health-care professionals (LHPs). Results demonstrate that clinical assessments by FLWs using MEDSINC had a specificity correlation between 84% and 99% to LHPs, except for two outlier assessments (63% and 75%) at one study site, in which local survey prevalence data indicated that MEDSINC outperformed LHPs. In addition, MEDSINC triage recommendation distributions were highly correlated with those of LHPs, whereas usability and feasibility responses from LHP/FLW were collectively positive for ease of use, learning, and job performance. These results indicate that the MEDSINC platform could significantly increase pediatric health-care capacity in LMICs by improving FLWs' ability to accurately assess health status and triage of children, facilitating early life-saving therapeutic interventions.

Development of a cost-effective high-throughput process of microsatellite analysis involving miniaturized multiplexed PCR amplification and automated allele identification.

BACKGROUND: Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing. RESULTS: To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software. CONCLUSIONS: In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.

VDJ recombinase-mediated TCR ß locus gene usage and coding joint processing in peripheral T cells during perinatal and pediatric development.

The generation of TCR proteins is the result of V(D)J recombinase-mediated genomic rearrangements at recombination signal sequences (RSS) in human lymphocytes. V(D)J recombinase can also mediate rearrangements at nonimmune or "cryptic" RSS in normal and leukemic human peripheral T cells. We previously demonstrated age- and gender-specific developmental differences in V(D)J coding joint processing at cryptic RSS within the HPRT locus in peripheral T cells from healthy children (Murray et al. 2006. J. Immunol. 177: 5393-5404). In this study, we investigated developmentally specific V(D)J recombinase TCRß immune gene rearrangements and coding joint processing at RSS in peripheral T cells in the same pediatric population. This approach provided a unique opportunity to investigate site-specific V(D)J recombinase rearrangements and coding joint processing at immune and nonimmune genes from the same individual T cell population. We determined the genomic sequence of 244 TCRß coding junctions from 112 (63 male, 49 female) subjects from the late stages of fetal development through 9 y of age. We observed both age- and gender-specific V(D)J recombinase-mediated TCRß gene usage and coding joint processing at immune RSS. To the best of our knowledge, these data represent the first description of age- and gender-specific developmental differences in TCR gene usage and coding joint processing that could directly influence TCR diversity and immune specificity. It will be important for future studies to ascertain the mechanistic etiology of these developmental and gender differences in TCR diversity and specificity, as well as their importance with respect to the age and gender risks for infectious and autoimmune diseases in humans.

Mutagenicity and potential carcinogenicity of thiopurine treatment in patients with inflammatory bowel disease.

The thiopurines azathioprine and 6-mercaptopurine (6-MP) are effective immune modulators and cytotoxic agents extensively used in the treatment of autoimmune diseases, graft rejection, and cancer. There is compelling epidemiologic evidence that thiopurine treatment increases the risk for a variety of tumors by mechanisms that are unclear. We investigated the in vivo mutagenicity of long-term thiopurine treatment by determining the frequency and spectra of somatic mutation events at the hypoxanthine phosphoribosyltransferase (HPRT) locus in peripheral T lymphocytes as well as the prevalence of mutant clonal proliferation in a cross-sectional analysis of data from 119 children and adults with inflammatory bowel disease (IBD). ANOVA and regression were performed to assess relationships among the frequency and spectra of HPRT mutations with disease, duration of illness, duration of treatment, and total therapeutic dose of azathioprine and 6-MP. We observed a significant increase in the frequency of somatic mutations in 56 subjects treated with thiopurines for IBD compared with 63 subjects not treated with thiopurines. This increase was related to both total dose (P < 0.001) and duration of treatment (P < 0.001). Comparative mutation spectra analysis of 1,020 mutant isolates revealed a significant increase in the proportion of all transitions (P < 0.001), particularly G:C to A:T transitions (P < 0.001). Combined analyses of two signatures for mutant clonality, HPRT mutation, and T-cell receptor beta CDR3 region unique gene sequence also showed a significant thiopurine-dependent increase in mutant cell clonal proliferation (P < 0.001). These findings provide in vivo evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Induction of V(D)J-mediated recombination of an extrachromosomal substrate following exposure to DNA-damaging agents.

