anti-HMGB1 mAb Research Articles

Therapeutic time window of anti-high mobility group box-1 antibody administration in mouse model of spinal cord injury

Spinal cord injury (SCI) is a devastating neurologic disorder that often leads to permanent disability, and there is no effective treatment for it. High mobility group box-1 (HMGB1) is a damage-associated molecular protein that triggers sterile inflammation upon injuries. We have previously shown that two administrations of neutralizing monoclonal antibody (mAb) against HMGB1 (immediately after (0 h) and 6 h after) SCI dramatically improves functional recovery after SCI in mice. However, when considering clinical application, 0 h after SCI is not practical. Therefore, in this study, we examined the therapeutic time window of the mAb administration. Injection at 3 h after SCI significantly improved the functional recovery comparably to injection immediately after SCI, while injection at 6 h was less effective, and injection at 9 or 12 h had no therapeutic effect. We also found beneficial effects of injection at 3 h after injury on blood-spinal cord barrier maintenance, inflammatory-related gene expression and preservation of the damaged spinal cord tissue. Taken together, our results suggest that a single administration of anti-HMGB1 mAb within a proper time window could be a novel and potential therapeutic strategy for SCI.

Evidence of a role for spinal HMGB1 in ischemic stress-induced mechanical allodynia in mice

We have previously showed that spinal high-mobility group box-1 (HMGB1) plays an important role in the induction of central post-stroke pain (CPSP). It has been reported that HMGB1 exacerbates inflammation and pain via TLR4 or RAGE. Furthermore, the relationship between glial cells, such as microglia and astrocytes, involved in pain exacerbation and HMGB1 has also attracted attention. In this study, we investigated whether the interaction between spinal glial cells and HMGB1 signaling, including its receptors TLR4 or RAGE, is directly involved in the induction of CPSP.Spinal HMGB1 expression increased on day 3 after bilateral carotid artery occlusion (BCAO), and spinal microglia and astrocytes were clearly activated. HMGB1 colocalized with neurons, but not with microglia and astrocytes after BCAO. Intrathecal (i.t.) injection of lipopolysaccharides from Rhodobacter sphaeroides (LPS-RS, a TLR4 antagonist) and low-molecular-weight heparin (LMWH, a RAGE antagonist) significantly blocked mechanical allodynia on day 3 after BCAO. BCAO-induced activation of spinal microglia and astrocyte were suppressed by i.t. anti-HMGB1 monoclonal antibody (mAb) and LPS-RS administration. In addition, i.t. injection of NG-nitro-l-arginine methyl ester [a nonselective nitric oxide synthetase (NOS) inhibitor] significantly blocked mechanical allodynia on day 3 after BCAO and i.t. administration of anti-HMGB1 mAb, LPS-RS, and LMWH significantly inhibited the increase of NOS activity in the spinal cord on day 3 after BCAO.These results showed that the interaction between spinal glial cells and HMGB1/TLR4/NOS or HMGB1/RAGE/NOS is directly involved in the induction of CPSP.

HMGB1 as a representative DAMP and anti-HMGB1 antibody therapy

High mobility group box-1 (HMGB1), a representative damage-associated molecular patterns (DAMPs), has been reported to be involved in many inflammatory diseases. To validate HMGB1 as a target molecule of inflammatory diseases and to examine the effects of inhibition of HMGB1 on the diseases, we raised anti-HMGB1 monoclonal antibody (mAb) neutralizing extracellular HMGB1 and characterized it. We report the effects of anti-HMGB1 mAb on brain infarction, brain hemorrhage, brain trauma, epilepsy and neuropathic pain using animal models. In these wide range of disease conditions, we found a common event; the injury-induced translocation of HMGB1 from nuclei to extracellular space, especially in neurons. Released HMGB1 disrupt the integrity of blood-brain barrier (BBB) and increased the permeability of BBB, associated with inflammatory responses including the induction of pro-inflammatory cytokines. The intravenous injection of anti-HMGB1 mAb inhibited translocation of HMGB1, BBB disruption, expression of inflammatory molecules and improved neurological symptoms of different kinds of disease models. These results as a whole indicated that HMGB1 may be a very sensitive factor which is mobilized readily by different injurious insults to the brain and that HMGB1 release may be present most upstream of cascade of events triggering BBB disruption and brain inflammation. Thus, HMGB1 may be an excellent target for the treatment of above-mentioned diseases. Anti-HMGB1 mAb provide a novel and potential therapy for these severe disease conditions.

