Complex biological patterns of soluble cytokines and CD163 in childhood necessitating age-specific reference intervals for evidence-based clinical interpretation.

BACKGROUND: Cytokine measurements to support clinical laboratory and research investigations have become increasingly common in pediatrics. However, there is a paucity of accurate pediatric reference intervals (RIs) essential to the interpretation of cytokine results. To address this gap, here, we establish age- and sex-specific pediatric reference values for clinically relevant inflammatory markers including CD163, and the cytokines IL-1ß, IL-6, IL-10, IL-18, TNF-α, IFN-γ, and CXCL-9. METHODS: Healthy children and adolescents (n = 311, 1-19 years) were recruited as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) study. Multi-analyte measurements in plasma and analytical performance verification were conducted on the ProteinSimple® Ella™ automated immunoassay platform (Bio-Techne, MN, USA). Age- and sex-specific RIs were calculated based on Clinical and Laboratory Standards Institute guidelines. Additionally, 75th and 95th percentile cut-offs were determined. RESULTS: Three types of reference value distributions were observed: (a) consistent levels throughout age and sex: IL-6, and IFN-γ, (b) gradual decline in concentration with age: CD163, TNF-α, CXCL-9, and IL-10, (c) significantly higher concentrations during ages 4-14 years than earlier and later ages: IL-1ß and IL-18. Reference values for CXCL-9, IL-10, and TNF-α under 8 years of age differed significantly from older children. CD163, IL-18 and IL-1ß required three age partitions. CD163 demonstrated significant sex differences in ages 8-13 years. CONCLUSION: The circulating profile of cytokines in children is complex and can vary by age and sex. This necessitates careful interpretation of test results based on age and/or sex specific RIs facilitating more accurate clinical decision making.

Multiplexed Digital Detection of B-Cell Acute Lymphoblastic Leukemia Fusion Transcripts Using the NanoString nCounter System.

Chromosomal rearrangements resulting in fusion transcripts have been reported in precursor B-cell acute lymphoblastic leukemia (B-ALL). The identification of fusion events is crucial in the diagnosis of B-ALL. In this study, we used NanoString nCounter technology to design, validate, and evaluate a multiplex panel for the detection of B-ALL fusion transcripts. Fifty-one B-ALL fusion transcripts reported in children in the literature were included in the design of the NanoString panel. Twenty-six fusion transcripts were validated using 64 positive-control samples and 74 negative-control samples with 100% sensitivity and 99% specificity in comparison to RT-PCR. Our results support a potential role of NanoString's technology as a robust and cost-effective technique that could be used in the detection of fusion transcripts and implemented into the diagnostic algorithm of B-ALL.

Biologically active human GM-CSF produced in the seeds of transgenic rice plants.

Rice flour is a well-known and characterized source of pharmaceutical ingredients, which are gluten-free and incorporated in many drug delivery applications such as excipient starch. To further exploit this uniqueness, the synthetic capacity of rice endosperm tissue, the basis of rice flour, was extended by genetic transformation. Recombinant human GM-CSF, a cytokine used in treating neutropenia and with other potential clinical applications, has been expressed in transgenic rice seeds using a rice glutelin promoter. Rice seeds accumulated human GM-CSF to a level of 1.3% of total soluble protein. The rice seed-produced human GM-CSF was found to be biologically active when tested using a human cell line TF-1. Use of rice as a host plant offers not only attractive features of safe production in seeds but also self-containment of foreign genes, as rice is primarily a self-pollinated crop plant.

A role for DHX32 in regulating T-cell apoptosis.

DHX32 is a novel putative RNA helicase with an activation-dependent pattern of expression in T-cells. To gain insight into the role of DHX32, Jurkat-DHX32 cells, a stable Jurkat T-cell line with constitutive DHX32 expression, were generated by retroviral gene transfer. There were no significant differences between control and Jurkat-DHX32 cells in terms of proliferation and response to several chemotherapeutic agents. There was an altered response of Jurkat-DHX32 cells to Fas signaling associated with down-regulation of the anti-apoptotic protein c-FLIP short. In normal peripheral blood lymphocytes, a correlation between DHX32 and c-FLIP short expression was detected in response to different T-cell specific and non-specific activation stimuli. Our results suggest that DHX32 might be involved in regulating T-cell response to certain apoptotic stimuli.

Characterization of murine Dhx32.

