Comparison of the Simplexa GBS Direct and ARIES GBS assays for the detection of S. agalactiae in broth-enriched swab specimens.

We conducted a comparative evaluation of the FDA-cleared Simplexa GBS Direct and ARIES GBS molecular assays for the detection of Streptococcus agalactiae (Group B Streptococcus, GBS) in 386 prospectively collected, broth-enriched vaginal/rectal swab specimens. The sensitivity of each test was 96.2% and specificity was ≥98.7% when compared to a combined direct and enriched culture method using chromogenic culture medium. A total of four specimens were called positive by both molecular assays but negative by culture, likely representing specimens with a low burden of GBS in these specimens. Two specimens were reported positive by culture but negative by both molecular assays. One of these specimens demonstrated atypically colored colonies on chromogenic agar; the other yielded typically colored colonies only observed after broth enrichment. Our data demonstrate equivalent performance of Simplexa and ARIES molecular assays for the detection of GBS in clinical specimens.IMPORTANCEClinical laboratories often face decisions regarding which of the multiple available molecular platforms would best fit their needs based on cost, workflow, menu, and diagnostic performance. Therefore, objective clinical comparisons of similar molecular tests are valuable resources to aid these decisions. We provide a clinical comparison of two FDA-cleared tests to routine culture and to each other that can be used by clinical laboratories when determining which of the available molecular platforms would best fit their laboratory in terms of workflow, cost, and performance.

Stat5 induces androgen receptor (AR) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling.

Androgen receptor (AR) drives prostate cancer (PC) growth and progression, and targeting AR signaling is the mainstay of pharmacological therapies for PC. Resistance develops relatively fast as a result of refueled AR activity. A major gap in the field is the lack of understanding of targetable mechanisms that induce persistent AR expression in castrate-resistant PC (CRPC). This study uncovers an unexpected function of active Stat5 signaling, a known promoter of PC growth and clinical progression, as a potent inducer of AR gene transcription. Stat5 suppression inhibited AR gene transcription in preclinical PC models and reduced the levels of wild-type, mutated, and truncated AR proteins. Pharmacological Stat5 inhibition by a specific small-molecule Stat5 inhibitor down-regulated Stat5-inducible genes as well as AR and AR-regulated genes and suppressed PC growth. This work introduces the concept of Stat5 as an inducer of AR gene transcription in PC. Pharmacological Stat5 inhibitors may represent a new strategy for suppressing AR and CRPC growth.

Second-Generation Jak2 Inhibitors for Advanced Prostate Cancer: Are We Ready for Clinical Development?

Androgen deprivation therapy (ADT) for metastatic and high-risk prostate cancer (PC) inhibits growth pathways driven by the androgen receptor (AR). Over time, ADT leads to the emergence of lethal castrate-resistant PC (CRPC), which is consistently caused by an acquired ability of tumors to re-activate AR. This has led to the development of second-generation anti-androgens that more effectively antagonize AR, such as enzalutamide (ENZ). However, the resistance of CRPC to ENZ develops rapidly. Studies utilizing preclinical models of PC have established that inhibition of the Jak2-Stat5 signaling leads to extensive PC cell apoptosis and decreased tumor growth. In large clinical cohorts, Jak2-Stat5 activity predicts PC progression and recurrence. Recently, Jak2-Stat5 signaling was demonstrated to induce ENZ-resistant PC growth in preclinical PC models, further emphasizing the importance of Jak2-Stat5 for therapeutic targeting for advanced PC. The discovery of the Jak2V617F somatic mutation in myeloproliferative disorders triggered the rapid development of Jak1/2-specific inhibitors for a variety of myeloproliferative and auto-immune disorders as well as hematological malignancies. Here, we review Jak2 inhibitors targeting the mutated Jak2V617F vs. wild type (WT)-Jak2 that are currently in the development pipeline. Among these 35 compounds with documented Jak2 inhibitory activity, those with potency against WT-Jak2 hold strong potential for advanced PC therapy.

Prospects for Clinical Development of Stat5 Inhibitor IST5-002: High Transcriptomic Specificity in Prostate Cancer and Low Toxicity In Vivo.

