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BACKGROUND:Hepatic encephalopathy (HE) is a major neurological condition that occurs due to acute and chronic liver failure following drug toxicity, viral hepatitis, or exposure to various hepatotoxins. Acute HE (Type A HE) is associated with cerebral edema, increased intracranial pressure, coma and death. Chronic HE (Type C HE) is characterized by mental confusion, behavioral changes, and motor disturbances. It is currently unclear whether HE is reversible or irreversible. We therefore examined whether acute or chronic liver failure is reversible or irreversible in the thioacetamide (TAA) rat model of acute and chronic HE. Accordingly, rats were treated with the liver toxin TAA and brain edema, neurobehavioral, cognitive and motor deficits were measured.METHODS:Rats were treated with the liver toxin TAA and brain edema, neurobehavioral, cognitive and motor deficits were measured.RESULTS:Treatment of rats with high doses of TAA (to induce acute HE) led to brain edema within 2-3 days. Rats following TAA treatment were examined for longer time-periods. Rats did not exhibit any brain edema, although they expressed neurobehavioral, cognitive and motor deficits without any changes documented in blood and brain ammonia levels, as well as liver failure markers, as compared to normal rats. However, rats that underwent chronic liver failure for 10 days displayed neurobehavioral, cognitive and motor deficits, along with brain structural and molecular events, including reduced levels of astrocytic matricellular proteins, as well as reduced levels of neuronal proteins. We also found a reduction in synaptic density and in dendritic complexity. These changes correlated-well with increased blood/brain ammonia levels and with liver failure markers. However, when TAA treatment was withdrawn (after 10 days), and the rats monitored for longer time periods still showed neurobehavioral, cognitive and motor deficits, as well as defective neuronal integrity, even though blood and brain ammonia levels, as well as liver biochemical and molecular parameters were reversed.CONCLUSIONS:These findings strongly suggest that neurobehavioral, cognitive and motor deficits in HE cannot be reversed even when acute or chronic liver failure had been corrected.

Swelling of astrocytes represents a major component of the brain edema associated with many neurological conditions, including acute hepatic encephalopathy (AHE), traumatic brain injury (TBI) and ischemia. It has previously been reported that exposure of cultured astrocytes to ammonia (a factor strongly implicated in the pathogenesis of AHE), oxygen/glucose deprivation, or to direct mechanical trauma results in an increase in cell swelling. Since dietary polyphenols have been shown to exert a protective effect against cell injury, we examined whether resveratrol (RSV, 3,5,4′-trihydroxy-trans-stilbene, a stilbenoid phenol), has a protective effect on astrocyte swelling following its exposure to ammonia, oxygen–glucose deprivation (OGD), or trauma in vitro. Ammonia increased astrocyte swelling, and pre- or post-treatment of astrocytes with 10 and 25 µM RSV displayed an additive effect, while 5 µM did not prevent the effect of ammonia. However, pre-treatment of astrocytes with 25 µM RSV slightly, but significantly, reduced the trauma-induced astrocyte swelling at earlier time points (3 h), while post-treatment had no significant effect on the trauma-induced cell swelling at the 3 h time point. Instead, pre- or post-treatment of astrocytes with 25 µM RSV had an additive effect on trauma-induced astrocyte swelling. Further, pre- or post-treatment of astrocytes with 5 or 10 µM RSV had no significant effect on trauma-induced astrocyte swelling. When 5 or 10 µM RSV were added prior to, or during the process of OGD, as well as post-OGD, it caused a slight, but not statistically significant decline in cell swelling. However, when 25 µM RSV was added during the process of OGD, as well as after the cells were returned to normal condition (90 min period), such treatment showed an additive effect on the OGD-induced astrocyte swelling. Noteworthy, a higher concentration of RSV (25 µM) exhibited an additive effect on levels of phosphorylated forms of ERK1/2, and p38 MAPK , as well as an increased activity of the Na + –K + –Cl − co-transporter-1 (NKCC1), factors known to induce astrocytes swelling, when the cells were treated with ammonia or after trauma or ischemia. Further, inhibition of ERK1/2, and p38 MAPK diminished the RSV-induced exacerbation of cell swelling post-ammonia, trauma and OGD treatment. These findings strongly suggest that treatment of cultured astrocytes with RSV enhanced the ammonia, ischemia and trauma-induced cell swelling, likely through the exacerbation of intercellular signaling kinases and ion transporters. Accordingly, caution should be exercised when using RSV for the treatment of these neurological conditions, especially when brain edema is also suspected.