Anti-HMGB1 mAb therapy for intracerebral hemorrhage-induced brain injury in rats

Anti-HMGB1 mAb therapy for intracerebral hemorrhage-induced brain injury in rats

Therapeutic effects of anti-HMGB1 monoclonal antibody on pilocarpine-induced status epilepticus in mice

Inflammatory processes in brain tissue have been described in human epilepsy of various etiologies and in experimental models of seizures. High mobility group box-1 (HMGB1) is now recognized as representative of damage-associated molecular patterns (DAMPs). In the present study, we focused on whether anti-HMGB1 antibody treatment could relieve status epilepticus- triggered BBB breakdown and inflammation response in addition to the seizure behavior itself. Pilocarpine and methyl-scopolamine were used to establish the acute seizure model. Anti-HMGB1 mAb showed inhibitory effects on leakage of the BBB, and on the HMGB1 translocation induced by pilocarpine. The expression of inflammation-related factors, such as MCP-1, CXCL-1, TLR-4, and IL-6 in hippocampus and cerebral cortex were down-regulated by anti-HMGB1 mAb associated with the number of activated astrocytes, microglial cells as well as the expression of IL-1β. Both hematoxylin & eosin and TUNEL staining showed that the apoptotic cells could be reduced after anti-HMGB1 mAb treatment. The onset and latency of Racine stage five were significantly prolonged in the anti-HMGB1 mAb group. These results suggested that anti-HMGB1 mAb prevented the BBB permeability, reduced HMGB1 translocation while inhibiting the expression of inflammation-related factors, protected against neural cell apoptosis and prolonged Racine stage 5 seizure onset and latency.

Anti-high mobility group box-1 (HMGB1) antibody inhibits hemorrhage-induced brain injury and improved neurological deficits in rats

As one of the most lethal stroke subtypes, intracerebral hemorrhage (ICH) is acknowledged as a serious clinical problem lacking effective treatment. Available evidence from preclinical and clinical studies suggests that inflammatory mechanisms are involved in the progression of ICH-induced secondary brain injury. High mobility group box-1 (HMGB1) is a ubiquitous and abundant nonhistone DNA-binding protein, and is also an important proinflammatory molecule once released into the extracellular space from the nuclei. Here, we show that treatment with neutralizing anti-HMGB1 mAb (1 mg/kg, i.v. twice) remarkably ameliorated ICH-injury induced by local injection of collagenase IV in the striatum of rats. Administration of anti-HMGB1 mAb inhibited the release of HMGB1 into the extracellular space in the peri-hematomal region, reduced serum HMGB1 levels and decreased brain edema by protecting blood-brain barrier integrity, in association with decreased activated microglia and the expression of inflammation-related factors at 24 h after ICH. Consequently, anti-HMGB1 mAb reduced the oxidative stress and improved the behavioral performance of rats. These results strongly indicate that HMGB1 plays a critical role in the development of ICH-induced secondary injury through the amplification of plural inflammatory responses. Intravenous injection of neutralizing anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy

Brain inflammation is a major factor in epilepsy, and the high mobility group box-1 (HMGB1) protein is known to contribute significantly to the generation of seizures. Here, we investigated the therapeutic potential of an anti-HMGB1 monoclonal antibody (mAb) in epilepsy. anti-HMGB1 mAb attenuated both acute seizure models (maximal electroshock seizure, pentylenetetrazole-induced and kindling-induced), and chronic epilepsy model (kainic acid-induced) in a dose-dependent manner. Meanwhile, the anti-HMGB1 mAb also attenuated seizure activities of human brain slices obtained from surgical resection from drug-resistant epilepsy patients. The mAb showed an anti-seizure effect with a long-term manner and appeared to be minimal side effects at even very high dose (no disrupted physical EEG rhythm and no impaired basic physical functions, such as body growth rate and thermoregulation). This anti-seizure effect of mAb results from its inhibition of translocated HMGB1 from nuclei following seizures, and the anti-seizure effect was absent in toll-like receptor 4 knockout (TLR4−/−) mice. Interestingly, the anti-HMGB1 mAb also showed a disease-modifying anti-epileptogenetic effect on epileptogenesis after status epileptics, which is indicated by reducing seizure frequency and improving the impaired cognitive function. These results indicate that the anti-HMGB1 mAb should be viewed as a very promising approach for the development of novel therapies to treat refractory epilepsy.

