Transcriptional signatures as a disease-specific and predictive inflammatory biomarker for type 1 diabetes.

The complex milieu of inflammatory mediators associated with many diseases is often too dilute to directly measure in the periphery, necessitating development of more sensitive measurements suitable for mechanistic studies, earlier diagnosis, guiding therapeutic decisions and monitoring interventions. We previously demonstrated that plasma samples from recent-onset type 1 diabetes (RO T1D) patients induce a proinflammatory transcriptional signature in freshly drawn peripheral blood mononuclear cells (PBMCs) relative to that of unrelated healthy controls (HC). Here, using cryopreserved PBMC, we analyzed larger RO T1D and HC cohorts, examined T1D progression in pre-onset samples, and compared the RO T1D signature to those associated with three disorders characterized by airway infection and inflammation. The RO T1D signature, consisting of interleukin-1 cytokine family members, chemokines involved in immunocyte chemotaxis, immune receptors and signaling molecules, was detected during early pre-diabetes and found to resolve post-onset. The signatures associated with cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, patients with confirmed bacterial pneumonia, and subjects with H1N1 influenza all reflected immunological activation, yet each were distinct from one another and negatively correlated with that of T1D. This study highlights the remarkable capacity of cells to serve as biosensors capable of sensitively and comprehensively differentiating immunological states.

Thymocyte maturation: selection for in-frame TCR alpha-chain rearrangement is followed by selection for shorter TCR beta-chain complementarity-determining region 3.

Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR beta-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which a-chains are selected has also been identified using beta-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. alpha selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR beta-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.

T cell immunobiology of alloimmune thrombocytopenias.

Allorecognition and generation of alloantisera depend on T cell help. Here we summarize our recent work on identifying the T cells that recognize a specific platelet alloantigen generated by a Pro to Leu polymorphism in beta-chain of the platelet integrin alphaIIb beta3. The ability to generate alloantibodies is restricted by HLA-DRB3*0101. By measuring peptide binding to HLA-DRB3*0101, we have shown that the polymorphism controls the generation of the T cell epitope. This is not a result of the polymorphism changing a T cell contact residue, but rather by its generating a peptide anchor. The result is a directional antibody response in which only the beta-chain that is antigenic is the one that produces a peptide that binds to the HLA-DR. This mechanism of generating alloantibodies may be a paradigm for a whole class of responses.

Concurrent or sequential delta and beta TCR gene rearrangement during thymocyte development: individual thymi follow distinct pathways.

Analysis of TCR rearrangement profiles of well-defined thymocyte populations in a number of individual thymi provides evidence for a new pathway of lineage commitment. In all of the thymi analyzed, alphabeta thymocytes have rearrangements in the delta locus that are enhanced for out-of-frame rearrangements. Thus, not only did alphabeta thymocytes pass through a stage in differentiation that included delta rearrangement, but they also constitute a population that was relatively unsuccessful at these rearrangements. Interestingly, in some thymi, gammadelta thymocytes have out-of-frame beta rearrangements. This represents a novel pathway in which delta and beta rearrangement happens concurrently. In this pathway, selection favors whichever gene is in frame, thus improving the chances of generating useful T cells. In other thymi, gammadelta cells showed no obvious beta gene rearrangement, indicating a sequential rearrangement. Thus, alphabeta/gammadelta lineage commitment can follow at least two distinct pathways in different individuals.

Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia.

Neonatal alloimmune thrombocytopenia induced by the human platelet alloantigen 1a (HPA1a) is characterized by generation of alloantibodies by a mother who is homozygous for the HPA1b alloantigen and almost always HLA-DRB3*0101. The disease is viewed as B cell mediated but the linkage with HLA is indicative of a role for T cells. The HPA1a and HPA1b allotypes are defined, respectively, by Leu and Pro at amino acid 33 of the beta-chain of the platelet integrin GPIIbIIIa (alpha(IIb)beta3). Under the assumption that the same polymorphism may control both the B cell epitope and constitute the MHC-bound peptide, we restimulated PBMC from a woman with an affected child with a synthetic peptide from this polymorphic region. Molecular analysis of the responding T cell repertoire identified two T cells which predominated in cultures stimulated with the alloantigen peptide and which were absent in cultures with the autoantigen peptide. In spite of the use of different V families, sequence of the CDR3 region of the T cell receptor (TCR) beta-chain revealed the presence of a shared motif, L-P-S/T. Oligonucleotide probes specific for the CDR3 sequence indicated that these T cells were present in the PBMC at the highest levels immediately after delivery of the affected infant and their frequency dropped at later times.

