Responses In Normal Mice Research Articles

Functional OCT angiography reveals early retinal neurovascular dysfunction in diabetes with capillary resolution.

Altered retinal neurovascular coupling may contribute to the development and progression of diabetic retinopathy (DR) but remains highly challenging to measure due to limited resolution and field of view of the existing functional hyperemia imaging. Here, we present a novel modality of functional OCT angiography (fOCTA) that allows a 3D imaging of retinal functional hyperemia across the entire vascular tree with single-capillary resolution. In fOCTA, functional hyperemia was evoked by a flicker light stimulation, recorded by a synchronized time-lapse OCTA (i.e., 4D), and extracted precisely from each capillary segment (space) and stimulation period (time) in the OCTA time series. The high-resolution fOCTA revealed that the retinal capillaries, particularly the intermediate capillary plexus, exhibited apparent hyperemic response in normal mice, and significant functional hyperemia loss (P < 0.001) at an early stage of DR with few overt signs of retinopathy and visible restoration after aminoguanidine treatment (P < 0.05). Retinal capillary functional hyperemia has strong potential to provide sensitive biomarkers of early DR, and retinal fOCTA would provide new insights into the pathophysiology, screening and treatment of early DR.

Bupi Yichang Pill alleviates dextran sulfate sodium-induced ulcerative colitis in mice by regulating the homeostasis of follicular helper T cells

BackgroundFollicular helper T (Tfh) cells-based therapy represents a new treatment option for inflammatory bowel disease. Bupi Yichang Pill (BPYCP), a traditional Chinese formula for the treatment of dysentery and diarrhea, exhibits potential anti-inflammatory activities in treating various kinds of inflammation. However, its anti-inflammatory effect on colitis and the underlying mechanisms remain unknown. PurposeTo explore the protective role and underlying immunomodulatory effects of BPYCP in the treatment of UC. MethodsThe dextran sodium sulfate (DSS) free-drinking method induced UC in C57BL/6 mice, and BPYCP was orally administrated at a dosage of 1.5, 3.0, or 6.0 g/kg/day. Throughout the experimental period, the effects of BPYCP on DSS-induced clinical symptoms and disease activity index (DAI) were monitored and analyzed. Hematoxylin-eosin staining was used to observe the histopathological injury of the colon. Flow cytometry was used to detect the levels of Tfh cells, Tfh cell subpopulations, and memory Tfh cells. ELISA, Western blot, and qRT-PCR were used to detect the expression of inflammatory cytokines and Tfh cell-related biomarkers. ResultsMedium and high dosages of BPYCP effectively alleviated DSS-induced experimental colitis with increased body weight, survival rate and colonic length, and decreased DAI, colonic weight, and colonic index, as well as less ulcer formation and inflammatory cell infiltration, increased anti-inflammatory cytokine IL-10, and decreased pro-inflammatory cytokines IL-17A, IL-6, and TNF-ɑ. Moreover, BPYCP administration significantly decreased the percentage of Tfh cells and the expression of Tfh markers ICOS, PD-1 and Bcl-6 in the mesenteric lymph nodes of colitis mice. In addition, BPYCP treatment obviously decreased the percentages of Tfh1, Tfh17 and Tem-Tfh cells and upregulated Tfr cells in colitis mice. However, there were no significant regulatory effects of BPYCP on Tfh cell response in normal mice. ConclusionTaken together, these results demonstrated a protective effect of BPYCP against DSS-induced experimental colitis by regulating Tfh cell homeostasis.

Swine Influenza Virus Infection Decreases the Protective Immune Responses of Subunit Vaccine Against Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is the primary pathogen of porcine circovirus diseases and porcine circovirus associated diseases. Immunization with a vaccine is considered an effective measure to control these diseases. However, it is still unknown whether PCV2 vaccines have protective immune responses on the animals infected with swine influenza virus (SIV), a pandemic virus in swine herds. In this study, we first compared the effects of 2 different PCV2 vaccines on normal mice and SIV-infected mice, respectively. The results showed that these two vaccines had protective immune responses in normal mice, and the subunit vaccine (vaccine S) had better effects. However, the inactivated vaccine (vaccine I) instead of vaccine S exhibited more immune responses in the SIV-infected mice. SIV infection significantly decreased the immune responses of vaccine S in varying aspects including decreased PCV2 antibody levels and increased PCV2 replication. Mechanistically, further studies showed that SIV infection increased IL-10 expression and M2 macrophage percentage, but decreased TNF-α expression and M1 macrophage percentage in the mice immunized with vaccine S; on the contrary, macrophage depleting by using clodronate-containing liposomes significantly alleviated the SIV infection-induced decrease in the protective immune responses of vaccine S against PCV2. This study indicates that SIV infection decreases the protective immune responses of vaccine S against PCV2. The macrophage polarization induced by SIV infection might facilitate decreased immune responses to vaccine S, which provides new insight into vaccine evaluation and a reference for the analysis of immunization failure.

