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Despite the current success of immunotherapies, only microsatellite instable (MSI) Colorectal Cancer (CRC) typically respond to T cell-driven immunotherapies while microsatellite stable (MSS) CRC displays poor response. Our goal is to identify resistance mechanisms in MSS-CRC to uncover novel therapeutic targets. Current syngeneic CRC models are largely immunogenic and lack complex structure. Therefore, we developed novel 3D humanized CRC mouse models which form complex human-like tumors which are typically absent in syngeneic systems. We investigated molecular profiles of human MSI-versus MSS-CRC tumor xenografts following Keytruda immunotherapy and identified an immunosuppressive profile in MSS models that was not observed in MSI-CRC. We evaluated the therapeutic potential of blocking inflammation in combination with other immuno-modulatory therapies in both cold and hot CRC models and identified a significant regression of tumor burden in MSS-CRC mice. Our studies provide a novel CRC in vivo model and a unique opportunity to better define how immune cells interact in human MSS- and MSI-CRC microenvironments, to enable clinical therapeutic implementation.

Objective Anti–citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthritis (RA). However, their presence years before the onset of clinical RA is perplexing. Although multiple putative citrullinated antigens have been identified, no studies have demonstrated the specific capacity of these antigens to initiate inflammatory arthritis. This study was undertaken to recapitulate the transition from preclinical to clinical RA and to demonstrate the capacity of local citrullination to facilitate this transition. Methods We performed proteomic analysis of activated human neutrophils to identify citrullinated proteins, including those targeted as part of the RA immune response. Using enzyme-linked immunosorbent assay, we compared RA and osteoarthritis synovial fluid for levels of citrullinated histone H2B and its immune complex. Using macrophage activation assays, we assessed the effect of histone citrullination on immunostimulatory capacity and evaluated the stimulatory capacity of native and citrullinated H2B immune complexes. Finally, we assessed the potential for anti–citrullinated H2B antibodies to mediate arthritis in vivo. Results We identified robust targeting of neutrophil-derived citrullinated histones by the ACPA immune response. More than 90% of the RA patients had anti–citrullinated H2B antibodies. Histone citrullination increased innate immunostimulatory capacity, and immune complexes containing citrullinated histones activated macrophage cytokine production and propagated neutrophil activation. Finally, we demonstrated that immunization with H2B was arthritogenic, but only in the setting of underlying articular inflammation. Conclusion Our findings indicate that citrullinated histones, specifically citrullinated H2B, are an antigenic target of the ACPA immune response. Furthermore, local generation of citrullinated antigen during low-grade articular inflammation provides a mechanistic model for the conversion from preclinical autoimmunity to inflammatory arthritis.

Objective The co-occurrence of rheumatoid factor (RF) and anti–citrullinated protein antibody (ACPA) positivity in rheumatoid arthritis (RA) is well described. However, the mechanisms underlying the potential interaction between these 2 distinct autoantibodies have not been well defined. The aim of this study was to evaluate the epidemiologic and molecular interaction of ACPAs and RF and its association with both disease activity and measures of RA-associated inflammation. Methods In a cohort of 1,488 US veterans with RA, measures of disease activity and serum levels of cytokines and multiplex ACPAs were compared between the following groups of patients: double-negative (anti–cyclic citrullinated peptide [anti-CCP]−/RF−), anti-CCP+/RF−, anti-CCP−/RF+, or double-positive (anti-CCP+/RF+). Additional studies were performed using an in vitro immune complex (IC) stimulation assay in which macrophages were incubated with ACPA ICs in the presence or absence of monoclonal IgM-RF, and tumor necrosis factor α production measured as a readout of macrophage activation. Results Compared with the double-negative subgroup (as well as each single-positive subgroup), the double-positive subgroup exhibited higher disease activity as well as higher levels of C-reactive protein and inflammatory cytokines (all P < 0.001). In vitro stimulation of macrophages by ACPA ICs increased cytokine production, and the addition of monoclonal IgM-RF significantly increased macrophage tumor necrosis factor α production (P = 0.003 versus ACPA ICs alone). Conclusion The combined presence of ACPAs and IgM-RF mediates increased proinflammatory cytokine production in vitro and is associated with increased systemic inflammation and disease activity in RA. Our data suggest that IgM-RF enhances the capacity of ACPA ICs to stimulate macrophage cytokine production, thereby providing a mechanistic link by which RF enhances the pathogenicity of ACPA ICs in RA.