Anti-high mobility group box-1 (HMGB1) antibody attenuates delayed cerebral vasospasm and brain injury after subarachnoid hemorrhage in rats.

Although delayed cerebral vasospasm (DCV) following subarachnoid hemorrhage (SAH) is closely related to the progression of brain damage, little is known about the molecular mechanism underlying its development. High mobility group box-1 (HMGB1) plays an important role as an initial inflammatory mediator in SAH. In this study, an SAH rat model was employed to evaluate the effects of anti-HMGB1 monoclonal antibody (mAb) on DCV after SAH. A vasoconstriction of the basilar artery (BA) associated with a reduction of nuclear HMGB1 and its translocation in vascular smooth muscle cells were observed in SAH rats, and anti-HMGB1 mAb administration significantly suppressed these effects. Up-regulations of inflammation-related molecules and vasoconstriction-mediating receptors in the BA of SAH rats were inhibited by anti-HMGB1 mAb treatment. Anti-HMGB1 mAb attenuated the enhanced vasocontractile response to thrombin of the isolated BA from SAH rats and prevented activation of cerebrocortical microglia. Moreover, locomotor activity and weight loss recovery were also enhanced by anti-HMGB1 mAb administration. The vasocontractile response of the BA under SAH may be induced by events that are downstream of responses to HMGB1-induced inflammation and inhibited by anti-HMGB1 mAb. Anti-HMGB1 mAb treatment may provide a novel therapeutic strategy for DCV and early brain injury after SAH.

Treatment with Anti-HMGB1 Monoclonal Antibody Does Not Affect Lupus Nephritis in MRL/lpr Mice.

High mobility group box 1 (HMGB1) is a nuclear DNA binding protein that acts as an alarmin when secreted. HMGB1 is increased in systemic lupus erythematosus and might represent a potential therapeutic target. We investigated whether treatment with an anti-HMGB1 antibody affects the development of lupus nephritis in MRL/lpr mice. Seven-week-old MRL/lpr mice were injected intraperitoneally twice weekly for 10 wks with 50μg monoclonal anti-HMGB1 (2G7, mouse IgG2b) (n = 12) or control antibody (n = 11). Control MRL/MPJ mice (n = 10) were left untreated. Every 2 wks, blood was drawn and urine was collected at wk 7, 11 and 17. Mice were sacrificed at 17 wks for complete disease evaluation. Plasma HMGB1 and anti-HMGB1 levels were increased in MRL/lpr mice compared with control MRL/MPJ mice. There were no differences in albuminuria, urine HMGB1 and plasma levels of complement C3, anti-dsDNA and proinflammatory cytokines between untreated and treated mice at any time point. Lupus nephritis of mice treated with anti-HMGB1 monoclonal antibody (mAb) was classified as class III (n = 3) and class IV (n = 9), while mice treated with control mAb were classified as class II (n = 4), class III (n = 2) and class IV (n = 5). IgG and C3 deposits in kidneys were similar in mice treated with anti-HMGB1 mAb or control mAb. In conclusion, treatment with monoclonal anti-HMGB-1 antibody 2G7 does not affect development of lupus nephritis, disease progression or proinflammatory cytokine levels in MRL/lpr mice. This result indicates that blocking of HMGB1 by this neutralizing antibody does not affect lupus nephritis in MRL/lpr mice.

Anti-high mobility group box-1 monoclonal antibody treatment provides protection against influenza A virus (H1N1)-induced pneumonia in mice.