A novel TLR7 agonist as adjuvant to stimulate high quality HBsAg-specific immune responses in an HBV mouse model

BackgroundThe global burden of hepatitis B virus (HBV) infection in terms of morbidity and mortality is immense. Novel treatments that can induce a protective immune response are urgently needed to effectively control the HBV epidemic and eventually eradicate chronic HBV infection.MethodsWe designed and evaluated an HBV therapeutic vaccine consisting of a novel Toll-like receptor 7 (TLR7) agonist T7-EA, an Alum adjuvant and a recombinant HBsAg protein. We used RNA-seq, ELISA and hTLR7/8 reporting assays to characterize T7-EA in vitro and real-time PCR to evaluate the tissue-retention characteristics in vivo. To evaluate the adjuvant potential, we administrated T7-EA intraperitoneally in a formulation with an Alum adjuvant and HBsAg in normal and HBV mice, then, we evaluated the HBsAg-specific immune responses by ELISA and Elispot assays.ResultsT7-EA acted as an hTLR7-specific agonist and induced a similar gene expression pattern as an unmodified TLR7 ligand when Raw 264.7 cells were exposed to T7-EA; however, T7-EA was more potent than the unmodified TLR7 ligand. In vivo studies showed that T7-EA had tissue-retaining activity with stimulating local cytokine and chemokine expression for up to 7 days. T7-EA could induce Th1-type immune responses, as evidenced by an increased HBsAg-specific IgG2a titer and a T-cell response in normal mice compared to mice received traditional Alum-adjuvant HBV vaccine. Importantly, T7-EA could break immune tolerance and induce persistent HBsAg-specific antibody and T-cell responses in an HBV mouse model.ConclusionsT7-EA might be a candidate adjuvant in a prophylactic and therapeutic HBV vaccine.

Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

We previously reported that syndecan-2 expression is increased on the colonic epithelium during chronic inflammation. Here, we report that syndecan-2 exhibits a different pattern of site-specific colonic expression during acute inflammation. Syndecan-2 expression was up-regulated predominantly in the proximal colon of dextran sulfate sodium-induced colitis mice. The colitis-associated up-regulation of syndecan-2 was barely detected in Rag-1-/- (recombination activating gene 1 knockout) mice under colitis-inducing conditions. Increased syndecan-2 expression correlated with increased levels of infiltrated CD4+ IL-17A+ T cells in the proximal colon. Serum levels of IL-17A were increased during the acute inflammatory response in normal mice but not Rag-1-/- mice. IL-17A directly induced IL-17 receptor (IL-17RA) and syndecan-2 expression in ex vivo-cultured proximal colon tissues and adenoma cell lines from proximal colon. IL-17RA knockdown reduced the IL-17A-mediated syndecan-2 expression in SNU1235 cells. No elevation of syndecan-2 or IL-17RA was observed in colonic tissues from IL-17A-/- mice during colitis induction. Finally, increased expression of syndecan-2 and IL-17RA was observed in the proximal colons of cecal ligation and puncture-induced sepsis mice and infectious pan colitis patients. Together, these data suggest that acute inflammation induces syndecan-2 expression predominantly in the proximal colon via IL-17A-IL-17RA signaling during the early stage of the inflammatory response and that proximal colonic syndecan-2 might be a biomarker for acute inflammation.-Hong, H., Song, H.-K., Hwang, E. S., Lee, A. R., Han, D. S., Kim, S.-E., Oh, E.-S. Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

Tetrandrine inhibits differentiation of proinflammatory subsets of T helper cells but spares de novo differentiation of iTreg cells