Minocycline is an antibiotic now recognized to have antiapoptotic and antiinflammatory properties. Because of these properties, minocycline may be of benefit in reducing neuronal apoptosis from ischemia and subsequent postischemic inflammation if administered soon after a stroke. We now explore the feasibility of using (99m)Tc-annexin V, an in vivo marker of apoptosis, with SPECT to monitor the antiapoptotic effects of minocycline therapy.CB6/F1 adult male mice underwent unilateral distal middle cerebral artery occlusion (dMCA) occlusion and were imaged and sacrificed at 1, 3, 7, or 30 d after injury. Animals were given minocycline (or vehicle) 30 min and 12 h after dMCA occlusion and then given 22.5 mg/kg twice daily for up to 7 d. Before imaging, behavioral tests were performed to evaluate the neurologic function. After imaging, brains were collected for histology and assessed for the degree of apoptosis and microglial activation.(99m)Tc-Annexin V uptake in injured hemispheres was significantly decreased 2- to 3-fold by minocycline at all time points. Minocyline reduced infarct size as seen histologically and improved behavioral indices as late as 30 d. Infarct volume as seen histologically correlated with radiolabeled annexin V uptake seen by SPECT. In situ fluorescent microscopy demonstrated that annexin V bound primarily to neurons at 1 and 3 d, with a shift toward microglia by 7 and 30 d.We found that minocycline significantly reduces neuronal apoptosis and infarct size and improves neurologic outcome in mice after acute focal cortical ischemia.

The first aim of the study was to determine whether 99mTc-HYNIC-annexin V, a marker of cellular stress and apoptosis, can detect ischemic injury in patients with acute stroke. Secondly, we wished to test radiolabeled annexin’s ability to monitor therapy in a rodent model of focal ischemic injury. SPECT imaging of patients was performed between 1 and 2 h after intravenous injection of 30 mCi (1,110 MBq) of tracer. Eight MFL4 (anti-FasL) antibody-treated (400 μg i.p. days 0 and 3) and 21 control adult male Sprague-Dawley rats underwent small animal SPECT imaging with 5–10 mCi (185–370 MBq) of tracer, 1 and 6 days after a 2-h intraluminal thread occlusion of the left middle cerebral artery. Two patients with acute stroke had regions of multifocal annexin uptake that correlated with sites of restricted diffusion on MRI. Anti-FasL antibody treatment significantly reduced annexin uptake by 92% with a 60% decrease in the number of caspase-8 staining (apoptotic) neurons on day 1. On day 6, treated animals had an 80% reduction in tracer uptake with a 75% decrease in infarct size as compared with controls. Annexin uptake in controls and treated animals (day 6) linearly correlated with infarct size (r 2=0.603, p=0.0036) and the number of TUNEL-positive (apoptotic) nuclei (r 2=0.728, p=0.00084). Annexin imaging shows foci of increased uptake at sites of ischemic injury in patients with acute stroke. Annexin imaging can assess the effects of therapy for ischemic cerebral injury in rats, suggesting its potential as a non-invasive indicator of drug efficacy in future clinical trials.