IntroductionProvision for the emergence of an influenza pandemic is an urgent issue. The discovery of a novel anti-influenza therapeutic approach would increase the effectiveness of traditional virus-based strategies. This study was undertaken to evaluate the therapeutic effects of anti-high mobility group box-1 (HMGB1) monoclonal antibody (mAb) treatment on influenza A virus (H1N1)-induced pneumonia in mice.MethodsNine-week-old male C57BL/6 mice were inoculated with H1N1, then anti-HMGB1 mAb or control mAb were administered intravenously at 1, 24 and 48 hours after H1N1 inoculation and the survival rate was analyzed. Lung lavage and histopathological analysis were performed on days 3, 5, 7 and 10 after inoculation.ResultsAnti-HMGB1 mAb significantly improved the survival rate of H1N1-inoculated mice (1 out of 15 versus 8 out of 15 deaths in the anti-HMGB1 mAb-treated group versus the control mAb-treated group, p < 0.01), although the treatment did not affect virus propagation in the lungs. The treatment also significantly attenuated histological changes and neutrophil infiltration in the lungs of H1N1-inoculated mice. This was associated with inhibition of HMGB1 and suppression of inflammatory cytokine/chemokine expression and oxidative stress enhancement, which were observed in H1N1-inoculated mice. The expression of receptor for advanced glycation end products and nuclear factor κB was attenuated by the treatment.ConclusionsAnti-HMGB1 mAb may provide a novel and effective pharmacological strategy for severe influenza virus infection in humans by reducing the inflammatory responses induced by HMGB1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0983-9) contains supplementary material, which is available to authorized users.

Anti-high mobility group box 1 antibody exerts neuroprotection in a rat model of Parkinson's disease

The high mobility group box-1 (HMGB1) exists as an architectural nuclear protein in the normal state, but displays an inflammatory cytokine-like activity in the extracellular space under pathological condition. Inflammation in the pathogenesis of Parkinson's disease (PD) has been documented. In this study, we investigated the involvement of HMGB1 in the pathology and the neuroprotective effects of neutralizing anti-HMGB1 monoclonal antibody (mAb) on an animal model of PD. Adult female Sprague–Dawley rats were initially injected with 6-hydroxydopmaine (6-OHDA, 20μg/4μl) into the right striatum, then anti-HMGB1 mAb (1mg/kg), or control mAb was intravenously administered immediately, at 6 and 24h after 6-OHDA injection. The treatment with anti-HMGB1 mAb significantly preserved dopaminergic neurons in substantia nigra pars compacta and dopaminergic terminals inherent in the striatum, and attenuated PD behavioral symptoms compared to the control mAb-treated group. HMGB1 was retained in the nucleus of neurons and astrocytes by inhibiting the proinflammation-induced oxidative stress in the anti-HMGB1 mAb-treated group, whereas HMGB1 translocation was observed in neurons at 1day and astrocytes at 7days after 6-OHDA injection in the control mAb-treated group. Anti-HMGB1 mAb inhibited the activation of microglia, disruption of blood–brain-barrier (BBB), and the expression of inflammation cytokines such as IL-1β and IL-6. These results suggested that HMGB1 released from neurons and astrocytes was at least partly involved in the mechanism and pathway of degeneration of dopaminergic neurons induced by 6-OHDA exposure. Intravenous administration of anti-HMGB1 mAb stands as a novel therapy for PD possibly acting through the suppression of neuroinflammation and the attenuation of disruption of BBB associated with the disease.

Anti–high mobility group box 1 monoclonal antibody improves ischemia/reperfusion injury and mode of liver regeneration after partial hepatectomy

BackgroundThe purpose of this study is to determine the effects of anti–high mobility group box 1 (HMGB1) monoclonal antibody (mAb) on ischemia/reperfusion injury (IRI) and the mode of liver regeneration. MethodsRats underwent 70% hepatectomy with IRI caused by clamping the hepatoduodenal ligament for 20 minutes, followed by the administration of anti-HMGB1 mAb immediately before declamping the hepatoduodenal ligament. Five animals were used for each time point. We then evaluated IRI, regeneration parameters and the status of HMGB1 in remnant livers. ResultsThe anti-HMGB1 mAb significantly ameliorated the degree of IRI in the remnant livers in association with the downregulation of HMGB1 protein. The ratio of Ki67-positive hepatocytes at 48 hours after 70% hepatectomy was significantly improved. Mean hepatocyte size was significantly reduced and cyclin-dependent kinase inhibitor 1 expression was significantly attenuated. ConclusionsAnti-HMGB1 mAb ameliorated IRI and improved the mode of liver regeneration after IRI followed by 70% hepatectomy in rats.

HMGB1 Neutralizing Antibody Attenuates Cardiac Injury and Apoptosis Induced by Hemorrhagic Shock/Resuscitation in Rats.

