Translational crossroads for biomarkers.

A group of investigators met at a Specialized Programs of Research Excellence Workshop to discuss key issues in the translation of biomarker discovery to the development of useful laboratory tests for cancer care. Development and approval of several new markers and technologies have provided informative examples that include more specific markers for prostate cancer, more sensitive tests for ovarian cancer, more objective analysis of tissue architecture and an earlier indication of response to treatment in breast cancer. Although there is no clear paradigm for biomarker development, several principles are clear. Marker development should be driven by clinical needs, including early cancer detection, accurate pretreatment staging, and prediction of response to treatment, as well as monitoring disease progression and response to therapy. Development of a national repository that uses carefully preserved, well-annotated tissue specimens will facilitate new marker development. Reference standards will be an essential component of this process. Both hospital-based and commercial laboratories can play a role in developing biomarkers from discovery to test validation. Partnering of academe and industry should occur throughout the process of biomarker development. The National Cancer Institute is in a unique position to bring together academe, industry, and the Food and Drug Administration to (a) define clinical needs for biomarkers by tumor type, (b) establish analytic and clinical paradigms for biomarker development, (c) discuss ways in which markers from different companies might be evaluated in combination, (d) establish computational methods to combine data from multiple biomarkers, (e) share information regarding promising markers developed in National Cancer Institute-supported programs, and (f) exchange data regarding new platforms and techniques that can accelerate marker development.     

The fluid content of the human intervertebral disc. Comparison between fluid content and swelling pressure profiles of discs removed at surgery and those taken postmortem.

The fluid content of the disc, which governs its mechanical response and biological behavior, varies with external load. Because load on the disc changes after death, the fluid content and swelling pressure profiles of human discs taken at autopsy were measured, and compared with discs removed during surgical procedures. In general, discs taken at surgery had a lower fluid content in the nucleus and a higher fluid content in the outer anulus than discs removed at autopsy. In discs removed at surgery, the swelling pressure of the nucleus was higher than that of the anulus, whereas in autopsy discs the swelling pressure profile was flat. These changes are though to result from changes in load after death, and could influence the results of in vitro mechanical tests on the disc.     

Effects of radiation on fixation of non-cemented porous-coated implants in a canine model

A non-weight-bearing porous-coated rod was implanted bilaterally in the proximal part of the humerus in thirty-five adult male mongrel dogs. In all of the animals, one limb was treated with radiation and the opposite limb served as the control. In twenty-one animals, the dose was 1000 centigrays (rads) and in fourteen, it was 500 centigrays. The strength of fixation and the volume fraction of ingrowth of bone were determined two, four, and eight weeks after the operation in the group that received 1000 centigrays and two and four weeks after the operation in the group that received 500 centigrays. Treatment with 500 centigrays had no significant effect on the strength of fixation or the amount of ingrowth of bone. In contrast, at two weeks, treatment with 1000 centigrays had reduced the strength of fixation to 50 per cent of the control value (p less than 0.01), although, at four and eight weeks, the strength of fixation was not significantly different than that in the control limb. The amount of ingrowth of bone in the irradiated limb was significantly reduced at two weeks (30 per cent of the control value) (p less than 0.01), four weeks (70 per cent of the control value) (p less than 0.05), and eight weeks (56 per cent of the control value) (p less than 0.05).     

Identification of N2 as a metabolite of acetylhydrazine in the rat

 In the pathogenesis of isoniazid-induced hepatic injury, cytochrome P450-dependent metabolic activation of the metabolite, acetylhydrazine (AcHz), is the crucial step. Exhalation of [¹⁴C]-carbon dioxide has previously been used to quantify indirectly this pathway. In contrast, according to the current concept of AcHz bioactivation, molecular nitrogen is produced directly, but has not yet been identified. Here, we measured [¹⁵N]-nitrogen and ¹⁴CO₂ exhalation, after the administration of [¹⁵N₂]-[¹⁴C]-AcHz, in rats. Laser magnetic resonance (LMR) spectroscopy, a new sensitive and specific technique for the measurement of ¹⁵N and ¹⁴N in gas samples, was used. To demonstrate the involvement of cytochrome P450, rats were treated with phenobarbital (PB) or PB + cobalt(II) chloride (CoCl₂) (n=3 in each group). Time-dependent ¹⁵N₂ exhalation differed significantly between treatment groups (p<0.001). At 240 min, cumulative exhalation of ¹⁵N was 1.92±0.43% (mean±SE) of the dose in the control group, 2.53±0.23% in the PB group, and 1.00±0.15% in the PB+CoCl₂ group (p<0.05 compared to controls, p<0.01 compared to PB). Cumulative exhalation of ¹⁴CO₂ in 24 h ranged from 15.1 to 21.9%, with no significant difference between treatment groups. In conclusion, N₂ is a metabolite of AcHz. N₂ formation reflects the cytochrome P450-mediated activation of AcHz and can be used as an index of this pathway. Generally, LMR spectroscopy is valuable for monitoring any N₂-liberating process in vivo. Received: 14 March 1995/Accepted: 15 August 1995     

