Unsaturated or saturated dietary fat-mediated steatosis impairs hepatic regeneration following partial hepatectomy in mice.

BACKGROUND: Partial hepatectomy is a preferred treatment option for many patients with hepatocellular carcinoma however, pre-existing pathological abnormalities originating from hepatic steatosis can alter the decision to perform surgery or postoperative outcomes as a consequence of the impact steatosis has on liver regeneration. AIM: The aim of this study was to investigate the role of a saturated or unsaturated high fat diet-mediated steatosis on liver regeneration following partial hepatectomy. METHODS: Mice were fed a low-fat control diet (CD, 13% fat), lard-based unsaturated (LD, 60% fat) or milk-based saturated high fat diet (MD, 60% fat) for 16 weeks at which time partial hepatectomy (approx. 70% resection) was performed. At days-2 and 7 post hepatectomy, one hour prior to euthanization, mice were injected with 5-bromo-2'-deoxyuridine in order to monitor hepatic regeneration. Serum was collected and assessed for levels of ALT and AST. Resected and regenerated liver tissue were examined for inflammation-indicative markers employing RT-PCR, Western blots, and histological methods. RESULTS: Mice fed LD or MD exhibited higher NAFLD scores, increased expression of inflammatory cytokines, neutrophil infiltration, macrophage accumulation, increased apoptosis, and elevated levels of serum ALT and AST activities, a decrease in the number of BrdU-incorporated-hepatocytes in the regenerated livers compared to the mice fed CD. Mice fed MD showed significantly lower percent of BrdU-incorporated hepatocytes and a higher trend of inflammation compared to the mice fed LD. CONCLUSION: A diet rich in saturated or unsaturated fat results in NASH with decreased hepatic regeneration however unsaturated fat diet cause lower inflammation and higher regeneration than the saturated fat diet following partial hepatectomy in mice.

Methods of Low-Density Lipoprotein-Cholesterol Measurement: Analytical and Clinical Applications.

Among the five major classes of lipoprotein particles, low-density lipoprotein-cholesterol (LDL-C) is the primary lipoprotein risk factor for the development of cardiovascular diseases (CVD) through the promotion of atherosclerotic pathogenesis. Therefore, it is of paramount importance to accurately measure the plasma concentration of LDL-C using an appropriate method to examine the risk of CVD and determine the efficacy of therapeutic interventions to reduce the cholesterol level and examine the risk assessment strategy. At present, there is a wide variety of methods available for LDL-C measurement. In this review, we have outlined the commonly used methods of LDL-C measurement. These methods have been classified into non-automated analytical methods, calculation methods, and automated direct measurement of LDL-C. We have also described some recently proposed promising calculation methods which are being considered for clinical adoption. This current review could assist the clinicians to have a better understanding regarding the measurement techniques and comparative utilities of different methods of LDL-C measurement and guide them to select an appropriate method based on accuracy, turnaround time, and cost of test.

Vascular and immunopathological role of Asymmetric Dimethylarginine (ADMA) in Experimental Autoimmune Encephalomyelitis.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor/uncoupler inducing vascular pathology. Vascular pathology is an important factor for the development and progression of CNS pathology of MS, yet the role of ADMA in MS remains elusive. Patients with multiple sclerosis (MS) are reported to have elevated blood levels of ADMA, and mice with experimental autoimmune encephalomyelitis (EAE, an animal model of MS) generated by auto-immunization of myelin oligodendrocyte glycoprotein (MOG) and blood-brain barrier (BBB) disruption by pertussis toxin also had increased blood ADMA levels in parallel with induction of clinical disease. To explore the role of ADMA in EAE pathogenesis, EAE mice were treated with a daily dose of ADMA. It is of special interest that ADMA treatment enhanced the BBB disruption in EAE mice and exacerbated the clinical and CNS disease of EAE. ADMA treatment also induced the BBB disruption and EAE disease in MOG-immunized mice even without pertussis toxin treatment, suggesting the role of ADMA in BBB dysfunction in EAE. T-cell polarization studies also documented that ADMA treatment promotes TH 1- and TH 17-mediated immune responses but without affecting Treg-mediated immune response in EAE mice as well as in in vitro T-cell culture. Taken together, these data, for the first time, document the vascular and immunopathogenic roles of ADMA in EAE, thus pointing to the potential of ADMA-mediated mechanism as a new target of potential therapy for MS.

Regulation of B cell functions by S-nitrosoglutathione in the EAE model.

