Secondary mechanisms of injury and viable pathophysiological targets in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) can be divided into a primary and secondary phase. In the primary phase, hematoma volume is evaluated and therapies are focused on reducing hematoma expansion. In the secondary, neuroprotective phase, complex systemic inflammatory cascades, direct cellular toxicity, and blood-brain barrier disruption can result in worsening perihematomal edema that can adversely affect functional outcome. To date, all major randomized phase 3 trials for ICH have targeted primary phase hematoma volume and incorporated clot evacuation, intensive blood pressure control, and hemostasis. Reasons for this lack of clinical efficacy in the major ICH trials may be due to the lack of therapeutics involving mitigation of secondary injury and inflexible trial design that favors unilateral mechanisms in a complex pathophysiology. Potential pathophysiological targets for attenuating secondary injury are highlighted in this review and include therapies increasing calcium, antagonizing microglial activation, maintaining macrophage M1 versus M2 balance by decreasing M1 signaling, aquaporin inhibition, NKCCl inhibition, endothelin receptor inhibition, Sur1-TRPM4 inhibition, matrix metalloproteinase inhibition, and sphingosine-1-phosphate receptor modulation. Future clinical trials in ICH focusing on secondary phase injury and, potentially implementing adaptive trial design approaches with multifocal targets, may improve insight into these mechanisms and provide potential therapies that may improve survival and functional outcome.

T Cell-Mediated Antitumor Immunity Cooperatively Induced By TGFßR1 Antagonism and Gemcitabine Counteracts Reformation of the Stromal Barrier in Pancreatic Cancer.

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.

Hepatitis B Virus Infection of Normal Hepatic Stem/Progenitor Cells.

BACKGROUND/AIMS: Whether the hepatitis B virus (HBV) infects normal hepatic stem/progenitor cells (NSCs) and if so, whether such infections play a role in the pathogenesis of HBV-induced chronic liver disease (CLD) and/or hepatocellular carcinoma (HCC) remains to be determined. The objectives of this study were to determine whether HBV infects NSCs and whether such infections alter NSC activity in a manner likely to contribute to the development of CLD and/or HCC. METHODS: Liver biopsies from five hepatitis B surface antigen (HBsAg) positive patients were co-stained for HBcAg and HBx and the stem cell markers EpCAM, Oct-4 and Nanog. In addition, primary NSCs derived from healthy human livers were exposed to HBV contaminated serum in vitro. Supernatant and/or cellular HBsAg, HBcAg and HBV-DNA expression were documented over the subsequent 30 days of culture. Pro- and anti-inflammatory cytokine expression, membrane potential differences (PDs), proliferative and telomerase activities of HBV-infected NSCs were also documented. RESULTS: Markers of HBV infection were present within the NSC population of all five biopsy specimens. In vitro, HBV markers appeared within three days of exposure, peaked in expression after 10-15 days and remained positive thereafter for the duration of cell viability. There were no consistent changes in HBV-infected NSC pro- or anti-inflammatory cytokine expression, membrane PDs, proliferative or telomerase activities. CONCLUSIONS: Although the results of this study need to be confirmed, they suggest that HBV infects human NSCs but in the short term, do not alter those NSC features or activities associated with CLD and/or HCC.

Cancer Stem Cells are Depolarized Relative to Normal Stem Cells Derived from Human Livers

ABSTRACT Introduction and aim. The inability to distinguish cancer (CSCs) from normal stem cells (NSCs) has hindered attempts to identify safer, more effective therapies for hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potential differences (PDs) of CSCs and NSCs derived from human HCC and healthy livers respectively and determine whether altered GABAergic innervation could explain the differences. Material and methods. Epithelial cell adhesion molecule (EpCAM) positive stem cells were isolated from human liver tissues by magnetic bead separations. Cellular PDs were recorded by microelectrode impalement of freshly isolated cells. GABAA receptor subunit expression was documented by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. Results. CSCs were significantly depolarized (-7.0 ± 1.3 mV) relative to NSCs (-23.0 ± 1.4 mV, p < 0.01). The depolarized state was associated with different GABAA receptor subunit expression profiles wherein phasic transmission, represented by GAGAA α3 subunit expression, was prevalent in CSCs while tonic transmission, represented by GABAA α6 subunit expression, prevailed in NSCs. In addition, GABAA subunits α3, β3, γ3 and δ were strongly expressed in CSCs while GABAA π expression was dominant in NSCs. CSCs and NSCs responded similarly to GABAA receptor agonists (ΔPD: 12.5 ± 1.2 mV and 11.0 ± 3.5 mV respectively). Conclusion. The results of this study indicate that CSCs are significantly depolarized relative to NSCs and these differences are associated with differences in GABAA receptor subunit expression. Together they provide new insights into the pathogenesis and possible treatment of human HCC.

Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.

Mutations in isocitrate dehydrogenase (IDH) are the most prevalent genetic abnormalities in lower grade gliomas. The presence of these mutations in glioma is prognostic for better clinical outcomes with longer patient survival. In the present study, we found that defects in oxidative metabolism and 2-HG production confer chemosensitization in IDH1-mutated glioma cells. In addition, temozolomide (TMZ) treatment induced greater DNA damage and apoptotic changes in mutant glioma cells. The PARP1-associated DNA repair pathway was extensively compromised in mutant cells due to decreased NAD+ availability. Targeting the PARP DNA repair pathway extensively sensitized IDH1-mutated glioma cells to TMZ. Our findings demonstrate a novel molecular mechanism that defines chemosensitivity in IDH-mutated gliomas. Targeting PARP-associated DNA repair may represent a novel therapeutic strategy for gliomas. Cancer Res; 77(7); 1709-18. ©2017 AACR.

Cancer Stem Cells are Depolarized Relative to Normal Stem Cells Derived from Human Livers.

INTRODUCTION AND AIM: The inability to distinguish cancer (CSCs) from normal stem cells (NSCs) has hindered attempts to identify safer, more effective therapies for hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potential differences (PDs) of CSCs and NSCs derived from human HCC and healthy livers respectively and determine whether altered GABAergic innervation could explain the differences. MATERIAL AND METHODS: Epithelial cell adhesion molecule (EpCAM) positive stem cells were isolated from human liver tissues by magnetic bead separations. Cellular PDs were recorded by microelectrode impalement of freshly isolated cells. GABAA receptor subunit expression was documented by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. RESULTS: CSCs were significantly depolarized (-7.0 ± 1.3 mV) relative to NSCs (-23.0 ± 1.4 mV, p &lt; 0.01). The depolarized state was associated with different GABAA receptor subunit expression profiles wherein phasic transmission, represented by GAGAA α3 subunit expression, was prevalent in CSCs while tonic transmission, represented by GABAA α6 subunit expression, prevailed in NSCs. In addition, GABAA subunits α3, ß3, ϒ3 and δ were strongly expressed in CSCs while GABAA π expression was dominant in NSCs. CSCs and NSCs responded similarly to GABAA receptor agonists (ΔPD: 12.5 ± 1.2 mV and 11.0 ± 3.5 mV respectively). CONCLUSION: The results of this study indicate that CSCs are significantly depolarized relative to NSCs and these differences are associated with differences in GABAA receptor subunit expression. Together they provide new insights into the pathogenesis and possible treatment of human HCC.

Evidence of Hepatitis B Virus Infection in Cancer and Noncancer Stem Cells Associated with Human Hepatocellular Carcinoma.

Both the hepatitis B virus (HBV) and cancer stem cells (CSCs) have been independently implicated in the pathogenesis of hepatocellular carcinoma (HCC). To date, there have been no reports describing HBV infection within CSCs. In this report we describe HBV core (HBcAg) and HBx protein expression within CSCs associated with human HCC. HBV markers were also identified in nonmalignant stem cells present in adjacent nontumor tissue. These findings provide new insights into the pathogenesis of HBV-induced HCC and are potentially relevant to the treatment of both HCC and chronic HBV.

Membrane potential differences and GABAA receptor expression in hepatic tumor and non-tumor stem cells.

The ability to differentiate tumor initiating stem cells (TISCs) from healthy, normal stem cells (NSCs) could have important diagnostic and therapeutic implications for patients with hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potentials (PDs) and GABAA receptor subunit expression in hepatic TISCs and NSCs. PD values were determined in CD133(+) Huh-7 TISCs and CD133(+) WBF344 NSCs by single channel microelectrode impalement. GABAA receptor subunit expression was documented using immunohistochemistry (IH) in both cell lines as well as surgically resected HCC and healthy liver tissues. TISCs were significantly depolarized compared with NSCs (-4.0 ± 1.8 versus -11.0 ± 2.4 mV, respectively; p < 0.05). GABAA α6 subunit expression was either absent or markedly attenuated, while γ3 subunit expression was abundant in TISCs and HCC compared with NSCs and healthy liver tissues. Exposure to the GABAA receptor agonist muscimol caused hyperpolarization of TISCs (Δ -4.4 ± 1.1) but depolarization of NSCs (Δ + 5.2 ± 2.3) and attenuation of TISC proliferative activity. We conclude that TISCs and NSCs have significantly different cell membrane potentials and these differences are associated with differences in GABAA receptor subunit expression.

Requirement of neuronal connexin36 in pathways mediating presynaptic inhibition of primary afferents in functionally mature mouse spinal cord.