Electrophysiological and pharmacological evidence in favor of amino acid neurotransmission in fimbria-fornix fibers innervating the lateral septal complex of rats

Summary Electrical stimulation of fimbria-fornix (fifx) fibers monosynaptically activated many of the neurons tested in the lateral septal complex (LSC) of the rat. The orthodromically activated LSC neurons were classified as strongly orthodromically activated (SOA) or weakly orthodromically activated (WOA) cells according to their threshold for eliciting a response, stability of the response latency, frequency following and the stimulus-response ratio.Microiontophoretically applied glutamate (GLU) could excite both SOA and WOA neurons. However, the expelling currents needed to activate the SOA cells were often considerably lower than those necessary to excite the WOA cells suggesting higher sensitivity to GLU of those cells which receive a strong fi-fx innervation. Iontophoretically administered glutamic acid diethylester (GDEE) in general reversibly attenuated excitatory responses of LSC cells to GLU but not to acetylcholine. GDEE was also effective in blocking the synaptic responses of SOA septal cells to fi-fx stimuli. In addition, GDEE administered topically reversibly suppressed the field potential induced in the LSC by fi-fx stimulation.These electrophysiological and pharmacological results support recent biochemical observations suggesting that the excitatory innervation of LSC neurons by fi-fx fibers is mediated by GLU or a closely related excitatory amino acid.The investigations were supported by the Foundation for Medical Research FUNGO which is subsidized by the Netherlands Organization for Advancement of Pure Research (Z.W.O.), grant 13-31-045 awarded to I.J. A. Urban     

Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR

Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.     

Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

Requirements for the development of IL-4-producing T cells during intestinal nematode infections: what it takes to make a Th2 cell in vivo

Summary: Components of the type 2 immune response may mediate host protection against both helminthic parasites and harmful allergic responses. A central player in this response is the T‐helper 2 (Th2) effector cell, which produces interleukin (IL)‐4, IL‐5, IL‐13, and other Th2 cytokines during the primary and memory response. Specific aspects of the parasite that trigger Th2‐cell differentiation are not yet defined. Furthermore, the cell types and cell surface and secreted molecules that provide the immune milieu required for the development of Th2 effector cells and also Th2 memory cells are not well understood. They will probably vary with the particular helminth or other antigen inducing the Th2 response. We have used third stage larvae of intestinal nematode parasites as adjuvants to promote naïve nonparasite antigen‐specific T cells to differentiate into Th2 cells. This model system avoids possible parasite antigen‐specific T‐cell clones or cross‐reactive memory T cells that may preferentially differentiate into Th2 effector cells during the course of infection and confound the stereotypical components of parasite‐induced Th2 cell differentiation. We have found that these parasites have a potent adjuvant effect and have used our model system to begin to investigate the events that lead to the development of polarized Th2 cells in vivo.     

Heligmosomoides polygyrus inhibits established colitis in IL-10-deficient mice

Inflammatory bowel disease (IBD) is prevalent in industrialized countries, but rare in less-developed countries. Helminths, common in less-developed countries, may induce immunoregulatory circuits protective against IBD. IL-10(-/-) mice given piroxicam develop severe and persistent colitis. Lamina propria mononuclear cells from colitic IL-10(-/-) mice released IFN-gamma and IL-12. The ongoing piroxicam-induced colitis could be partially blocked with anti-IL-12 monoclonal antibody suggesting that the inflammation was at least partly IL-12 dependent. Colonization of piroxicam-treated colitic IL-10(-/-) mice with Heligmosomoides polygyrus (an intestinal helminth) suppressed established inflammation and inhibited mucosal IL-12 and IFN-gamma production. H. polygyrus augmented mucosal IL-13, but not IL-4 or IL-5 production. Transfer of mesenteric lymph node (MLN) T cells from IL-10(-/-) animals harboring H. polygyrus into colitic IL-10(-/-) recipients inhibited colitis. MLN T cells from worm-free mice did not. Foxp3 (scurfin) drives regulatory T cell function. H. polygyrus enhanced Foxp3 mRNA expression in MLN T cells that had regulatory activity. This suggests that H. polygyrus inhibits ongoing IL-10(-/-) colitis in part through blocking mucosal Th1 cytokine production. Resolution of inflammation is associated with increased IL-13 production and can be adoptively transferred by MLN T cells.