B cells play both protective and pathogenic roles in T cell-mediated autoimmune diseases by releasing regulatory vs. pathogenic cytokines. B cell-depleting therapy has been attempted in various autoimmune diseases but its efficacy varies and can even worsen symptoms due to depletion of B cells releasing regulatory cytokines along with B cells releasing pathogenic cytokines. Here, we report that S-nitrosoglutathione (GSNO) and GSNO-reductase (GSNOR) inhibitor N6022 drive upregulation of regulatory cytokine (IL-10) and downregulation of pathogenic effector cytokine (IL-6) in B cells and protected against the neuroinflammatory disease of experimental autoimmune encephalomyelitis (EAE). In human and mouse B cells, the GSNO/N6022-mediated regulation of IL-10 vs. IL-6 was not limited to regulatory B cells but also to a broad range of B cell subsets and antibody-secreting cells. Adoptive transfer of B cells from N6022 treated EAE mice or EAE mice deficient in the GSNOR gene also regulated T cell balance (Treg > Th17) and reduced clinical disease in the recipient EAE mice. The data presented here provide evidence of the role of GSNO in shifting B cell immune balance (IL-10 > IL-6) and the preclinical relevance of N6022, a first-in-class drug targeting GSNOR with proven human safety, as therapeutics for autoimmune disorders including multiple sclerosis.

Hypoxia-inducible factor-1 drives divergent immunomodulatory functions in the pathogenesis of autoimmune diseases.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric (HIF-1α/ HIF-1ß) transcription factor in which the oxygen-sensitive HIF-1α subunit regulates gene transcription to mediate adaptive tissue responses to hypoxia. HIF-1 is a key mediator in both regulatory and pathogenic immune responses, because ongoing inflammation in localized tissues causes increased oxygen consumption and consequent hypoxia within the inflammatory lesions. In autoimmune diseases, HIF-1 plays complex and divergent roles within localized inflammatory lesions by orchestrating a critical immune interplay sponsoring the pathogenesis of the disease. In this review, we have summarized the role of HIF-1 in lymphoid and myeloid immunomodulation in autoimmune diseases. HIF-1 drives inflammation by controlling the Th17/Treg /Tr1 balance through the tipping of the differentiation of CD4+ T cells in favour of pro-inflammatory Th17 cells while suppressing the development of anti-inflammatory Treg /Tr1 cells. On the other hand, HIF-1 plays a protective role by facilitating the expression of anti-inflammatory cytokine IL-10 in and expansion of CD1dhi CD5+ B cells, known as regulatory B cells or B10 cells. Apart from lymphoid cells, HIF-1 also controls the activation of macrophages, neutrophils and dendritic cells, thus eventually further influences the activation and development of effector/regulatory T cells by facilitating the creation of a pro/anti-inflammatory microenvironment within the autoinflammatory lesions. Based on the critical immunomodulatory roles that HIF-1 plays, this master transcription factor seems to be a potent druggable target for the treatment of autoimmune diseases.

GSNOR and ALDH2 alleviate traumatic spinal cord injury.

Traumatic spinal cord injury (SCI) enhances the activity of S-nitrosoglutathione reductase (GSNOR) and inhibits the mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, resulting in prolonged and sustained pain and functional deficits. This study's objective was to test the hypotheses that GSNOR's specific inhibitor N6022 mitigates pain and improves functional recovery in a mouse model of SCI. Furthermore, the degree of recovery is enhanced and the rate of recovery is accelerated by an ALDH2 activator Alda-1. Using both wild-type and GSNOR-/- mice, the SCI model deployed for groups was contusion at the T9-T10 vertebral level. The enzymatic activity of GSNOR and ALDH2 was measured, and the expression of GSNOR and ALDH2 was determined by western blot analysis. Functional improvements in experimental animals were assessed with locomotor, sensorimotor, and pain-like behavior tests. Wild-type SCI animals had enhanced GSNOR activity and decreased ALDH2 activity, leading to neurovascular dysfunction, edema, and worsened functional outcomes, including locomotor deficits and pain. Compared to wild-type SCI mice, GSNOR-/- mice had better functional outcomes. Monotherapy with either GSNOR inhibition by N6022 or enhanced ALDH2 activity by Alda-1 correlated well with functional recovery and lessened pain. However, combination therapy provided synergistic pain-relieving effects and more significant functional recovery compared with monotherapy. Conclusively, dysregulations in GSNOR and ALDH2 are among the causative mechanisms of SCI injury. Either inhibiting GSNOR or activating ALDH2 ameliorates SCI. Combining the specific inhibitor of GSNOR (N6022) with the selective activator of ALDH2 (Alda-1) provides greater protection to the neurovascular unit and confers greater functional recovery. The study is novel, and the combination therapy (N6022 + Alda-1) possesses translational potential.