Stat5 is of significant interest in the search for new therapeutics for prostate cancer (PC) and hematopoietic disorders. We evaluated the transcriptomic specificity of the Stat5a/b inhibitor IST5-002 (IST5) in PC, defined more closely its mechanisms of action, and investigated the in vivo toxicity of IST5 for further optimization for clinical development. The transcriptomic specificity of IST5 vs. genetic Stat5 knockdown was evaluated by RNA-seq analysis, which showed high similarity with the Pearson correlation coefficient ranging from 0.98-0.99. The potency of IST5 vs. its derivative lacking the phosphate group in suppressing Stat5 was evaluated in two separate but complementary assays. The inhibitory activity of IST5 against kinases was investigated in cell-free assays followed by more focused evaluation in a cell-based assay. IST5 has no specific inhibitory activity against 54 kinases, while suppressing Stat5 phosphorylation and subsequent dimerization in PC cells. The phosphate group was not critical for the biological activity of IST5 in cells. The acute, sub-chronic and chronic toxicity studies of IST5 were carried out in mice. IST5 did not cause any significant toxic effects or changes in the blood profiles. The present work supports further optimization of IST5 for oral bioavailability for clinical development for therapies for solid tumors, hematological and myeloproliferative disorders.

Cytokines, JAK-STAT Signaling and Radiation-Induced DNA Repair in Solid Tumors: Novel Opportunities for Radiation Therapy.

A number of solid tumors are treated with radiation therapy (RT) as a curative modality. At the same time, for certain types of cancers the applicable doses of RT are not high enough to result in a successful eradication of cancer cells. This is often caused by limited pharmacological tools and strategies to selectively sensitize tumors to RT while simultaneously sparing normal tissues from RT. We present an outline of a novel strategy for RT sensitization of solid tumors utilizing Jak inhibitors. Here, recently published pre-clinical data are reviewed which demonstrate the promising role of Jak inhibition in sensitization of tumors to RT. A wide number of currently approved Jak inhibitors for non-malignant conditions are summarized including Jak inhibitors currently in clinical development. Finally, intersection between Jak/Stat and the levels of serum cytokines are presented and discussed as they relate to susceptibility to RT.

Presenilin 1 Regulates NF-κB Activation via Association with Breakpoint Cluster Region and Casein Kinase II.

We recently reported that NF-κB-mediated inflammation caused by breakpoint cluster region (BCR) is dependent on the α subunit of casein kinase II (CK2α) complex. In the current study, we demonstrate that presenilin 1 (Psen1), which is a catalytic component of the γ-secretase complex and the mutations of which are known to cause familial Alzheimer disease, acts as a scaffold of the BCR-CK2α-p65 complex to induce NF-κB activation. Indeed, Psen1 deficiency in mouse endothelial cells showed a significant reduction of NF-κB p65 recruitment to target gene promoters. Conversely, Psen1 overexpression enhanced reporter activation under NF-κB responsive elements and IL-6 promoter. Furthermore, the transcription of NF-κB target genes was not inhibited by a γ-secretase inhibitor, suggesting that Psen1 regulates NF-κB activation in a manner independent of γ-secretase activity. Mechanistically, Psen1 associated with the BCR-CK2α complex, which is required for phosphorylation of p65 at serine 529. Consistently, TNF-α-induced phosphorylation of p65 at serine 529 was significantly decreased in Psen1-deficient cells. The association of the BCR-CK2α-p65 complex was perturbed in the absence of Psen1. These results suggest that Psen1 functions as a scaffold of the BCR-CK2α-p65 complex and that this signaling cascade could be a novel therapeutic target for various chronic inflammation conditions, including those in Alzheimer disease.

Breakpoint Cluster Region-Mediated Inflammation Is Dependent on Casein Kinase II.

The breakpoint cluster region (BCR) is known as a kinase and cause of leukemia upon fusing to Abl kinase. In this study, we demonstrate that BCR associated with the α subunit of casein kinase II (CK2α), rather than BCR itself, is required for inflammation development. We found that BCR knockdown inhibited NF-κB activation in vitro and in vivo. Computer simulation, however, suggested that the putative BCR kinase domain has an unstable structure with minimal enzymatic activity. Liquid chromatography-tandem mass spectrometry analysis showed that CK2α associated with BCR. We found the BCR functions are mediated by CK2α. Indeed, CK2α associated with adaptor molecules of TNF-αR and phosphorylated BCR at Y177 to establish a p65 binding site after TNF-α stimulation. Notably, p65 S529 phosphorylation by CK2α creates a p300 binding site and increased p65-mediated transcription followed by inflammation development in vivo. These results suggest that BCR-mediated inflammation is dependent on CK2α, and the BCR-CK2α complex could be a novel therapeutic target for various inflammatory diseases.