Tetrandrine (TET) is an anti-inflammatory compound isolated from Chinese herb Stephania tetrandra S. Moore. It was reported recently that the differentiation of Th17 cells was inhibited, while the generation of induced Treg cells (iTregs) was promoted, by TET treatment. We therefore carefully examined the effect of TET on the differentiation of four major subsets of T helper cells. The results showed that in vitro treatment with TET potently inhibited the differentiation of Th1, Th2 and Th17 cells. Administration of LPS resulted in a mixed Th1, Th2 and Th17 responses in normal mice, and such effect of LPS was inhibited by in vivo TET treatment as well. In contrast, TET did not promote or inhibit the in vitro generation of iTregs from naïve CD4+CD25−Foxp3/gfp− T cells. Furthermore, spontaneous and rapamycin-induced conversion of naïve CD4+CD25−Foxp3/gfp− T cells into Foxp3-expressing iTregs in congenic mice was not affected by TET treatment. Thus, TET had the capacity to inhibit the differentiation of proinflammatory Th1, Th2 and Th17 cells, while sparing the generation of Tregs. As a Treg-friendly and broad spectrum anti-inflammatory agent, the molecular mechanism and the therapeutic potential of TET in various human inflammatory diseases should be further studied.

Alpha-phellandrene-induced apoptosis in mice leukemia WEHI-3 cells in vitro.

Although reports have shown that α-phellandrene (α-PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α-PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α-PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α-PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI-3 cells in vitro. Results indicated that α-PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub-G1 phase (apoptosis) in WEHI-3 cells. α-PA increased the productions of reactive oxygen species (ROS) and Ca2+ and decreased the levels of mitochondrial membrane potential (ΔΨm ) in dose- and time-dependent manners in WEHI-3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α-PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI-3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α-PA induces cell cycle arrest and apoptosis in WEHI-3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640-1651, 2016.

Crude extract of Polygonum cuspidatum stimulates immune responses in normal mice by increasing the percentage of Mac-3-positive cells and enhancing macrophage phagocytic activity and natural killer cell cytotoxicity.

Polygonum cuspidatum is a natural plant that is used in traditional Chinese herbal medicine. The crude extract of Polygonum cuspidatum (CEPC) has numerous biological effects; however, there is a lack of studies on the effects of CEPC on immune responses in normal mice. The aim of the present study was to determine the in vivo effects of CEPC on immune responses in normal mice. CEPC (0, 50, 100, 150 and 200 mg/kg) was orally administered to BALB/c mice for three weeks, following which blood, liver, and spleen samples were collected. CEPC did not significantly affect the total body weight, or tissue weights of the liver or spleen, as compared with the control mice. CEPC increased the percentages of CD3 (T-cell marker), 11b (monocytes) and Mac-3 (macrophages) positive-cells, and reduced the percentage of CD19-positive cells (B-cell marker), as compared with the control mice. CEPC (100 mg/kg) stimulated macrophage phagocytosis of blood samples but did not affect macrophage phagocytosis in the peritoneum. Activity of the splenic natural killer cells was increased in response to CEPC (50 mg/kg) treatment. Furthermore, CEPC inhibited T- and B-cell proliferation when the cells were stimulated with concanavalin A and lipopolysaccharide, respectively.

Sildenafil alters retinal function in mouse carriers of Retinitis Pigmentosa

Sildenafil, the active ingredient in Viagra®, has been reported to cause transient visual disturbance from inhibition of phosphodiesterase 6 (PDE6), a key enzyme in the visual phototransduction pathway. This study investigated the effects of sildenafil on the rd1+/− mouse, a model for carriers of Retinitis Pigmentosa which exhibit normal vision but may have a lower threshold for cellular stress caused by sildenafil due to a heterozygous mutation in PDE6. Sildenafil caused a dose-dependent decrease in electroretinogram (ERG) responses of normal mice which mostly recovered two days post administration. In contrast, rd1+/− mice exhibited a significantly reduced photoreceptor and a supernormal bipolar cell response to sildenafil within 1 h of treatment. Carrier mice retinae took two weeks to return to baseline levels suggesting sildenafil has direct effects on both the inner and outer retina and these effects differ significantly between normal and carrier mice. Anatomically, an increase in expression of the early apoptotic marker, cytochrome C in rd1+/− mice indicated that the effects of sildenafil on visual function may lead to degeneration. The results of this study are significant considering approximately 1 in 50 people are likely to be carriers of recessive traits leading to retinal degeneration.