Molecular defects in TCRBV genes preclude thymic selection and limit the expressed TCR repertoire.

A prerequisite for the assembly of a functional TCR is the rearrangement of gene segments to result in in-frame transcripts that can vary in length across the CDR3 region. Selection for in-frame 3-bp spaced rearrangements is observed for functional TCRB genes in thymocyte DNA and mRNA transcripts from PBMC. Previous analyses of the expressed human TCRBV gene repertoire have suggested that BV10S1 and BV19S1 gene segments may be expressed at very low levels or not at all in some individuals. CDR3 size analysis for BV10 and BV19 transcripts and thymic DNA rearrangements revealed no such selection of in-frame 3-bp spaced rearrangements. Comparison of the BV19 leader intron sequence with consensus 5'-splice signal sequences suggested that the mature mRNA for this gene would contain the unspliced leader intron. Sequencing of BV19 transcripts from PBMC confirmed that the intron was not spliced, resulting in a predicted translation product that terminates prematurely. Both genomic DNA and mRNA were analyzed for the BV10 gene. The leader sequence contained a single extra base, which would result in a shift in the V region reading frame upon conventional mRNA splicing. This gene is predicted to be nonfunctional due to the presence of a stop codon in the V gene segment just after the splice signal. A splice variant that uses an alternative 3'-splice site further downstream in the V region was also detected. This variant is predicted to be nonfunctional due to the presence of an in-frame stop codon in the V region. These processing defects are sufficient to abrogate positive selection. Therefore, the conclusions drawn from previous studies of the expressed T cell repertoire in normal and disease states based on the presumed functional status of these two genes need to be reassessed.

Molecular analysis of T cell repertoires. Spectratypes generated by multiplex polymerase chain reaction and evaluated by radioactivity or fluorescence.

The analysis of T-cell repertoires has been facilitated by the introduction of methods in which the length heterogeneity of the third complementarity region (CDR3) is used to further refine V-family-specific PCR. We call our implementation of this technique T-cell spectratyping. This method is especially important in analysis of specific expansion or retention of T cells in human immune system function. Current methodologies are cumbersome in the number of PCR reactions and gels needed for complete analysis of TCR BV repertoires. We describe here the optimized conditions for using 11 TCR BV primer pairs in multiplex PCR which allow for a more compact analysis. In addition, the two primers act as controls for each other in the PCR. The use of these primers is shown using either fluorescent or radiolabeled constant primers. The two labeling methods give comparable results. Fluorescent primers avoid the difficulties associated with use of radioactivity. Autoradiography with 32P-labeled primers is simpler, requiring less instrumentation.

Analysis of reconstituting T cell receptor repertoires in bone marrow transplant recipients.

Allogeneic bone marrow transplantation (BMT) has become the treatment of choice for a variety of hematopoietic disorders. An important factor limiting the use of allogeneic BMT is delayed restoration of immune function. A quantitative understanding of immune reconstitution of the T cell compartment based on an efficient method of analysis would be of benefit. Distinct lineages of T cells which have resulted from previous and ongoing clonal expansion can be identified by their unique T cell receptors (TCR). Thus, TCR complexity can be used as a measure of repertoire complexity. Here we use a polymerase chain reaction-based approach which visualizes the size heterogeneity of the third complementarity determining region (CDR3) to study T cell reconstitution in adult bone marrow transplant recipients. We find that repertoire complexity, as determined by the number of bands of different length for each V family, reflects the general immune status of individuals tested. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. This approach provides a new tool in the analysis of reconstitution of alpha/beta T cell repertoires and it can be also be applied to B cells and gamma/delta T cells.

Physical, neuromotor and intellectual impairment in non-cretinous schoolchildren with iodine deficiency.

Thyroid status and neurologic, psychometric and auditory functions were evaluated in presumably normal schoolchildren aged 6 to 16 years from three areas of iodine deficiency in Iran. The subjects from each area were identified as members of groups A, B or C. In group A there were retarded growth, high prevalence of visible goiter (93%), low T4 (39%) and high TSH (70%). In group B 66% had a visible goiter and 7% had high serum TSH. In group C visible goiter was present in 22% of the subjects but they had normal thyroid function. Urinary iodine excretion was low in all three groups. Head circumference was less in groups A and B, as compared to C. Pyramidal signs occurred in over half of the subjects in Group A (hyperreflexia in 39% and crossed adductor reflex in 29%). The glabellar sign was present in 50% of group A and 20% of group B. Forty-four percent of the subjects in group A and 17% in group B had hearing deficits as shown by audiometry. Psychomotor examination was performed using the Bender Gestalt test. A higher number of errors was evident in groups A and B their psychomotor age was below their chronological age. The results of the Raven test showed mild impairment of IQ in group A, with 55% having an IQ below 91 and 15% less than 70. The subjects in group B had lower IQ than group C, but higher than group A. There was a negative correlation between serum TSH and free thyroid indices and a positive correlation between TSH and the number of pyramidal signs. This study demonstrates that mild to moderate growth retardation and neurological, auditory and psychomotor impairments occur in apparently normal subjects residing in areas of iodine deficiency.

Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status.

The analysis of the T cell repertoires involved in local or systemic immune responses is beginning to play an important role in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, alloimmunity including allograft rejection, and tumor immunity. Here we analyze circulating T cell repertoires by determining TCR beta-chain gene complexity using a modification of V beta family-specific PCR. This approach, called CDR3 size spectratyping, uses the size heterogeneity of the CDR3 as a further source of specificity in TCR analysis. It has been used here to analyze the complexity and stability of circulating T cell repertoires in normal adults, including bone marrow donors, and bone marrow transplant recipients. Normal spectratypes are both complex and stable. The repertoire complexity of marrow recipients correlates with their state of immune function. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. Spectratype analysis is applicable to other studies of specific repertoire skewing such as may be associated with immunodeficiency or found at sites of immune activity.

Analysis of human T-cell receptor alpha-chain cDNAs isolated from peripheral blood mononuclear cells.

T-cell receptor alpha-chain cDNA were generated from unstimulated peripheral blood mononuclear cells of a DR2,3,52a individual using a modified anchor PCR method. Fifty-six cDNA clones were identified representing 47 distinct T-cell receptor clonotypes and 26 VA loci. This analysis identified a new VA gene family VA30, and a new member of the VA6 gene family.

Survey of iodine deficiency in a rural area near Tehran: association of food intake and endemic goitre.

Goitre is endemic in many parts of Iran. This study was undertaken first to obtain the base line data and identify the nature of the problem and then to take measures to ameliorate it. Shahryar, an endemic area near the capital city, was selected as the pilot field. The sample size was 368 families. Sixty-six per cent of females and 54 per cent of males were goitrous. A food consumption survey revealed that 38 per cent and 62 per cent of the families had failed to receive at least 80 per cent of their requirement for energy and vitamin A, respectively. Free T4 index was normal but significantly lower in grade 2 goitre than the non-goitrous subjects (P less than 0.01). Significant reverse correlations were found between goitre prevalence and vitamin A and protein intakes (P less than 0.05). Supplementation with 40 micrograms iodine/g salt raised urinary iodine excretion significantly (P less than 0.001). We conclude that although iodine deficiency plays the key role in goitre, the association of other dietary factors observed in this study needs further consideration.

Newborns vitamin A in relation to sex and birth weight.

Cord serum vitamin A values were determined in 256 male and 294 female neonates born in Tehran. The mean cord serum vitamin A values (micrograms/dl +/- SD) was 24.04 +/- 6.87 and ranged from 3.16 to 49.71 micrograms/dl. Males had significantly lower mean cord serum vitamin A values than females (P less than 0.001), and the prevalence of low serum vitamin A (below 20 micrograms/dl) was higher in male neonates than female ones (35 and 21 per cent, respectively). Serum retinol values increased gradually with birth weight. The mean serum vitamin A for premature neonates was significantly lower than term neonates. A significant r value for the linear correlation between cord serum retinol and parity was obtained for mothers aged more than 35 years.

Vitamin A status and endemic goiter.

Serum vitamin A levels were determined in two groups of subjects living in an endemic goiter area in Iran. The first group consisted of 242 non-goitrous subjects, and the second, contained 603 subjects with different grades of goiter. Serum vitamin A concentrations were correlated with goiter, sex, and age. Serum retinol values were not statistically different between goitrous and non-goitrous females, but goitrous male subjects especially those under 13 years of age, had lower serum retinol values than non-goitrous male subjects. The prevalence of low vitamin A levels was higher in goitrous boys. In both groups the prevalence of low serum vitamin A decreased with the increase in age. The prevalence of goiter was not statistically different between boys and girls under 18 years, but was more prevalent in women over 18 years, than men in the same age group (P less than 0.001). This difference was in accordance with serum vitamin A status between women and men after the age of 18 (P less than 0.001). Serum vitamin A increased with age up to 18 years, in both sexes and remained unchanged afterwards. The increase was gradual in girls under 18 years but was sharper in boys during puberty years. Although the values for serum vitamin A in each sex covered wide overlapping ranges, but a general superiority of male serum vitamin A levels over the females was observed.