Strong TCR-mediated signals suppress integrated stress responses induced by KDELR1 deficiency in naive T cells.

KDEL receptor 1 (KDELR1) regulates integrated stress responses (ISR) to promote naive T-cell survival in vivo. In a mouse line having nonfunctional KDELR1, T-Red (naive T-cell reduced) mice, polyclonal naive T cells show excessive ISR and eventually undergo apoptosis. However, breeding T-Red mice with TCR-transgenic mice bearing relatively high TCR affinity rescued the T-Red phenotype, implying a link between ISR-induced apoptosis and TCR-mediated signaling. Here, we showed that strong TCR stimulation reduces ISR in naive T cells. In mice lacking functional KDELR1, surviving naive T cells expressed significantly higher levels of CD5, a surrogate marker of TCR self-reactivity. In addition, higher TCR affinity/avidity was confirmed using a tetramer dissociation assay on the surviving naive T cells, suggesting that among the naive T-cell repertoire, those that receive relatively stronger TCR-mediated signals via self-antigens survive enhanced ISR. Consistent with this observation, weak TCR stimulation with altered peptide ligands decreased the survival and proliferation of naive T cells, whereas stimulation with ligands having higher affinity had no such effect. These results suggest a novel role of TCR-mediated signals in the attenuation of ISR in vivo.

KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR.

KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

In this study, we investigated the relationship between several growth factors and inflammation development. Serum concentrations of epiregulin, amphiregulin, betacellulin, TGF-α, fibroblast growth factor 2, placental growth factor (PLGF), and tenascin C were increased in rheumatoid arthritis patients. Furthermore, local blockades of these growth factors suppressed the development of cytokine-induced arthritis in mice by inhibiting chemokine and IL-6 expressions. We found that epiregulin expression was early and followed by the induction of other growth factors at different sites of the joints. The same growth factors then regulated the expression of epiregulin at later time points of the arthritis. These growth factors were increased in patients suffering from multiple sclerosis (MS) and also played a role in the development of an MS model, experimental autoimmune encephalomyelitis. The results suggest that the temporal expression of growth factors is involved in the inflammation development seen in several diseases, including rheumatoid arthritis and MS. Therefore, various growth factor pathways might be good therapeutic targets for various inflammatory diseases.

Naïve T Cell Homeostasis Regulated by Stress Responses and TCR Signaling.

The survival of naïve T cells is believed to require signals from TCR-pMHC interactions and cytokines such as IL-7. In contrast, signals that negatively impact naïve T cell survival are less understood. We conducted a forward genetic screening of mice and found a mutant mouse line with reduced number of naïve T cells (T-Red mice). T-Red mice have a point mutation in the Kdelr1 gene, and their naïve T cells show enhanced integrated stress response (ISR), which eventually induces their apoptosis. Therefore, naïve T cells require a KDEL receptor-mediated mechanism that efficiently relieves cellular stress for their survival in vivo. Interestingly, naïve T cells expressing TCR with higher affinity/avidity to self-antigens survive in T-Red mice, suggesting the possible link between TCR-mediated survival and ISR-induced apoptosis. In this article, we discuss the regulation of naïve T cell homeostasis, keeping special attention on the ISR and TCR signal.

The Gateway Reflex, which is mediated by the inflammation amplifier, directs pathogenic immune cells into the CNS.

The brain-blood barrier (BBB) tightly limits immune cell migration into the central nervous system (CNS), avoiding unwanted inflammation under the normal state. However, immune cells can traverse the BBB when inflammation occurs within the CNS, suggesting a certain signal that creates a gateway that bypasses the BBB might exist. We revealed the inflammation amplifier as a mechanism of this signal, and identified dorsal vessels of the fifth lumber (L5) spinal cord as the gateway. The inflammation amplifier is driven by a simultaneous activation of NF-κB and STATs in non-immune cells, causing the production of a large amount of inflammatory chemokines to open the gateway at L5 vessels. It was found that the activation of the amplifier can be modulated by neural activation and artificially operated by electric pulses followed by establishment of new gateways, Gateway Reflex, at least in mice. Furthermore, genes required for the inflammation amplifier have been identified and are highly associated with various inflammatory diseases and disorders in the CNS. Thus, physical and/or pharmacological manipulation of the inflammation amplifier holds therapeutic value to control neuro-inflammation.

The reverse-direction method links mass experimental data to human diseases.