High-mobility group box 1 (HMGB1) and its natural receptor, Toll-like receptor-4 (TLR4), are involved in various infectious or noninfectious diseases including hemorrhagic shock. HMGB1 neutralizing antibody (anti-HMGB1 monoclonal antibody (mAb)) treatment was shown to alleviate ischemia-reperfusion injury effectively. The aim of this study was to explore whether and by what mechanisms anti-HMGB1 mAb attenuates hemorrhagic shock and resuscitation (HS/R)-induced cardiac injury. Employing rat HS/R models, we found that anti-HMGB1 mAb treatment improved HS/R-induced cardiac function deterioration, attenuated cardiac enzyme elevation, improved ATP loss, and protected cardiac tissue. Anti-HMGB1 mAb also inhibited the production of inflammatory factors interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). Moreover, anti-HMGB1 mAb reduced apoptotic responses by suppressing activated caspase-3 and reversing apoptotic gene expression of capase-3, Bax, and Bcl-2 in rat cardiac tissue. Moreover, anti-HMGB1 mAb decreased HS/R-induced HMGB1 and TLR4 expression elevation. We further confirmed that anti-HMGB1 mAb inhibited lipopolysaccharide-activated HGMB1 and TLR4 expression and decreased inflammatory factors IL-1β, IL-6, and TNF-α at the cellular level. It was concluded that anti-HMGB1 mAb treatment protects rats from cardiac injury induced by HS/R, and the beneficial effects may be related to its inhibitory effects on the HMGB1-TLR4 axis.

HMGB1 Is a Key Modulator Of Stress Erythropoiesis During Sepsis

▪Severe sepsis is a leading cause of death and disability. Anemia in sepsis survivors affects close to 100% of patients after the third day of in-hospital stay, regardless of blood levels on admission. Circulating levels of Erythropoietin (Epo) are low; paradoxically, administration of recombinant Epo is ineffective, and related to increased morbidity. During sepsis, bone Marrow is hypoproliferative. While transfusions can improve outcome in the short term, its use increases the risk of infection and mortality without any sustained beneficial effect. The pathogenesis of anemia during sepsis is unclear. High mobility group box 1 (HMGB1), a cytokine that is a critical mediator of sepsis, is released into circulation a few days after sepsis onset, remaining increased for 8 weeks after severe sepsis. HMGB1 levels are increased for at least 8 weeks in murine models of sepsis survival.To induce severe sepsis, cecal ligation and puncture (CLP) was performed in BALB/c mice. Three days after CLP, mice developed persistent anemia, represented by a significant reduction in hematocrit (Sham=49.8±3.2 vs. CLP=29.7±6.7%; p≤0.001), hemoglobin (16.7±1.2 vs. 9.9±2.4mg/dL; p≤0.001), and red blood cells mass (10.2±0.7 vs. 5.4±1.7 x106/µL; p≤0.001). Anemia persisted for at least 25 days after CLP. In CLP survivors, reticulocyte counts were erratic, and insufficient to the degree and duration of anemia (8.2±0.8 vs. 6.6±2.1%; p=ns). Analysis of terminal erythroid differentiation using CD44 and Ter119 or CD44 and FSC as markers demonstrated a significant decrease in all erythroid progenitors, from proerythroblast to orthochromatic erythroblast. Concomitantly, mice surviving CLP developed splenomegaly. Splenic architecture was disrupted after CLP, with expansion of the red pulp, characteristic of stress erythropoiesis. Analysis of terminal erythroid differentiation demonstrated an increase in the quantity of erythroid progenitors.An anti-HMGB1 mAb (2G7) was administered after CLP. Strikingly, 2G7-treated septic mice were significantly protected from developing anemia, and had levels of hemoglobin and hematocrit similar to sham-operated mice. These results highlight a critical role for HMGB1 as key modulator of stress erythropoiesis in a murine model of sepsis survivors.To get further insight into the function of HMGB1 and translate our findings to the pathophysiology of human erythropoiesis, we used CD34+ cells derived from cord blood.Cord blood-derived CD34+ cells were incubated in MethoCult in the presence or not of HMGB1. HMGB1 induced a dose dependent decrease in CFU-E.In murine sepsis, there is a stepwise elevation of different redox forms of HMGB1, with an early increase in all-thiol (inflammatory), followed by a partially oxidized before a fully oxidized (with no known inflammatory activity) appears. At day 7, all-thiol HMGB1 reduced significantly the number of CFU-E, while the fully oxidized had no significant effect. At day 14, the number of BFU-E was reduced in the presence of HMGB1, and further decreased with all-thiol HMGB1.In conclusion, our findings suggest that CLP is a reproducible model to study anemia of sepsis. In mice surviving sepsis, stress erythropoiesis is consistently found. Administration of anti-HMGB1 monoclonal antibody reverses anemia of murine sepsis, demonstrating that HMGB1 can be a potential target in the anemia of sepsis survivors. Translating the findings to the human system, we found that HMGB1 impairs differentiation of CD34+ cells towards the BFU-E and CFU-E stages in colony formation assays, implying that HMGB1 might play a role early during differentiation. The redox status of HMGB1 is critical for its biological function, since its effects are not retrieved when HMGB1 is fully oxidized. Disclosures:No relevant conflicts of interest to declare.