V(D)J recombinase normally mediates recombination signal sequence (RSS) directed rearrangements of variable (V), diversity (D), and joining (J) germline gene segments that lead to the generation of diversified T cell receptor or immunoglobulin proteins in lymphoid cells. Of significant clinical importance is that V(D)J-recombinase-mediated rearrangements at immune RSS and nonimmune cryptic RSS (cRSS) have been implicated in the genomic alterations observed in lymphoid malignancies. There is growing evidence that exposure to DNA-damaging agents can increase the frequency of V(D)J-recombinase-mediated rearrangements in vivo in humans. In this study, we investigated the frequency of V(D)J-recombinase-mediated rearrangements of an extrachromosomal V(D)J plasmid substrate following exposure to alkylating agents and ionizing radiation. We observed significant dose- and time-dependent increases in V(D)J recombination frequency (V(D)J RF) following exposure to ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) but not a nonreactive analogue, methylsulfone (MeSulf). We also observed a dose-dependent increase in V(D)J RF when cells were exposed to gamma radiation. The induction of V(D)J rearrangements following exposure to DNA-damaging agents was not associated with an increase in the expression of RAG 1/2 mRNA compared to unexposed controls or an increase in expression of the DNA repair Ku70, Ku80 or Artemis proteins of the nonhomologous end joining pathway. These studies demonstrate that genotoxic alkylating agents and ionizing radiation can induce V(D)J rearrangements through a cellular response that appears to be independent of differential expression of proteins involved with V(D)J recombination.

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development.

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.

Analysis of genetic alterations and clonal proliferation in children treated for acute lymphocytic leukemia.

The development of risk-directed treatment protocols over the last 25 years has resulted in an increase in the survival rates of children treated for cancer. As a consequence, there is a growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapy is unknown. We previously reported that children treated for acute lymphocytic leukemia have significantly elevated somatic mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in their peripheral T cells. To understand the molecular etiology of the increase in mutant frequencies following chemotherapy, we investigated the HPRT mutation spectra and the extent of clonal proliferation in 562 HPRT T cell mutant isolates of 87 blood samples from 47 subjects at diagnosis, during chemotherapy, and postchemotherapy. We observed a significant increase in the proportion of CpG transitions following treatment (13.6-23.3%) compared with healthy controls (4.0%) and a significant decrease in V(D)J-mediated deletions following treatment (0-6.8%) compared with healthy controls (17.0%). There was also a significant change in the class type percentage of V(D)J-mediated HPRT deletions following treatment. In addition, there was a >5-fold increase in T cell receptor gene usage-defined mean clonal proliferation from diagnosis compared with the completion of chemotherapeutic intervention. These data indicate that unique genetic alterations and extensive clonal proliferation are occurring in children following treatment for acute lymphocytic leukemia that may influence long-term risks for multifactorial diseases, including secondary cancers.

V(D)J recombinase mediated inter-chromosomal HPRT alterations at cryptic recombination signal sequences in peripheral human T cells.

The V(D)J recombinase enzyme complex is responsible for the development of a diverse immune system by catalyzing intra-molecular rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes at specific recombination signal sequences (RSSs). This enzyme complex has also been implicated in mediating pathologic and non-pathologic intra- and inter-molecular genomic rearrangements at cryptic (Psi) RSSs outside the immune system loci in lymphoid cells. We describe here two V(D)J recombinase mediated genomic rearrangements resulting in alterations at the HPRT locus in human T-cells. These are inter-chromosomal insertions in which DNA fragments are inserted at breakpoints generated by V(D)J recombinase cleavage at Psi RSS sites in the HPRT locus at Xq26. In the first, a TCR signal ended segment from chromosome 14q11 is inserted at a Psi RSS in intron 1 of the HPRT locus. In the second, a DNA fragment from 9q22 is integrated between the coding ends generated by a V(D)J recombinase mediated HPRT deletion. Identification of these in vivo V(D)J mediated inter-chromosomal insertions at Psi RSSs in the HPRT gene supports the accumulating evidence that V(D)J recombinase can mediate mutagenic rearrangements in humans with potential pathologic consequences.