Detoxification of Reactive Aldehydes by Alda-1 Treatment Ameliorates Experimental Autoimmune Encephalomyelitis in Mice.

Reactive aldehydes are generated as a toxic end-product of lipid peroxidation under inflammatory oxidative stress condition which is a well-established phenomenon in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Alda-1, a selective agonist of mitochondrial aldehyde dehydrogenase 2 (ALDH2), is known to detoxify the reactive aldehydes. In this study, we investigated the effect of Alda-1 on CNS myelin pathology associated with reactive aldehydes and mitochondrial/peroxisomal dysfunctions in a mouse model of EAE. Daily treatment of EAE mice with Alda-1, starting at the peak of disease, ameliorated the clinical manifestation of disease along with the improvement of motor functions. Accordingly, Alda-1 treatment improved demyelination and neuroaxonal degeneration in EAE mice. EAE mice had increased levels of reactive aldehyde species, such as 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), and acrolein (ACL) in the spinal cords and these levels were significantly reduced in Alda-1-treated EAE mice. Furthermore, Alda-1 treatment improved the loss of mitochondrial (OXPHOS) and peroxisomal (PMP70 and catalase) proteins as well as mitochondrial/peroxisomal proliferation factors (PGC-1α and PPARs) in the spinal cords of EAE mice. Taken together, this study demonstrates the therapeutic efficacy of ALDH2-agonist Alda-1 in the abatement of EAE disease through the detoxification of reactive aldehydes, thus suggesting Alda-1 as a potential therapeutic intervention for MS.

Targeting GSNOR for functional recovery in a middle-aged mouse model of stroke.

The nitric oxide (NO) metabolome and the NO metabolite-based neurovascular protective pathways are dysregulated after stroke. The major NO metabolite S-nitrosoglutahione (GSNO) is essential for S-nitrosylation-based signaling events and the inhibition of S-nitrosoglutahione (GSNO)-metabolizing enzyme GSNO reductase (GSNOR) provides protective effects following cardiac ischemia. However, the role of GSNOR and GSNOR inhibition-mediated increased GSNO/S-nitrosylation is not understood in neurovascular diseases such as stroke. Because age is the major risk factor of stroke and recovery in aged stroke patients is low and slow, we investigated the efficacy of GSNOR inhibition using a GSNOR selective inhibitor N6022 in a clinically relevant middle-aged cerebral ischemia and reperfusion (IR) mouse model of stroke. N6022 (5 mg/kg; iv) treatment of IR mice at 2 h after reperfusion followed by the treatment of the same dose daily for 3 days reduced the infarct volume and decreased the neurological score. Daily treatment of IR animals with N6022 for 2 weeks significantly improved neurological score, brain infarctions/atrophy, survival rate, motor (measured by cylinder test) and cognitive (evaluated by novel object recognition test) functions which paralleled the decreased activity of GSNOR, reduced levels of peroxynitrite and decreased neurological score. These results are the first evidence of a new pathway for the treatment of stroke via the inhibition of GSNOR. Based on the efficacy of N6022 in the stroke animal model and its use in human therapeutic studies without toxicity, we submit that GSNOR is a druggable target, and N6022 is a promising drug candidate for human stroke therapy.

Peroxisomal footprint in the pathogenesis of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a form of fatty liver disease where benign hepatic steatosis leads to chronic inflammation in the steatotic liver of a patient without any history of alcohol abuse. Mechanisms underlying the progression of hepatic steatosis to NASH have long been investigated. This review outlines the potential role of peroxisomal dysfunctions in exacerbating the disease in NASH. Loss of peroxisomes as well as impaired peroxisomal functions have been demonstrated to occur in inflammatory conditions including NASH. Because peroxisomes and mitochondria co-operatively perform many metabolic functions including O2 and lipid metabolisms, a compromised peroxisomal biogenesis and function can potentially contribute to defective lipid and reactive oxygen species metabolism which in turn can lead the progression of disease in NASH. Impaired peroxisomal biogenesis and function may be due to the decreased expression of peroxisomal proliferator-activated receptor-α (PPAR-α), the major transcription factor of peroxisomal biogenesis. Recent studies indicate that the reduced expression of PPAR-α in NASH is correlated with the activation of the toll-like receptor-4 pathway (TLR-4). Further investigations are required to establish the mechanistic connection between the TLR-4 pathway and PPAR-α-dependent impaired biogenesis/function of peroxisomes in NASH.

IFN-ß Facilitates Neuroantigen-Dependent Induction of CD25+ FOXP3+ Regulatory T Cells That Suppress Experimental Autoimmune Encephalomyelitis.

