Jason M. Rosenzweig

Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-α-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52–90%)] was higher than that of CA125 alone [65% (95% CI, 43–84%)] at a matched specificity of 97% (95% CI, 89–100%). When compared at a fixed sensitivity of 83% (95% CI, 61–95%), the specificity of the model [94% (95% CI, 85–98%)] was significantly better than that of CA125 alone [52% (95% CI, 39–65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.

Serum diagnosis of pancreatic adenocarcinoma using surface-enhanced laser desorption and ionization mass spectrometry.

Purpose: Each year in the United States, ∼ 30,000 people die from pancreatic cancer. Fewer than 5% of patients survive >5 years after diagnosis, because most patients present with advanced disease. Early diagnosis may improve the prognosis of patients with pancreatic cancer. Experimental Design: In an attempt to improve on current approaches to the serological diagnosis of pancreatic cancer, we analyzed serum samples from patients with and without pancreatic cancer using surface-enhanced laser desorption and ionization (SELDI) protein chip mass spectrometry. Using a case-control study design, serum samples from 60 patients with resectable pancreatic adenocarcinoma were compared with samples from 60 age- and sex-matched patients with nonmalignant pancreatic diseases, as well as 60 age- and sex-matched healthy controls. To increase the number of proteins potentially identifiable, serum was fractionated using anion exchange and profiled on two ProteinChip surfaces (metal affinity capture and weak cation exchange). Results: We determined a minimum set of protein peaks able to discriminate between patient groups and used the unified maximum separability algorithm to compare the performance of the individual marker panels alone or in conjunction with CA19–9. Among the peaks identified by SELDI profiling that had the ability to distinguish between patient groups, the 2 most discriminating protein peaks could differentiate patients with pancreatic cancer from healthy controls with a sensitivity of 78% and specificity of 97%. These 2 markers performed significantly better than the current standard serum marker, CA19–9 (P < 0.05). The diagnostic accuracy of the 2 markers was improved by using them in combination with CA 19-9. Similarly, a combination of 3 SELDI markers and CA19–9 was superior to CA19–9 alone in distinguishing individuals with pancreatic cancer from the combined pancreatic disease controls and healthy subject groups (P = 0.078). SELDI markers were also better than CA19–9 in distinguishing patients with pancreatic cancer from those with pancreatitis. Conclusion: SELDI profiling of serum can be used to accurately differentiate patients with pancreatic cancer from those with other pancreatic diseases and from healthy controls.

Proteomic Approaches to Tumor Marker Discovery

CONTEXT Current tumor markers for ovarian cancer still lack adequate sensitivity and specificity to be applicable in large populations. High-throughput proteomic profiling and bioinformatics tools allow for the rapid screening of a large number of potential biomarkers in serum, plasma, or other body fluids. OBJECTIVE To determine whether protein profiles of plasma can be used to identify potential biomarkers that improve the detection of ovarian cancer. DESIGN We analyzed plasma samples that had been collected between 1998 and 2001 from patients with sporadic ovarian serous neoplasms before tumor resection at various International Federation of Gynecology and Obstetrics stages (stage I [n = 11], stage II [n = 3], and stage III [n = 29]) and from women without known neoplastic disease (n = 38) using proteomic profiling and bioinformatics. We compared results between the patients with and without cancer and evaluated their discriminatory performance against that of the cancer antigen 125 (CA125) tumor marker. RESULTS We selected 7 biomarkers based on their collective contribution to the separation of the 2 patient groups. Among them, we further purified and subsequently identified 3 biomarkers. Individually, the biomarkers did not perform better than CA125. However, a combination of 4 of the biomarkers significantly improved performance (P < or =.001). The new biomarkers were complementary to CA125. At a fixed specificity of 94%, an index combining 2 of the biomarkers and CA125 achieves a sensitivity of 94% (95% confidence interval, 85%-100.0%) in contrast to a sensitivity of 81% (95% confidence interval, 68%-95%) for CA125 alone. CONCLUSIONS The combined use of bioinformatics tools and proteomic profiling provides an effective approach to screen for potential tumor markers. Comparison of plasma profiles from patients with and without known ovarian cancer uncovered a panel of potential biomarkers for detection of ovarian cancer with discriminatory power complementary to that of CA125. Additional studies are required to further validate these biomarkers.