BACKGROUND:Chronic hepatic encephalopathy (CHE) is a major neurological condition that occurs following chronic liver failure (CLF) following drug-induced hepatotoxicity, viral hepatitis, or exposure to various hepatotoxins. CHE is characterized by mental confusion, behavioral changes, and motor disturbances. The molecular basis for CHE remains elusive. The presence of Alzheimer type II astrocytes (AT2A) is the only histopathological findings observed in CHE. However, little is currently known regarding its development, and its involvement in the pathogenesis of CHE.METHODS:Astrocytic glia maturation factor and other inflammatory factors were measured by western blots, immunohistochemistry, and by ELISA. AT2A was analyzed by histopathology.RESULTS:We identified increased levels of astrocytc glia maturation factor (GMF), a factor strongly implicated in neuroinflammation and in the overexpression of various inflammatory factors (IL-1β, TNF-α, IL-6, chemokine (C-X-C motif) ligand 1 (CXCL1), matrix metalloproteinase-3 (MMP-3), prostaglandin E2 (PGE2) and cyclooxygenase 2 (COX2), as well as reduced levels of α-tubulin and glial fibrillary acidic protein, along with increased levels of aggregated nuclear protein lamin a/c in the thioacetamide-induced rat model of CHE. Further, we identified reduced levels of neuronal proteins, PSD95, synaptophysin, and NMDA-nr1. Moreover, synaptic density and dendritic complexity are reduced post-CLF. Since elevated blood/brain ammonia levels have been strongly implicated in the pathogenesis of CHE, while exposure of cultured astrocytes to ammonia was shown to develop AT2A, we utilized this in vitro system to delineate mechanisms by which ammonia contributes to the development of AT2A. We found increased levels of GMF, aggregated nuclear protein lamin a/c, and inflammatory factors (IL-1β, TNF-α, IL-6, CXCL1, MMP-3, PGE2 and COX2), as well as reduced levels and oxidized forms of α-tubulin in astrocytes post-ammonia treatment, which was similar to that found in vivo. Further, exposure of cultured neurons to conditioned media (CM) from ammonia-treated astrocytes (AT2A), but not ammonia per se, resulted in reduced levels of neuronal PSD95, synaptophysin and NMDA-nr1, as well as reduced synaptic density and dendritic complexity. Noteworthy, pharmacological inhibition of GMF, or silencing GMF by CRISPR reversed the defective neuronal integrity post-exposure of neurons to CM from ammonia-treated astrocytes in vitro.CONCLUSIONS:These findings strongly suggest that increased levels of GMF post-CLF may negatively impact neuronal integrity that may ultimately contribute to the neurobehavioral/cognitive and motor deficits observed in CHE. We anticipate that our studies aimed at a better understanding of the molecular mechanisms involved in the development of AT2A, and its impact on neuronal integrity in CHE, will greatly facilitate the identification of agents capable of ameliorating this debilitating condition.

The COVID-19 pandemic, which emerged in early 2020, has had a profound and lasting impact on global health, resulting in over 7.0 million deaths and persistent challenges. In addition to acute concerns, there is growing attention being given to the long COVID health consequences for survivors of COVID-19 with documented cases of cardiovascular abnormalities, liver disturbances, lung complications, kidney issues, and noticeable cognitive deficits. Recent studies have investigated the physiological changes in various organs following prolonged exposure to murine hepatitis virus-1 (MHV-1), a coronavirus, in mouse models. One significant finding relates to the effects on the gastrointestinal tract, an area previously understudied regarding the long-lasting effects of COVID-19. This research sheds light on important observations in the intestines during both the acute and the prolonged phases following MHV-1 infection, which parallel specific changes seen in humans after exposure to SARS-CoV-2. Our study investigates the histopathological alterations in the small intestine following MHV-1 infection in murine models, revealing significant changes reminiscent of inflammatory bowel disease (IBD), celiac disease. Notable findings include mucosal inflammation, lymphoid hyperplasia, goblet cell hyperplasia, and immune cell infiltration, mirroring pathological features observed in IBD. Additionally, MHV-1 infection induces villous atrophy, altered epithelial integrity, and inflammatory responses akin to celiac disease and IBD. SPIKENET (SPK) treatment effectively mitigates intestinal damage caused by MHV-1 infection, restoring tissue architecture and ameliorating inflammatory responses. Furthermore, investigation into long COVID reveals intricate inflammatory profiles, highlighting the potential of SPK to modulate intestinal responses and restore tissue homeostasis. Understanding these histopathological alterations provides valuable insights into the pathogenesis of COVID-induced gastrointestinal complications and informs the development of targeted therapeutic strategies.

The COVID-19 pandemic has been one of the most impactful events in our lifetime, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Multiple SARS-CoV-2 variants were reported globally, and a wide range of symptoms existed. Individuals who contract COVID-19 continue to suffer for a long time, known as long COVID or post-acute sequelae of COVID-19 (PASC). While COVID-19 vaccines were widely deployed, both unvaccinated and vaccinated individuals experienced long-term complications. To date, there are no treatments to eradicate long COVID. We recently conceived a new approach to treat COVID in which a 15-amino-acid synthetic peptide (SPIKENET, SPK) is targeted to the ACE2 receptor binding domain of SARS-CoV-2, which prevents the virus from attaching to the host. We also found that SPK precludes the binding of spike glycoproteins with the receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) of a coronavirus, murine hepatitis virus-1 (MHV-1), and with all SARS-CoV-2 variants. Further, SPK reversed the development of severe inflammation, oxidative stress, tissue edema, and animal death post-MHV-1 infection in mice. SPK also protects against multiple organ damage in acute and long-term post-MHV-1 infection. Our findings collectively suggest a potential therapeutic benefit of SPK for treating COVID-19.