Analysis of mutagenic V(D)J recombinase mediated mutations at the HPRT locus as an in vivo model for studying rearrangements with leukemogenic potential in children.

Pediatric acute lymphocytic leukemia (ALL) is a multifactorial malignancy with many distinctive developmentally specific features that include age specific acquisition of deletions, insertions and chromosomal translocations. The analysis of breakpoint regions involved in these leukemogenic genomic rearrangements has provided evidence that many are the consequence of V(D)J recombinase mediated events at both immune and non-immune loci. Hence, the direct investigation of in vivo genetic and epigenetic features in human peripheral lymphocytes is necessary to fully understand the mechanisms responsible for the specificity and frequency of these leukemogenic non-immune V(D)J recombinase events. In this review, I will present the utility of analyzing mutagenic V(D)J recombinase mediated genomic rearrangements at the HPRT locus in humans as an in vivo model system for understanding the mechanisms responsible for leukemogenic genetic alterations observed in children with leukemia.

Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites.

The somatic mutant frequency (Mf) of the hypoxanthine phosphoribosyl transferase (HPRT) gene has been widely used as a biomarker for the genotoxic effects of exposure but few studies have found an association with environmental exposures. We measured background Mfs in 49 current and former residents of Dover Township, New Jersey, who were exposed during childhood to industrially contaminated drinking water. The exposed subjects were the siblings of children who developed cancer after residing in Dover Township, where the incidence of childhood cancer has been elevated since 1979. Mfs from this exposed group were compared to Mfs in 43 age-matched, presumably unexposed residents of neighboring communities with no known water contamination and no increased cancer incidence. Statistical comparisons were based on the natural logarithm of Mf (lnMF). The mean Mf for the exposed group did not differ significantly from the unexposed group (3.90 x 10(-6) vs. 5.06 x 10(-6); P = 0.135), but unselected cloning efficiencies were higher in the exposed group (0.55 vs. 0.45; P = 0.005). After adjustment for cloning efficiency, lnMf values were very similar in both groups and age-related increases were comparable to those previously observed in healthy children. The results suggest that HPRT Mf may not be a sensitive biomarker for the genotoxic effects of environmental exposures in children, particularly when substantial time has elapsed since exposure.

Analysis of microsatellite instability in children treated for acute lymphocytic leukemia with elevated HPRT mutant frequencies.

Survival rates of children treated for cancer have increased dramatically over the last 25 years following the development of risk-directed multi-modality treatment protocols. As a result, there is a rapidly growing population of children and young adult cancer survivors in which the long-term genotoxic effects of chemotherapeutic intervention is unknown. We have previously observed that children treated for acute lymphocytic leukemia (ALL) have significantly increased somatic mutant frequencies (Mfs) (30- to 1300-fold higher) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene in their non-malignant peripheral T cells compared with children at diagnosis or controls. In order to gain insight into the etiology of the observed dramatic increase in Mfs following antineoplastic therapy, we investigated the prevalence of microsatellite instability (MSI), reflective of a defect in DNA mismatch repair (MMR), in children with ALL at diagnosis, during and after chemotherapy and compared them with healthy age-matched controls. MSI analysis using five microsatellite markers was performed on 167 T cell isolates from 40 healthy children and on 842 T cell isolates from 50 patients treated for ALL. High-frequency MSI (MSI-high) was identified in 2 healthy children (5%) and in 2 of 20 ALL subjects at the time of disease recurrence (relapse) (10%). There was no statistically significant difference between the prevalence of MSI-high in patients at the time of ALL relapse and healthy children, nor between the children with ALL at other time points and healthy children. These data indicate that MMR defects, represented by MSI, are not a significant contributor to the elevated HPRT Mfs seen in children treated for ALL. However, in a small number of patients chemotherapy may play a role in the selection of cells with defects in MMR that may have long-term clinical implications.

Genotoxicity of therapeutic intervention in children with acute lymphocytic leukemia.