Primary retinal pathology in multiple sclerosis as detected by optical coherence tomography

Optical coherence tomography studies in multiple sclerosis have primarily focused on evaluation of the retinal nerve fibre layer. The aetiology of retinal changes in multiple sclerosis is thought to be secondary to optic nerve demyelination. The objective of this study was to use optical coherence tomography to determine if a subset of patients with multiple sclerosis exhibit primary retinal neuronopathy, in the absence of retrograde degeneration of the retinal nerve fibre layer and to ascertain if such patients may have any distinguishing clinical characteristics. We identified 50 patients with multiple sclerosis with predominantly macular thinning (normal retinal nerve fibre-layer thickness with average macular thickness < 5th percentile), a previously undescribed optical coherence tomography defined phenotype in multiple sclerosis, and compared them with 48 patients with multiple sclerosis with normal optical coherence tomography findings, 48 patients with multiple sclerosis with abnormal optical coherence tomography findings (typical for multiple sclerosis) and 86 healthy controls. Utilizing a novel retinal segmentation protocol, we found that those with predominant macular thinning had significant thinning of both the inner and outer nuclear layers, when compared with other patients with multiple sclerosis (P < 0.001 for both), with relative sparing of the ganglion cell layer. Inner and outer nuclear layer thicknesses in patients with non-macular thinning predominant multiple sclerosis were not different from healthy controls. Segmentation analyses thereby demonstrated extensive deeper disruption of retinal architecture in this subtype than may be expected due to retrograde degeneration from either typical clinical or sub-clinical optic neuropathy. Functional corroboration of retinal dysfunction was provided through multi-focal electroretinography in a subset of such patients. These findings support the possibility of primary retinal pathology in a subset of patients with multiple sclerosis. Multiple sclerosis-severity scores were also significantly increased in patients with the macular thinning predominant phenotype, compared with those without this phenotype (n = 96, P=0.006). We have identified a unique subset of patients with multiple sclerosis in whom there appears to be disproportionate thinning of the inner and outer nuclear layers, which may be occurring as a primary process independent of optic nerve pathology. In vivo analyses of retinal layers in multiple sclerosis have not been previously performed, and structural demonstration of pathology in the deeper retinal layers, such as the outer nuclear layer, has not been previously described in multiple sclerosis. Patients with inner and outer nuclear layer pathology have more rapid disability progression and thus retinal neuronal pathology may be a harbinger of a more aggressive form of multiple sclerosis.

Cutting Edge: The Transcription Factor Kruppel-Like Factor 4 Regulates the Differentiation of Th17 Cells Independently of RORγt

Th17 cells play a significant role in inflammatory and autoimmune responses. Although a number of molecular pathways that contribute to the lineage differentiation of T cells have been discovered, the mechanisms by which lineage commitment occurs are not fully understood. Transcription factors play a key role in driving T cells toward specific lineages. We have identified a role for the transcription factor Kruppel-like factor (KLF) 4 in the development of IL-17–producing CD4+ T cells. KLF4 was required for the production of IL-17, and further, chromatin immunoprecipation analysis demonstrated binding of KLF4 to the IL-17 promoter, indicating a direct effect on the regulation of IL-17. Further, KLF4-deficient T cells upregulated expression of retinoic acid-related orphan receptor γt similar to wild-type during the polarization process toward Th17, suggesting that these two transcription factors are regulated independently.

Mouse model of intrauterine inflammation: sex-specific differences in long-term neurologic and immune sequelae.

Preterm infants, especially those that are exposed to prenatal intrauterine infection or inflammation, are at a major risk for adverse neurological outcomes, including cognitive, motor and behavioral disabilities. We have previously shown in a mouse model that there is an acute fetal brain insult associated with intrauterine inflammation. The objectives of this study were: (1) to elucidate long-term (into adolescence and adulthood) neurological outcomes by assessing neurobehavioral development, MRI, immunohistochemistry and flow cytometry of cells of immune origin and (2) to determine whether there are any sex-specific differences in brain development associated with intrauterine inflammation. Our results have shown that prenatal exposure appeared to lead to changes in MRI and behavior patterns throughout the neonatal period and during adulthood. Furthermore, we observed chronic brain inflammation in the offspring, with persistence of microglial activation and increased numbers of macrophages in the brain, ultimately resulting in neuronal loss. Moreover, our study highlights the sex-specific differences in long-term sequelae. This study, while extending the growing literature of adverse neurologic outcomes following exposure to inflammation during early development, presents novel findings in the context of intrauterine inflammation.