A cheese-containing diet modulates immune responses and alleviates dextran sodium sulfate–induced colitis in mice

Diet has a significant effect on immune and inflammatory responses. To date, no studies have described how consumption of a diet containing a relatively high amount of cheese affects immune responses and the inflammatory status of the body. We examined these responses in normal mice and mice with dextran sodium sulfate (DSS)–induced colitis associated with increased inflammatory responses, using a diet containing approximately 44% of a whole cheese powder and a diet containing casein, lard, and corn oil as the control. In normal mice, consumption of the cheese-containing diet induced regulatory T cells (Treg), which regulate immune and inflammatory responses, and suppressed the production of IL-17, IL-4, and IL-10 in Peyer's patch cells from the intestine. The Treg population and cytokine production were not altered in spleen cells. In mice with DSS-induced colitis, consumption of the cheese-containing diet alleviated the symptoms of colitis, as evidenced by prevention of body weight loss and colon length shortening, and inhibition of an increase in the disease activity index, which includes diarrhea and fecal bleeding. This relief of clinical symptoms was also associated with decreased production of proinflammatory cytokines (IL-17 and IL-6) and increased production of the antiinflammatory cytokine transforming growth factor-β1 in Peyer's patch cells. The Treg population was reduced by consumption of the cheese-containing diet in Peyer's patch cells and spleen cells, which might reflect the alleviated symptoms of colitis. Consumption of the cheese-containing diet compared with the control diet enhanced antiinflammatory and immune regulatory responses in normal mice and in a DSS-colitis mouse model.

Butylated hydroxyanisole affects immunomodulation and promotes macrophage phagocytosis in normal BALB/c mice

Butylated hydroxyanisole (BHA), a synthetic antioxidant, has been used in fat and fatty foods to prevent oxidative deterioration. However, the functions of BHA on immune responses in normal mice remain elusive. The aim of the present study was to investigate the effects of oral treatment of BHA on immune responses in normal mice in vivo. BALB/c mice received various treatments. Blood samples were collected and analyzed. Flow cytometry was used to determine the levels of the cell markers. Results showed that BHA did not significantly affect the weight of the animal body and spleen in normal mice. BHA promoted macrophage phagocytosis from peripheral blood mononuclear cells, but did not alter this process in the peritoneal cavity. Furthermore, BHA did not influence natural-killer cell cytotoxicity in normal mice. Notably, BHA promoted the levels of CD3 (T cells) and decreased the level of CD19 (B cells), but did not significantly affect the levels of CD11b (monocytes) and macrophages (Mac-3) in normal mice. Based on these observations it can be concluded that BHA promotes immune responses by increasing T cells and activating phagocytosis by macrophages in normal mice. However, the molecular mechanisms require further investigation.

Effect of age and weight on upper airway function in a mouse model

Defects in pharyngeal mechanical and neuromuscular control are required for the development of obstructive sleep apnea. Obesity and age are known sleep apnea risk factors, leading us to hypothesize that specific defects in upper airway neuromechanical control are associated with weight and age in a mouse model. In anesthetized, spontaneously breathing young and old wild-type C57BL/6J mice, genioglossus electromyographic activity (EMG(GG)) was monitored and upper airway pressure-flow dynamics were characterized during ramp decreases in nasal pressure (Pn, cmH₂O). Specific body weights were targeted by controlling caloric intake. The passive critical pressure (Pcrit) was derived from pressure-flow relationships during expiration. The Pn threshold at which inspiratory flow limitation (IFL) developed and tonic and phasic EMG(GG) activity during IFL were quantified to assess the phasic modulation of pharyngeal patency. The passive Pcrit increased progressively with increasing body weight and increased more in the old than young mice. Tonic EMG(GG) decreased and phasic EMG(GG) increased significantly with obesity. During ramp decreases in Pn, IFL developed at a higher (less negative) Pn threshold in the obese than lean mice, although the frequency of IFL decreased with age and weight. The findings suggest that weight imposes mechanical loads on the upper airway that are greater in the old than young mice. The susceptibility to upper airway obstruction increases with age and weight as tonic neuromuscular activity falls. IFL can elicit phasic responses in normal mice that mitigate or eliminate the obstruction altogether.