The serine/threonine kinase, glycogen synthase kinase 3beta (GSK3beta), is abundant in CNS and is neuron specific. GSK3beta plays a pivotal role in the regulation of numerous cellular functions. GSK3beta phosphorylates and thereby regulates many metabolic, signaling, and structural proteins which can influence cell survival. Increased GSK3beta correlates with increased cell death, whereas reduced GSK3beta expression correlates with increased cell survival. We report that the GSK3beta inhibitor Chir025 is neuroprotective in vitro and in vivo. First, Chir025 reduced cultured hippocampal neuron death following glutamate exposure by 15-20% versus vehicle-treated controls. Second, Chir025 significantly reduced cultured cortical neuron death following oxygen-glucose deprivation (OGD) by approximately 50%. Third, Chir025 reduced infarct size following focal cerebral ischemia by nearly 20%. There were no significant differences in the number of TUNEL-positive neurons or in caspase-3 and -9 activities between Chir025- and vehicle-treated rats, although Chir025 elevated cytosolic Bcl-2 expression. These data show that Chir025-mediated inhibition of GSK3beta is neuroprotective and that the mechanism is probably not anti-apoptotic.

Apoptosis plays a critical role in many neurologic diseases, including stroke. Cytochrome c release and activation of various caspases are known to occur after focal and global ischemia. However, recent reports indicate that caspase-independent pathways may also be involved in ischemic damage. Apoptosis-inducing factor (AIF) is a novel flavoprotein that helps mediate caspase-independent apoptotic cell death. AIF translocates from mitochondria to nuclei where it induces caspase-independent DNA fragmentation. Bcl-2, a mitochondrial membrane protein, protects against apoptotic and necrotic death induced by different insults, including cerebral ischemia. In the present study, Western blots confirmed that AIF was normally confined to mitochondria but translocated to nuclei or cytosol 8, 24, and 48 hours after onset of ischemia. Overall, AIF protein levels also increased after stroke. Confocal microscopy further demonstrated that nuclear AIF translocation occurred in the peri-infarct region but not in the ischemic core where only some cytosolic AIF release was observed. Our data also suggest that AIF translocated into nuclei after cytochrome c was released into the cytosol. Bcl-2 transfection in the peri-infarct region blocked nuclear AIF translocation and improved cortical neuron survival.

Bcl-2 protects against both apoptotic and necrotic death induced by several cerebral insults. We and others have previously demonstrated that defective herpes simplex virus vectors expressing Bcl-2 protect against various insults in vitro and in vivo, including cerebral ischemia. Because the infarct margin may be a region that is most amenable to treatment, we first determined whether gene transfer to the infarct margin is possible using a focal ischemia model. Since ischemic injury with and without reperfusion may occur by different mechanisms, we also determined whether Bcl-2 protects against focal cerebral ischemic injury either with or without reperfusion in rats. Bax expression, cytochrome c translocation and activated caspase-3 expression were also assessed. Viral vectors overexpressing Bcl-2 were delivered to the infarct margin. Reperfusion resulted in larger infarcts than permanent occlusion. Bcl-2 overexpression significantly improved neuron survival in both ischemia models. Bcl-2 overexpression did not alter overall Bax expression, but inhibited cytosolic accumulation of cytochrome c and caspase-3 activation. Thus, we provide the first evidence that gene transfer to the infarct margin is feasible, that overexpression of Bcl-2 protects against damage to the infarct margin induced by ischemia with and without reperfusion, and that Bcl-2 overexpression using gene therapy attenuates apoptosis-related proteins. This suggests a potential therapeutic strategy for stroke.

Following a transient ischemic insult there is a marked increase in free radical (FR) production within the first 10-15 min of reperfusion and again at the peak of the inflammatory process. Hypothermia decreases lipid peroxidation following global ischemia, raising the possibility that it may act by reducing FR production early on and by maintaining or increasing endogenous antioxidant systems. By means of FR fluorescence, Western blot, immunohistochemistry, and enzymatic assay, we studied the effects of mild hypothermia on superoxide (O(-*)(2)) anion production, superoxide dismutase SOD expression, and activity following focal cerebral ischemia in rats. Mild hypothermia significantly reduced O(-*)(2) generation in the ischemic penumbra and corresponding contralateral region, but did not alter the bilateral SOD expression. SOD enzymatic activity in the ischemic core was slightly reduced in hypothermia-treated animals compared with normothermic controls. Our results suggest that the neuroprotective effect of mild hypothermia may be due, in part, to a reduction in neuronal and endothelial O(-*)(2) production during early reperfusion.