The survival rates of children treated for cancer have dramatically increased after the development of standardized multiple-modality treatment protocols. As a result, there is a rapidly growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapeutic intervention is unknown. To study the genotoxic effects of antineoplastic treatment in children, we performed a comparative analysis of the changes in the frequency of somatic mutations (Mfs) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT)-reporter gene in children treated for acute lymphocytic leukemia (ALL). We measured HPRT Mfs from 130 peripheral blood samples from 45 children with ALL (13, low risk; 22, standard risk; and 10, high risk) from the time of diagnosis, as well as during and after the completion of therapy. We observed a significant increase in mean HPRT Mfs during each phase of therapy (diagnosis, 1.4 x 10(-6); consolidation, 52.1 x 10(-6); maintenance, 93.2 x 10(-6); and off-therapy, 271.7 x 10(-6)) that were independent of the risk group treatment protocol used. This 200-fold increase in mean somatic Mf remained elevated years after the completion of therapy. We did not observe a significant difference in the genotoxicity of each risk group treatment modality despite differences in the compositional and clinical toxicity associated with these treatment protocols. These findings suggest that combination chemotherapy used to treat children with ALL is quite genotoxic, resulting in an increased somatic mutational load that may result in an elevated risk for the development of multi-factorial diseases, in particular second malignancies.

Comparative analysis of HPRT mutant frequency in children with cancer.

The link between exposure to environmental mutagens and the development of cancer is well established. Yet there is a paucity of data on the relationship between gene-environment interactions and the mechanisms associated with the somatic mutational events involved with malignant transformation, especially in children. To gain insight into somatic mutational mechanisms in children who develop cancer, we determined the background mutant frequency (Mf) in the hypoxanthine phosphoribosyl transferase (HPRT) reporter gene of peripheral blood lymphocytes from pediatric cancer patients at the time of diagnosis and prior to therapeutic intervention. We studied 23 children with hematologic malignancies and 31 children with solid tumors prior to initial therapeutic intervention. Children with solid tumors, specifically sarcomas, and Hodgkin's disease were significantly older and had elevated HPRT Mfs (6.1 x 10(-6) and 3.7 x 10(-6), respectively) at the time of diagnosis, compared to normal controls (2.3 x 10(-6)) and other pediatric tumor groups including children with acute lymphocytic leukemia and non-Hodgkin's lymphoma (ALL/NHL, 1.7 x 10(-6)), central nervous system tumors (CNS, 3.6 x 10(-6)), and neuroblastoma (1.9 x 10(-6)). Of importance is that the significant differences observed in HPRT Mfs between these groups no longer existed after correcting for the effects of age. These data demonstrate that in children who develop cancer there appears to be no significant increase in background HPRT Mf that would indicate significant exposure to genotoxic chemicals or an underlying DNA repair defect resulting in genomic instability. In addition, these data demonstrate the importance of correcting for the effect of age when comparing the frequency of somatic mutations in children and should provide baseline data for future longitudinal biomonitoring studies on the genetic effects of chemotherapy in children treated for cancer.

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes.

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.

Mutational spectral analysis at the HPRT locus in healthy children.

There is growing evidence linking somatic mutational events during fetal development and childhood to an increasing number of multifactorial human diseases. Despite this, little is known about the relationship between endogenous and environmentally induced exogenous mutations during human development. Here we describe a comparative spectral analysis of somatic mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene locus in healthy children. We observed an age-specific decrease in the proportion of large alterations and a corresponding increase in the proportion of small alterations with increasing age following birth (P<0.001). The age specific decrease in the proportion of large alterations (67-30%) was mainly due to a decrease in the proportion of aberrant variable (V), diversity (D) and joining (J) (V(D)J) recombinase mediated HPRT deletions (P<0.001). The increase in the proportion of small alterations with age (28-64%) was associated with an increase in transversions from 8% in children at the late stages of fetal development to 31% in children 12-16 years old (P=0.003). Transitions decreased with age, especially at CpG dinucleotides (P=0.010), as transversions increased (P=0.009). These patterns of mutations provide insight into important spontaneous, genotoxic, and site-specific recombinational somatic mutational events associated with the age-specific development of human disease in children as well as adults.