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.

A novel fluorinated stilbene exerts hepatoprotective properties in CCl(4)-induced acute liver damage.

There has been a recently increase in the development of novel stilbene-based compounds with in vitro anti-inflamatory properties. For this study, we synthesized and evaluated the anti-inflammatory properties of 2 fluorinated stilbenes on carbon tetrachloride (CCl4)-induced acute liver damage. To achieve this, CCl4 (4 g·kg(-1), per os) was administered to male Wistar rats, followed by either 2-fluoro-4'-methoxystilbene (FME) or 2,3-difluoro-4'-methoxystilbene (DFME) (10 mg·kg(-1), per os). We found that although both of the latter compounds prevented cholestatic damage (γ-glutamyl transpeptidase activity), only DFME showed partial but consistent results in the prevention of necrosis, as assessed by both alanine aminotransferase activity and histological analysis. Since inflammatory responses are mediated by cytokines, mainly tumour necrosis factor α (TNF-α), we used the Western blot technique to determine the action of FME and DFME on the expression level of this cytokine. The observed increase in the level of TNF-α caused by CCl4 administration was only prevented by treatment with DFME, in agreement with our biochemical findings. This result was confirmed by measuring interleukin-6 (IL-6) levels, since the expression of this protein depends on the level of TNF-α. In this case, DFME completely blocked the CCl4-induced increase of IL-6. Our results suggest that DFME possesses greater anti-inflammatory properties in vivo than FME. DFME constitutes a possible therapeutic agent for liver disease and could serve as a template for structure optimization.

Pre- and postsynaptic modulation of monosynaptic reflex by GABAA receptors on turtle spinal cord.

There is growing evidence that activation of high affinity extrasynaptic GABA(A) receptors in the brain, cerebellum and spinal cord substantia gelatinosa results in a tonic inhibition controlling postsynaptic excitability. The aim of the present study was to determine if GABA(A) receptors mediating tonic inhibition participate in the modulation of monosynaptic reflex (MSR) in the vertebrate spinal cord. Using an in vitro turtle lumbar spinal cord preparation, we show that conditioning stimulation of a dorsal root depressed the test monosynaptic reflex (MSR) at long condition-test intervals. This long duration inhibition is similar to the one seen in mammalian spinal cord and it is dependent on GABA(A) as it was completely blocked by 20 microm picrotoxin (PTX) or bicuculline (BIC) or 1 microm gabazine, simultaneously depressing the dorsal root potential (DRP) without MSR facilitation. Interestingly 100 microm picrotoxin or BIC potentiated the MSR, depressed the DRP, and produced a long lasting motoneurone after-discharge. Furosemide, a selective antagonist of extrasynaptic GABA(A) receptors, affects receptor subtypes with alpha(4/6) subunits, and in a similar way to higher concentrations of PTX or BIC, also potentiated the MSR but did not affect the DRP, suggesting the presence of alpha(4/6) GABA(A) receptors at motoneurones. Our results suggest that (1) the turtle spinal cord has a GABA(A) mediated long duration inhibition similar to presynaptic inhibition observed in mammals, (2) GABA(A) receptors located at the motoneurones and primary afferents might produce tonic inhibition of monosynaptic reflex, and (3) GABA(A) receptors modulate motoneurone excitability reducing the probability of spurious and inappropriate activation.

N- and P/Q-type Ca2+ channels regulate synaptic efficacy between spinal dorsolateral funiculus terminals and motoneurons.

Ca2+ influx through voltage-gated Ca2+ channels mediates synaptic transmission at numerous central synapses. However, electrophysiological and pharmacological evidence linking Ca+ channel activity with neurotransmitter release in the vertebrate mature spinal cord is scarce. In the current report, we investigated in a slice preparation from the adult turtle spinal cord, the effects of various Ca+ channel antagonists on neurotransmission at terminals from the dorsolateral funiculus synapsing motoneurons. Bath application of tetrodotoxin or NiCl2 prevented the monosynaptic excitatory postsynaptic potentials (EPSPs), and this effect was mimicked by exposure to a zero-Ca2+ solution. Application of polypeptide toxins that block N- and P/Q-type channels (omega-CTx-GVIA and omega-Aga-IVA) reduced the EPSP amplitude in a dose-dependent manner. By analyzing the input resistance and the EPSP time course, and using a paired pulse protocol we determined that both toxins act at presynaptic level to modulate neurotransmitter release. RT-PCR studies showed the expression of N- and P/Q-type channel mRNAs in the turtle spinal cord. Together, these results indicate that N- and P/Q-type Ca2+ channels may play a central role in the regulation of neurotransmitter release in the adult turtle spinal cord.