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-ß and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-ß, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-ß + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35-55 in proteolipid protein- and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-ß/Alum-based vaccination exhibited hallmarks of infectious tolerance, because IFN-ß + OVA in Alum-specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-ß + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-ß and MOG35-55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti-TGF-ß and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-ß + MOG-induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-ß + NAg in Alum vaccines, vaccination with TGF-ß + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-ß + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-ß has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs.

GM-CSF-neuroantigen fusion proteins reverse experimental autoimmune encephalomyelitis and mediate tolerogenic activity in adjuvant-primed environments: association with inflammation-dependent, inhibitory antigen presentation.

Single-chain fusion proteins comprised of GM-CSF and neuroantigen (NAg) are potent, NAg-specific inhibitors of experimental autoimmune encephalomyelitis (EAE). An important question was whether GMCSF-NAg tolerogenic vaccines retained inhibitory activity within inflammatory environments or were contingent upon steady-state conditions. GM-CSF fused to the myelin oligodendrocyte glycoprotein MOG35-55 peptide (GMCSF-MOG) reversed established paralytic disease in both passive and active models of EAE in C57BL/6 mice. The fusion protein also reversed EAE in CD4-deficient and B cell-deficient mice. Notably, GMCSF-MOG inhibited EAE when coinjected adjacent to the MOG35-55/CFA emulsion. GMCSF-MOG also retained dominant inhibitory activity when directly emulsified with MOG35-55 in the CFA emulsion in both C57BL/6 or B cell-deficient models of EAE. Likewise, when combined with proteolipid protein 139-151 in CFA, GM-CSF fused to proteolipid protein 139-151 peptide inhibited EAE in SJL mice. When deliberately emulsified in CFA with the NAg, GMCSF-NAg inhibited EAE even though NAg was present at >30-fold molar excess. In vitro studies revealed that the GM-CSF domain of GMCSF-MOG stimulated growth and differentiation of inflammatory dendritic cells (DC) and simultaneously targeted the MOG35-55 domain for enhanced presentation by these DC. These inflammatory DC presented MOG35-55 to MOG-specific T cells by an inhibitory mechanism that was mediated in part by IFN-γ signaling and NO production. In conclusion, GMCSF-NAg was tolerogenic in CFA-primed proinflammatory environments by a mechanism associated with targeted Ag presentation by inflammatory DC and an inhibitory IFN-γ/NO pathway. The inhibitory activity of GMCSF-NAg in CFA-primed lymphatics distinguishes GMCSF-NAg fusion proteins as a unique class of inflammation-dependent tolerogens that are mechanistically distinct from naked peptide or protein-based tolerogens.

Cytokine-neuroantigen fusion proteins as a new class of tolerogenic, therapeutic vaccines for treatment of inflammatory demyelinating disease in rodent models of multiple sclerosis.

Myelin-specific induction of tolerance represents a promising means to modify the course of autoimmune inflammatory demyelinating diseases such as multiple sclerosis (MS). Our laboratory has focused on a novel preclinical strategy for the induction of tolerance to the major encephalitogenic epitopes of myelin that cause experimental autoimmune encephalomyelitis (EAE) in rats and mice. This novel approach is based on the use of cytokine-NAg (neuroantigen) fusion proteins comprised of the native cytokine fused either with or without a linker to a NAg domain. Several single-chain cytokine-NAg fusion proteins were tested including GMCSF-NAg, IFNbeta-NAg, NAgIL16, and IL2-NAg. These cytokine-NAg vaccines were tolerogenic, therapeutic vaccines that had tolerogenic activity when given as pre-treatments before encephalitogenic immunization and also were effective as therapeutic interventions during the effector phase of EAE. The rank order of inhibitory activity was as follows: GMCSF-NAg, IFNbeta-NAg > NAgIL16 > IL2-NAg > MCSF-NAg, IL4-NAg, IL-13-NAg, IL1RA-NAg, and NAg. Several cytokine-NAg fusion proteins exhibited antigen-targeting activity. High affinity binding of the cytokine domain to specific cytokine receptors on particular subsets of APC resulted in the concentrated uptake of the NAg domain by those APC which in turn facilitated the enhanced processing and presentation of the NAg domain on cell surface MHC class II glycoproteins. For most cytokine-NAg vaccines, the covalent linkage of the cytokine domain and NAg domain was required for inhibition of EAE, thereby indicating that antigenic targeting of the NAg domain to APC was also required in vivo for tolerogenic activity. Overall, these studies introduced a new concept of cytokine-NAg fusion proteins as a means to induce tolerance and to inhibit the effector phase of autoimmune disease. The approach has broad application for suppressive vaccination as a therapy for autoimmune diseases such as MS.