A preformed scleral search coil for measuring mouse eye movements

Mice are excellent subjects for use of genetic-manipulation techniques to study the basis of pathological and normal physiology and behavior; however behavioral analyses of associated phenotypes is often limited. To improve the accuracy and specificity of repeated measurements of vestibular function, we developed a miniaturized, contact-lens scleral search coil to measure mouse eye movements. We describe the physical attributes and document its functionality by measuring vestibuloocular responses in normal mice. This coil should greatly improve the sensitivity and documentation of vestibular dysfunction in mouse models of pathology and dysfunction while allowing screening of significant numbers of subjects.

The effect of shark liver oil on the tumor infiltrating lymphocytes and cytokine pattern in mice

Ethnopharmacological relevance Shark Liver Oil (SLO) is a traditional medicine that has been widely used in Scandinavian folk to augment the immune response in some immune-related diseases, especially as an anti-cancer agent. Aim of the study The object of this project was to confirm the anti-cancer effect of SLO and the possible involving mechanisms. Materials and methods Using delayed-type hypersensitivity (DTH) response in normal mice, the optimal dose for stimulation of cellular immunity was obtained and injected intraperitoneally to the tumor-bearing mice. Cytokine pattern of splenic MNCs was tested by ELISA. The percentage of CD 4 + and CD 8 + lymphocytes in tumor-infiltrating lymphocytes was determined by flow cytometry. Also the rate of increase in tumor volume measured. Results Our findings indicated that SLO highly augments delayed-type hypersensitivity response against sheep Red Blood Cell (sRBC) in mice. Furthermore, intraperitoneal injection of SLO to tumor-bearing mice could increase T-cell infiltration into the tumor and lower the increasing rate of tumor's volume. Also, it changes the cytokine pattern of the splenic Mononuclear cells (MNCs) to Th1. Conclusion Increase in IFN-γ (resulting in enhanced cellular immunity) and increase in especially CD 8 + lymphocytes accompanied by a decrease in tumor size are among the signs of its anti-tumor effect. Accordingly, we suppose that SLO is a good candidate for further studies in cancer therapy.

Cancer-induced Expansion and Activation of CD11b+Gr-1+ Cells Predispose Mice to Adenoviral-triggered Anaphylactoid-type Reactions

Intravascular delivery (1.5 x 10(9) particles and higher) of recombinant adenovirus (rAd) induces myeloid cell mediated, self-limiting hemodynamic responses in normal mice. However, we observed anaphylactoid-type reactions and exacerbated hemodynamic events following rAd injection in mice bearing malignant 4T1 mammary carcinoma. Because 4T1 tumors induce significant CD11b(+)Gr-1(+) myeloid cell expansion and activation, we set to determine whether this causes rAd-induced exaggerated responses. When treated with a single intravenous dose (1 x 10(10) particles) of rAd, mice implanted with 4T1 carcinoma succumbed due to the anaphylactoid-type reactions. In contrast, normal mice and mice implanted with a related mammary carcinoma (66cl4) that does not induce CD11b(+)Gr-1(+) cell expansion, showed minimal responses. Depletion of phagocytic CD11b(+)Gr-1(+) cells prior to rAd delivery protected 4T1 tumor-bearing animals, whereas passive transfer of CD11b(+)Gr-1(+) cells from 4T1 tumor-bearing animals was sufficient to convey susceptibility to anaphylactoid-type reactions in normal animals. We further show that there is upregulation of nitric oxide and leukotriene signaling pathways in the 4T1 tumor-induced CD11b(+)Gr-1(+) myeloid cells and that pretreating mice with inhibitors of nitric oxide synthetase and leukotrienes can attenuate the anaphylactoid-type reactions. These data show that malignant tumor growth can alter CD11b(+)Gr-1(+) myeloid cells, rendering hosts susceptible to anaphylactoid-type reactions upon intravascular treatment with rAd.