In this report, we studied the expression of Dhx32, the murine counterpart of the human putative RNA helicase DHX32. Northern analysis showed widespread but variable level of expression among different tissues. RT-PCR detected the presence of two mRNA transcripts in several tissues that result from alternative usage of either exon 1 or exon 2 at the 5' UTR. Immunohistochemistry showed the presence of Dhx32 in all tissues examined including spleen, kidney and liver. The distribution of Dhx32 proteins was not uniform among different cell types within the same tissue, suggesting cell type-dependent regulation of expression. Finally, electron microscope immunocytochemistry detected the presence of Dhx32 in the mitochondria of hepatocytes.

Altered distribution of heat shock protein 60 (Hsp60) with dysregulated expression of DHX32.

DHX32 has an overall similarity to the DHX family of RNA helicases but with a novel helicase domain and nuclear and mitochondrial localizations. The expression of DHX32 is highly regulated during lymphocyte activation and is dysregulated in lymphoid malignancies. In this study, we report our finding of an altered subcellular localization of heat shock protein 60 (Hsp60) in Jurkat-DHX32 cell line in which DHX32 is constitutively expressed. Two-dimensional gel electrophoresis followed by mass spectrometry, electron microscopic immunocytochemistry, and immunoblot analysis showed mainly cytoplasmic localization of Hsp60 in Jurkat-DHX32 cells instead of its mainly mitochondrial localization in control cells. No significant changes were detected in the mitochondrial ultra-structure and the mitochondrial membrane potential activity as a result of dysregulated DHX32 expression. The subcellular distribution of several mitochondrial proteins including cytochrome c, peroxiredoxin 3, manganese superoxide dismutase, Leucine-Rich PentatricoPeptide Repeat Cassette and the 49-kDa subunit of the mitochondrial respiratory Complex I were similar in control and Jurkat-DHX32 cells ruling out non-specific cytoplasmic leakage of mitochondrial proteins. No significant changes in the expression of Hsp60 transcript or total cellular protein were detected. These findings suggest that dysregulated expression of DHX32 might lead to as of yet unknown changes in mitochondrial homeostasis manifested by cytoplasmic redistribution of the molecular chaperon Hsp60.

Nuclear and mitochondrial localization of the putative RNA helicase DHX32.

RNA helicases are involved in all aspects of RNA metabolism. We identified DHX32 as a novel gene with homology to the DHX family of RNA helicases. To gain insight into the role of DHX32 in RNA metabolism, we determined its subcellular localization. Electron microscopy immunocytochemistry detected DHX32 in the nucleus and mitochondria of the myeloid leukemia cell line HL-60. Confocal microscopy shows mitochondrial localization of DHX32 in HeLa cells. Double labeling showed close proximity of DHX32 to some of the newly synthesized RNA in the mitochondria. These results suggest that DHX32 is a putative RNA helicase that might be involved in regulating nuclear and mitochondrial gene expression.

Expression of DHX32 in lymphoid tissues.

DHX32 is a novel putative RNA helicase identified based on its downregulation in acute lymphoblastic leukemia. DHX32 gene has 12 exons, alternative usage of exons 1 and 2 results in the expression of two transcripts that differ in their 5' untranslated region (UTR), consistent with the involvement of two different promoters. In addition, exon 5 skipping results in the production of a full-length and alternatively spliced transcripts. We used transcript-specific primers in reverse transcriptase (RT)-PCR to show that the thymus and spleen express all of DHX32 transcripts. We also used immunohistochemistry to study the expression of DHX32 protein. The expression pattern of DHX32 protein in normal lymphoid tissues is variable but suggests association with state of lymphocyte activation and/or differentiation. Dysregulated expression of DHX32 is noted in some types of lymphoma compared to their normal counterpart.

The activation-induced expression of DHX32 in Jurkat T cells is specific and involves calcium and nuclear factor of activated T cells.

In this report, we studied DHX32 expression in human Jurkat T cells. Co-stimulation of CD3 and CD28 resulted in upregulation of DHX32. No significant changes in the expression of the closely related RNA helicases were seen. Ionomycin treatment alone was sufficient to upregulate the expression of DHX32 mRNA isoform transcribed from the proximal promoter. We cloned DHX32 proximal promoter and identified a 218bp fragment containing two potential binding sites for the transcription factor nuclear factor of activated T cells (NF-AT). Mutation of core sequence of NF-AT resulted in reduced transcriptional activity, with more reduction observed in the second NF-AT site. Electrophoretic mobility shift assay results were consistent with a specific binding of NF-AT from ionomycin stimulated nuclear extract of Jurkat cells to oligonucleotide probes from DHX32 proximal promoter. These results suggest that the DHX32 expression is modulated in Jurkat T cells via a pathway that involves NF-AT.

Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.

Biological activity of human granulocyte-macrophage colony stimulating factor is maintained in a fusion with seed glutelin peptide.

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.