Anti–high mobility group box‐1 antibody therapy for traumatic brain injury

High mobility group box-1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. In this study, we examined the involvement of HMGB1 in traumatic brain injury (TBI) and evaluated the ability of intravenously administered neutralizing anti-HMGB1 monoclonal antibody (mAb) to attenuate brain injury. Traumatic brain injury was induced in rats or mice by fluid percussion. Anti-HMGB1 mAb or control mAb was administered intravenously after TBI. Anti-HMGB1 mAb remarkably inhibited fluid percussion-induced brain edema in rats, as detected by T2-weighted magnetic resonance imaging; this was associated with inhibition of HMGB1 translocation, protection of blood-brain barrier (BBB) integrity, suppression of inflammatory molecule expression, and improvement of motor function. In contrast, intravenous injection of recombinant HMGB1 dose-dependently produced the opposite effects. Experiments using receptor for advanced glycation end product (RAGE)(-/-) , toll-like receptor-4 (TLR4)(-/-) , and TLR2(-/-) mice suggested the involvement of RAGE as the predominant receptor for HMGB1. Anti-HMGB1 mAb may provide a novel and effective therapy for TBI by protecting against BBB disruption and reducing the inflammatory responses induced by HMGB1.

HMGB1 mediates Splenomegaly, Anemia and Leukocytosis in Mice Surviving Sepsis (44.7)

Abstract Severe sepsis, a syndrome complicating infection or injury, is a leading cause of death. It also has long-term deleterious effects on survivors, including high mortality and diminished quality of life. High-mobility group box 1 (HMGB1) is a critical late mediator of acute sepsis that remains elevated after other inflammatory cytokines have normalized. Here we studied the kinetics of circulating HMGB1 in mice surviving severe abdominal sepsis, and the effects of administering neutralizing anti HMGB1 mAb. In a standardized model of murine sepsis, we observed that mice surviving sepsis developed splenomegaly for at least four weeks. Splenocytes from sepsis survivors produced significantly more cytokines following exposure to TLR ligands. Circulating HMGB1 levels are significantly increased for at least 4 weeks after sepsis. Treatment with anti-HMGB1 neutralizing monoclonal antibodies effectively prevented the development of splenomegaly, leukocytosis and anemia. Chronic administration of recombinant HMGB1 to healthy mice induced significant splenomegaly and leukocytosis. Recombinant HMGB1 also sensitized splenocytes and significantly increased the release of TNF and IL-6. In conclusion, serum HMGB1 levels are persistently elevated for at least 4 weeks in mice that survive lethal sepsis. Anti-HMGB1 mAb administration reverses the development of splenomegaly, leukocytosis and anemia, which may have implications for understanding the pathogenesis of post-sepsis complications.