Severe disease from SARS-CoV-2 infection often progresses to multi-organ failure and results in an increased mortality rate amongst these patients. However, underlying mechanisms of SARS- CoV-2-induced multi-organ failure and subsequent death are still largely unknown. Cytokine storm, increased levels of inflammatory mediators, endothelial dysfunction, coagulation abnormalities, and infiltration of inflammatory cells into the organs contribute to the pathogenesis of COVID-19. One potential consequence of immune/inflammatory events is the acute progression of generalized edema, which may lead to death. We, therefore, examined the involvement of water channels in the development of edema in multiple organs and their contribution to organ dysfunction in a Murine Hepatitis Virus-1 (MHV-1) mouse model of COVID-19. Using this model, we recently reported multi-organ pathological abnormalities and animal death similar to that reported in humans with SARS-CoV-2 infection. We now identified an alteration in protein levels of AQPs 1, 4, 5, and 8 and associated oxidative stress, along with various degrees of tissue edema in multiple organs, which correlate well with animal survival post-MHV-1 infection. Furthermore, our newly created drug (a 15 amino acid synthetic peptide, known as SPIKENET) that was designed to prevent the binding of spike glycoproteins with their receptor(s), angiotensin- converting enzyme 2 (ACE2), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (SARS-CoV-2 and MHV-1, respectively), ameliorated animal death and reversed altered levels of AQPs and oxidative stress post-MHV-1 infection. Collectively, our findings suggest the possible involvement of altered aquaporins and the subsequent edema, likely mediated by the virus-induced inflammatory and oxidative stress response, in the pathogenesis of COVID- 19 and the potential of SPIKENET as a therapeutic option.

The 5-year survival rate of patients with metastatic renal cell carcinoma (mRCC) is <12% due to treatment failure. Therapeutic strategies that overcome resistance to modestly effective drugs for mRCC, such as sorafenib (SF), could improve outcome in mRCC patients. SF is terminally biotransformed by UDP-glucuronosyltransferase-1A9 (A9) mediated glucuronidation, which inactivates SF. In a clinical-cohort and the TCGA-dataset, A9 transcript and/or protein levels were highly elevated in RCC specimens and predicted metastasis and overall-survival. This suggested that elevated A9 levels even in primary tumors of patients who eventually develop mRCC could be a mechanism for SF failure. 4-methylumbelliferone (MU), a choleretic and antispasmodic drug, downregulated A9 and inhibited SF-glucuronidation in RCC cells. Low-dose SF and MU combinations inhibited growth, motility, invasion and downregulated an invasive signature in RCC cells, patient-derived tumor explants and/or endothelial-RCC cell co-cultures; however, both agents individually were ineffective. A9 overexpression made RCC cells resistant to the combination, while its downregulation sensitized them to SF treatment alone. The combination inhibited kidney tumor growth, angiogenesis and distant metastasis, with no detectable toxicity; A9-overexpressing tumors were resistant to treatment. With effective primary tumor control and abrogation of metastasis in preclinical models, the low-dose SF and MU combinations could be an effective treatment option for mRCC patients. Broadly, our study highlights how targeting specific mechanisms that cause the failure of “old” modestly effective FDA-approved drugs could improve treatment response with minimal alteration in toxicity profile.

Hepatic encephalopathy (HE) is a neurological condition linked to liver failure. Acute HE (Type A) occurs with acute liver failure, while chronic HE (Type C) is tied to cirrhosis and portal hypertension. HE treatments lag due to gaps in understanding its development by gender and age. We studied how sex and age impact HE and its severity with combined liver toxins. Our findings indicate that drug-induced (thioacetamide, TAA) brain edema was more severe in aged males than in young males or young/aged female rats. However, adding alcohol (ethanol, EtOH) worsens TAA’s brain edema in both young and aged females, with females experiencing a more severe effect than males. These patterns also apply to Type A HE induced by azoxymethane (AZO) in mice. Similarly, TAA-induced behavioral deficits in Type C HE were milder in young and aged females than in males. Conversely, EtOH and TAA in young/aged males led to severe brain edema and fatality without noticeable behavioral changes. TAA metabolism was slower in aged males than in young or middle-aged rats. When TAA-treated aged male rats received EtOH, there was a slow and sustained plasma level of thioacetamide sulfoxide (TASO). This suggests that with EtOH, TAA-induced HE is more severe in aged males. TAA metabolism was similar in young, middle-aged, and aged female rats. However, with EtOH, young and aged females experience more severe drug-induced HE as compared to middle-aged adult rats. These findings strongly suggest that gender and age play a role in the severity of HE development and that the presence of one or more liver toxins may aggravate the severity of the disease progression.

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 μg/d; and a 18.4% reduction with 50 μg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κβ/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF). Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB), have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3), we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated) at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1) protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.