Mild hypothermia protects the brain against experimental ischemia, but the reasons are not well known. We examined whether the protective effects of mild hypothermia could be correlated with alterations in expression of Bcl-2, an anti-apoptotic protein in a rat model of transient global ischemia. Following 10 min of forebrain ischemia, hippocampal neurons were examined 72 h later for survival, expression of Bcl-2 family proteins and apoptosis. Intraischemic mild hypothermia was applied for 3 h (33 degrees C, isch-33) or normal body temperature was maintained (37 degrees C, isch-37). Survival of CA1 neurons was significantly improved in the isch-33 group compared to the isch-37 group (90 vs. 53% survival; P0.01). The proportion of Bcl-2-positive cells among surviving CA1 neurons in the isch-33 group was increased compared to that of sham and isch-37 groups (P0.01). Bax expression in CA1 was no different between sham and isch-33 groups, but was significantly decreased in isch-37 (P0.05). TUNEL staining was positive in many isch-37 CA1 neurons, but absent in isch-33. Utilizing electron microscopy, more cells meeting criteria for apoptosis were observed in the isch-37 than isch-33. These data suggest that mild hypothermia attenuates apoptotic death, and that this protection may be related to increases in Bcl-2.

We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.

Introduction: The serine/threonine kinase glycogen synthase kinase 3β (GSK3β) is abundant in the central nervous system and is neuron-specific. GSK3β plays a pivotal role in the regulation of numerous cellular functions (including phosphorylation) and, thereby, regulation of many metabolic, signaling, and structural proteins as well as transcription factors that can influence cell survival. This article reports that GSK3β expression following global cerebral ischemia (GCI) is altered by the neuroprotectant, mild hypothermia (33°C). Methods: Male Sprague-Dawley rats were anesthetized and subjected to GCI; arterial blood pressure was reduced to 30 mmHg by blood withdrawal via the jugular vein, and both common carotid arteries were occluded with aneurysm clips for 8 minutes. Hypothermia (33°C) was induced in half the rats 10 minutes prior to GCI and was maintained for 3 hours. Rats were killed 24 or 72 hours later to assess cell death and GSK3β expression. Results: At 72 hours post-GCI, levels of GSK3β expression were significantly lower in hypothermic rats than in normothermic rats. This reduction in GSK3β correlated with marked neuroprotection and reduced terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling staining in hippocampal CA1 neurons. No significant changes in phosphorylated GSK3β expression were observed. Conclusion: These data suggest that GSK3β plays a role in GCI pathology that can be altered by mild hypothermia.

Hypothermia is effective in preventing ischemic damage. A caspase-dependent apoptotic pathway is involved in ischemic damage, but how hypothermia inhibits this pathway after global cerebral ischemia has not been well explored. It was determined whether hypothermia protects the brain by altering cytochrome c release and caspase activity. Cerebral ischemia was produced by two-vessel occlusion plus hypotension for 10 mins. Body temperature in hypothermic animals was reduced to 33°C before ischemia onset and maintained for 3 h after reperfusion. Western blots of subcellular fractions revealed biphasic cytosolic cytochrome c release, with an initial peak at about 5 h after ischemia, which decreased at 12 to 24 h, and a second, larger peak at 48 h. Caspase-3 and −9 activity increased at 12 and 24 h. A caspase inhibitor, Z-DEVD-FMK, administered 5 and 24 h after ischemia onset, protected hippocampal CA1 neurons from injury and blocked the second cytochrome c peak, suggesting that caspases mediate this second phase. Hypothermia (33°C), which prevented CA1 injury, did not inhibit cytochrome c release at 5 h, but reduced cytochrome c release at 48 h. Caspase-3 and −9 activity was markedly attenuated by hypothermia at 12 and 24 h. Thus, biphasic cytochrome c release occurs after transient global ischemia and mild hypothermia protects against ischemic damage by blocking the second phase of cytochrome c release, possibly by blocking caspase activity.