Deleterious Role of Anti-high Mobility Group Box 1 Monoclonal Antibody in Retinal Ischemia-reperfusion Injury

Purpose: To investigate the effect of anti-high mobility group box 1 (HMGB1) monoclonal antibody (mAb) against ischemia-reperfusion injury in the rat retina.Materials and Methods: Retinal ischemia was induced by increasing and then maintaining intraocular pressure at 130 mmHg for 45 min. An intraperitoneal injection of anti-HMGB1 mAb was administered 30 min before ischemia. Retinal damage was evaluated at 7 days after the ischemia. Immunohistochemistry and image analysis were used to measure changes in the levels of reactive oxygen species (ROS) and the localization of anti-HMGB1 mAb. Dark-adapted full-field electroretinography (ERG) was also performed.Results: Pretreatment with anti-HMGB1 mAb significantly enhanced the ischemic injury of the retina. HMGB1 expression increased at 6–12 h after ischemia in the retina. After the ischemia, production of ROS was detected in retinal cells. However, pretreatment with anti-HMGB1 mAb increased the production of ROS. On the seventh postoperative day, the amplitudes of both the ERG a- and b-waves were significantly higher in the vehicle group than in the groups pretreated with anti-HMGB1 mAb.Conclusions: The current in vivo model of retinal injury demonstrated that anti-HMGB1 mAb plays a large deleterious role in ischemia-reperfusion injury. In order to develop neuroprotective therapeutic strategies for acute retinal ischemic disorders, further studies on anti-HMGB1 mAb function are needed.

Monoclonal Anti-HMGB1 (High Mobility Group Box Chromosomal Protein 1) Antibody Protection in Two Experimental Arthritis Models

High mobility group box chromosomal protein 1 (HMGB1) is a DNA-binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB1 promotes inflammation. Experimental studies demonstrate HMGB1 to be a pathogenic factor in many inflammatory conditions including arthritis. HMGB1-blocking therapies in arthritis models alleviate disease and confer significant protection against cartilage and bone destruction. So far, the most successful HMGB1-targeted therapies have been demonstrated with HMGB1-specific polyclonal antibodies and with recombinant A box protein, a fragment of HMGB1. The present study is the first to evaluate the potential of a monoclonal anti-HMGB1 antibody (2G7, mouse IgG2b) to ameliorate arthritis. Effects of repeated injections of this antibody have now been studied in two conceptually different models of arthritis: collagen type II-induced arthritis (CIA) in DBA/1 mice and in a spontaneous arthritis disease in mice with combined deficiencies for genes encoding for the enzyme DNase type II and interferon type I receptors. These mice are unable to degrade phagocytozed DNA in macrophages and develop chronic, destructive polyarthritis. Therapeutic intervention in CIA and prophylactic administration of anti-HMGB1 monoclonal antibody (mAb) in the spontaneous arthritis model significantly ameliorated the clinical courses. Anti-HMGB1 mAb therapy also partially prevented joint destruction, as demonstrated by histological examination. The beneficial antiarthritic effects by the anti-HMGB1 mAb in two diverse models of arthritis represent additional proof-of-concept, indicating that HMGB1 may be a valid target molecule to consider for development of future clinical therapy.

Anti-high Mobility Group Box-1 Monoclonal Antibody Protects the Blood–Brain Barrier From Ischemia-Induced Disruption in Rats

High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity in the extracellular space. We previously demonstrated that intravenous injection of anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain infarction induced by middle cerebral artery occlusion in rats. In the present study, we focused on the protective effects of the mAb on the marked translocation of HMGB1 in the brain, the disruption of the blood-brain barrier (BBB), and the resultant brain edema. Middle cerebral artery occlusion in the rat was used as the ischemia model. Rats were treated with anti-HMGB1 mAb or control IgG intravenously. BBB permeability was measured by MRI. Ultrastructure of the BBB unit was observed by transmission electron microscope. The in vitro BBB system was used to study the direct effects of HMGB1 in BBB components. HMGB1 was time-dependently translocated and released from neurons in the ischemic rat brain. The mAb reduced the edematous area on T2-weighted MRI. Transmission electron microscope observation revealed that the mAb strongly inhibited astrocyte end feet swelling, the end feet detachment from the basement membrane, and the opening of the tight junction between endothelial cells. In the in vitro reconstituted BBB system, recombinant HMGB1 increased the permeability of the BBB with morphological changes in endothelial cells and pericytes, which were inhibited by the mAb. Moreover, the anti-HMGB1 mAb facilitated the clearance of serum HMGB1. These results indicated that the anti-HMGB1 mAb could be an effective therapy for brain ischemia by inhibiting the development of brain edema through the protection of the BBB and the efficient clearance of circulating HMGB1.

Anti-HMGB1 mAb protects the blood-brain barrier from ischemia-induced disruption in rats

Anti-HMGB1 mAb protects the blood-brain barrier from ischemia-induced disruption in rats