KLF4 Modulates Expression of IL-6 in Dendritic Cells via Both Promoter Activation and Epigenetic Modification

Background: Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that influences immunity. Results: KLF4 binds to CACCC sequences in the IL-6 promoter, directly activates transcription, and impacts promoter acetylation. Conclusion: KLF4 modulates IL-6 production by dendritic cells via both a direct transcriptional activation and epigenetic modification of the promoter. Significance: KLF4 plays a significant and previously unrecognized role in regulation of IL-6. The initiation and maintenance of the immune response require a coordinated regulation of signal transduction pathways. Identifying the mechanisms by which these pathways are controlled and modulated is a significant goal of immunology. In the present report, we show a novel role for the zinc finger transcription factor Kruppel-like factor 4 (KLF4) in the modulation of the inflammatory immune response via its regulation of IL-6. We analyzed the role of KLF4 in the production of IL-6 by dendritic cells. Our data indicate that KLF4 can act in a dual function manner. It acts as a transcription factor in that it can bind to and activate the IL-6 promoter at specific binding sites. KLF4 also has a role in the chromatin remodeling of the IL-6 promoter in that cells deficient in KLF4 exhibited a relative hypoacetylation. These results indicate a molecular role for KLF4 in modulating the intensity of the inflammatory response and help to explain its pleiotropic role in different settings.

Functional Blockade of the Voltage-gated Potassium Channel Kv1.3 Mediates Reversion of T Effector to Central Memory Lymphocytes through SMAD3/p21cip1 Signaling

Background: The role of Kv1.3 in regulating T cell differentiation and memory is incompletely understood. Results: A dominant negative mutation of Kv1.3 mediates reversion of TEM into TCM through SMAD3-dependent cell cycle changes. Conclusion: Signaling through Kv1.3 is a mechanism by which TEM may revert to TCM. Significance: These findings suggest a novel role for Kv1.3 in T cell differentiation and memory responses. The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (TEM) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (TCM) into TEM. Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of TEM into TCM, via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21cip1 with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in TEM-mediated autoimmune diseases.

Interleukin-1 Receptor Blockade in Perinatal Brain Injury

Interleukin-1 (IL-1) is a potent inflammatory cytokine that can be produced by a variety of cell types throughout the body. While IL-1 is a central mediator of inflammation and response to infection, the role of IL-1 signaling in adult and pediatric brain injury is becoming increasingly clear. Although the mechanisms of IL-1 expression are largely understood, the downstream effects and contributions to excitotoxicity and oxidative stress are poorly defined. Here, we present a review of mechanisms of IL-1 signaling with a focus on the role of IL-1 in perinatal brain injury. We highlight research models of perinatal brain injury and the use of interleukin-1 receptor antagonist (IL-1RA) as an agent of therapeutic potential in preventing perinatal brain injury due to exposure to inflammation.

Expression of inwardly rectifying potassium channels by an inducible adenoviral vector reduced the neuronal hyperexcitability and hyperalgesia produced by chronic compression of the spinal ganglion

BackgroundA chronic compressed dorsal root ganglion (CCD) in rat produces pain behavior and an enhanced excitability of neurons within the compressed ganglion. Kir2.1 is an inwardly rectifying potassium channel that acts to stabilize the resting potential of certain cell types. We hypothesized that an inducible expression of Kir2.1 channels in CCD neurons might suppress neuronal excitability in the dorsal root ganglion (DRG) and reduce the associated pain behavior.ResultsWe delivered, by microinjection into the fourth lumbar (L4) DRG, an adenoviral vector containing a reporter gene encoding the enhanced green fluorescent protein (GFP) and a Kir2.1 channel (AdKir). At the same time the ganglion was compressed by implantation of a rod through the intervertebral foramen (CCD). The in vivo expression of the transferred gene was controlled by an ecdysone analog via an ecdysone-inducible promoter in the viral vector. In comparison with the effects of vehicle or a control vector containing only the GFP gene, AdKir significantly reduced the neuronal hyperexcitability after CCD. Electrophysiological recordings, in vivo, from nociceptive and non-nociceptive DRG neurons expressing the virally produced Kir2.1 channels revealed a hyperpolarized resting membrane potential, an increased rheobase, and lack of spontaneous activity. Inducing the Kir2.1 gene at the beginning of CCD surgery partially prevented the development of mechanical hyperalgesia. However, a delayed induction of the Kir2.1 gene (3 days after CCD surgery) produced no significant effect on the pain behavior.ConclusionsWe found that an inducible expression of Kir2.1 channels in chronically compressed DRG neurons can effectively suppress the neuronal excitability and, if induced at the beginning of CCD injury, prevent the development of hyperalgesia. We hypothesize that a higher level of neuronal hyperexcitability in the DRG is required to initiate than to maintain the hyperalgesia and that the hyperexcitability contributing to neuropathic pain is best inhibited as soon as possible after injury.