Background and Purpose— We previously showed that mild hypothermia protects against experimental stroke, even when cooling was delayed by 2 hours. Protection may be due in part to inhibiting inflammation. To clarify, we examined leukocyte infiltration, microglial activation, and adhesion molecule expression in models of stroke and pure brain inflammation. Methods— Rats underwent 2-hour middle cerebral artery occlusion (MCAO; n=36) or intravenous injection with 5 mg/kg lipopolysaccharide (LPS; n=22). Temperature was lowered to 33°C for 2 hours or kept at 37°C. In MCAO, cooling was applied intraischemically or on reperfusion (delayed). In the LPS model, cooling began after injection. One and 3 days later, brains were assessed for neutrophils, monocytes/microglia, major histocompatibility complex class II antigen, and intercellular adhesion molecule-1 (ICAM-1). Results— One day after MCAO, both intraischemic and delayed hypothermia decreased ICAM-1 (51% and 60%, respectively, versus normothermia; P P P P P P P P P Conclusions— Mild hypothermia decreases inflammatory responses in both brain inflammation and stroke, implicating a direct anti-inflammatory effect of cooling. This suggests that hypothermia can attenuate factors contributing to delayed ischemic injury.

Bcl-2 protects against both apoptotic and necrotic death induced by several cerebral insults. We and others have previously demonstrated that defective herpes simplex virus vectors expressing Bcl-2 protect against various insults in vitro and in vivo, including cerebral ischemia. Because the infarct margin may be a region that is most amenable to treatment, we first determined whether gene transfer to the infarct margin is possible using a focal ischemia model. Since ischemic injury with and without reperfusion may occur by different mechanisms, we also determined whether Bcl-2 protects against focal cerebral ischemic injury either with or without reperfusion in rats. Bax expression, cytochrome c translocation and activated caspase-3 expression were also assessed. Viral vectors overexpressing Bcl-2 were delivered to the infarct margin. Reperfusion resulted in larger infarcts than permanent occlusion. Bcl-2 overexpression significantly improved neuron survival in both ischemia models. Bcl-2 overexpression did not alter overall Bax expression, but inhibited cytosolic accumulation of cytochrome c and caspase-3 activation. Thus, we provide the first evidence that gene transfer to the infarct margin is feasible, that overexpression of Bcl-2 protects against damage to the infarct margin induced by ischemia with and without reperfusion, and that Bcl-2 overexpression using gene therapy attenuates apoptosis-related proteins. This suggests a potential therapeutic strategy for stroke.

Mild hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not well known. The authors previously found that hypothermia reduces the density of apoptotic cells, but it is not certain whether temperature alters associated biochemical events. Mitochondrial release of cytochrome c has recently been shown to be a key trigger in caspase activation and apoptosis via the intrinsic pathway. Using a model of transient focal cerebral ischemia, the authors determined whether mild hypothermia altered expression of Bcl-2 family proteins, mitochondrial release of cytochrome c, and caspase activation. Mild hypothermia significantly decreased the amount of cytochrome c release 5 hours after the onset of ischemia, but mitochondrial translocation of Bax was not observed until 24 hours. Mild hypothermia did not alter Bcl-2 and Bax expression, and caspase activation was not observed. The present study provides the first evidence that intraischemic mild hypothermia attenuates the release of cytochrome c in the brain, but does not appear to affect other biochemical aspects of the intrinsic apoptotic pathway. They conclude that necrotic processes may have been interrupted to prevent cytochrome c release, and that the ameliorative effect of mild hypothermia may be a result of maintaining mitochondrial integrity. Furthermore, the authors show it is unlikely that mild hypothermia alters the intrinsic apoptotic pathway.