Genome-wide analyses such as DNA microarray, RNA sequencing and RNA interference-based high-throughput screening are prevalent to decipher a biological process of interest, and provide a large quantity of data to be processed. An ultimate goal for researchers must be extrapolation of their data to human diseases. We have conducted functional genome-wide screenings to elucidate molecular mechanisms of the inflammation amplifier, a NFκB/STAT3-dependent machinery that potently drives recruitment of immune cells to promote inflammation. Using a public database of genome-wide association studies (GWAS), we recently reported the reverse-direction method by which our mass screening data were successfully linked to many human diseases. As an example, the epiregulin-epidermal growth factor receptor pathway was identified as a regulator of the inflammation amplifier, and associated with human diseases by GWAS. In fact, serum epiregulin levels were higher in patients with chronic inflammatory disorders. The reverse-direction method can be a useful tool to narrow mass data down to focus on human disease-related genes.

Inflammation amplifier, a new paradigm in cancer biology.

Tumor-associated inflammation can induce various molecules expressed from the tumors themselves or surrounding cells to create a microenvironment that potentially promotes cancer development. Inflammation, particularly chronic inflammation, is often linked to cancer development, even though its evolutionary role should impair nonself objects including tumors. The inflammation amplifier, a hyperinducer of chemokines in nonimmune cells, is the principal machinery for inflammation and is activated by the simultaneous stimulation of NF-κB and STAT3. We have redefined inflammation as local activation of the inflammation amplifier, which causes an accumulation of various immune cells followed by dysregulation of local homeostasis. Genes related to the inflammation amplifier have been genetically associated with various human inflammatory diseases. Here, we describe how cancer-associated genes, including interleukin (IL)-6, Ptgs2, ErbB1, Gas1, Serpine1, cMyc, and Vegf-α, are strongly enriched in genes related to the amplifier. The inflammation amplifier is activated by the stimulation of cytokines, such as TNF-α, IL-17, and IL-6, resulting in the subsequent expression of various target genes for chemokines and tumor-related genes like BCL2L11, CPNE7, FAS, HIF1-α, IL-1RAP, and SOD2. Thus, we conclude that inflammation does indeed associate with the development of cancer. The identified genes associated with the inflammation amplifier may thus make potential therapeutic targets of cancers.

The gateway theory: bridging neural and immune interactions in the CNS.

The central nervous system (CNS) is considered an immune-privileged tissue protected by a specific vessel structure, the blood-brain barrier (BBB). Upon infection or traumatic injury in the CNS, the BBB is breached, and various immune cells are recruited to the affected area. In the case of autoimmune diseases in the CNS like multiple sclerosis (MS), autoreactive T cells against some CNS-specific antigens can theoretically attack neurons throughout the CNS. The affected CNS regions in MS patients can be detected as multiple focal plaques in the cerebrum, thoracic cord, and other regions. Vision problems are often associated with the initial phase of MS, suggesting a disturbance in the optic nerves. These observations raise the possibility that there exist specific signals that direct autoreactive T cells past the BBB and into particular sites of the CNS. Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we recently defined the mechanism of the pathogenesis in which regional neural stimulations modulate the status of the blood vessel endothelium to allow the invasion of autoreactive T cells into specific sites of the CNS via the fifth lumbar cord. This gate for autoreactive T cells can be artificially manipulated by removing gravity forces on the hind legs or by electric pulses to the soleus muscles, quadriceps, and triceps of mice, resulting in an accumulation of autoreactive T cells in the intended regions via the activation of regional neurons. Gating blood vessels by regional neural stimulations, a phenomenon we call the gateway theory, has potential therapeutic value not only in preventing autoimmunity, but also in augmenting the effects of cancer immunotherapies.

Regulation of immune cell infiltration into the CNS by regional neural inputs explained by the gate theory.

The central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB), which consists of specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet. Despite the BBB, various immune and tumor cells can infiltrate the CNS parenchyma, as seen in several autoimmune diseases like multiple sclerosis (MS), cancer metastasis, and virus infections. Aside from a mechanical disruption of the BBB like trauma, how and where these cells enter and accumulate in the CNS from the blood is a matter of debate. Recently, using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found a "gateway" at the fifth lumber cord where pathogenic autoreactive CD4+ T cells can cross the BBB. Interestingly, this gateway is regulated by regional neural stimulations that can be mechanistically explained by the gate theory. In this review, we also discuss this theory and its potential for treating human diseases.