The effects of B‐cell depletion therapies on humoral immune responses in normal mice

Anti‐CD20 mediated B cell depletion is a therapy for treating rheumatoid arthritis and B‐cell malignancies. To examine how B cell depletion affects an individual's capacity to generate antibody responses we assessed in a mouse model primary and secondary antibody responses after treatment with anti‐mouse CD20 and/or BAFFR‐Ig. BALB/c mice dosed with anti‐CD20 one week prior to primary TNP‐KLH immunization had a 93% decrease in anti‐TNP IgG titers at day 10 compared to controls. Mice treated with anti‐CD20 one week prior to immunization with TNP‐Ficoll had a ~50% increase in TNP‐specific IgG and IgM titers. The secondary antibody response to TNP‐KLH was not significantly affected by anti‐CD20 treatment one week prior to immunization. BAFFR‐Ig treatment reduced the secondary response to TNP‐KLH by 57%. Treatment of mice with the combination of anti‐CD20 and BAFFR‐Ig dramatically reduced secondary antibody responses to either antigen. After B‐cell repopulation all treatment groups mounted a strong response to a tertiary challenge. Therefore, B cell depletion with anti‐CD20 results in a strong reduction in the primary antibody response to a neoantigen, however memory B cells appear resistant to anti‐CD20 treatment. Simultaneous anti‐CD20 and BAFFR‐Ig treatment results in elimination of the secondary response by blocking memory B cell activation but does not deplete memory B cells.

Cognition-enhancing and anxiolytic effects of memantine

Memantine, a moderate-affinity NMDA receptor antagonist, is clinically used for the treatment of Alzheimer's disease (AD). Both clinical and preclinical studies have shown that memantine, at doses producing a steady-state plasma level of 0.5–1 μM, is well tolerated and improves cognition. Here we tested the effects of chronic oral administration of memantine (10, 30 and 100 mg/kg per day) producing steady state plasma drug levels ranging between ∼0.5and 6 μM on motor, social, emotional and cognitive behavior in normal C57BL/6J mice. Memantine dose-dependently reduced escape latency (hidden platform) and decreased wall swimming tendency in the Morris water maze test, increased time spent in open arms in the elevated plus-maze test, and reduced the number of isolation-induced aggressive attacks, but did not affect exploratory activity in the open field. These data indicate that high, stable doses of memantine improved cognition and exhibited a potential anxiolytic response in normal mice.

Visual function testing: A quantifiable visually guided behavior in mice

A measure of improved vision remains the most meaningful way to demonstrate the efficacy of a therapy. Animal models allow us to describe the pathology of inherited retinal degenerations and to evaluate emerging therapies in specific disorders in ways not possible in human subjects. The potential use of mice in this role has been limited by the lack of a simple, unambiguous and practical test of an innate visually guided behavior. To begin to address this need, we have developed equipment and protocols to measure a performance enhancing effect of vision on use of a running wheel; a scotopic visually guided behavior termed positive masking. This assay is objective, quantitative, automated and can be adapted for in-depth studies of visual thresholds, longitudinal studies of visual pathology or treatment efficacy, and large scale screening programs. Proof-of-principle experiments show that our equipment and protocols are able to characterize the full range of masking responses in normal mice in an informative and efficient manner. A sustained activity increase across a range of dim light irradiances was consistent with scotopic visual guidance of behavior, while at higher irradiances a dose dependent suppression of activity was apparent. This study also describes for the first time the interaction of experience and vision in performing a task. Specifically, we identified an experience dependent acclimatization to wheel use in scotopic conditions; a performance reduction in complete darkness; and a partial but not complete recovery of performance levels with experience in complete darkness. This suggests that where visual guidance is performance enhancing but not essential, loss of the contribution of visual guidance to the tasks might be compensated for by experience or training.

LPS deacylation by a host enzyme shortens endotoxin tolerance (44.20)

Abstract Prior exposure to bacterial lipopolysaccharide (LPS) alters animals’ responses to a second LPS exposure. Although this reprogramming (also called endotoxin tolerance) may prevent harmful inflammatory reactions, it also may decrease resistance to subsequent infection. How animals recover from tolerance is not known. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates LPS by cleaving the secondary fatty acyl chains from lipid A. We found that 21 days after an initial dose of LPS, long after the responses of normal mice had returned to baseline, Aoah−/− mice had diminished serum IL-6 and TNF responses to LPS challenge. Ten days after receiving LPS i.p., macrophages explanted from Aoah−/− mice and stimulated ex vivo with LPS produced less IL-6 and TNFα than did macrophages from LPS-primed control mice. Whereas the LPS that was associated with peritoneal cells of Aoah+/+ mice was completely deacylated by 10 days, LPS remained undeacylated in Aoah−/− peritoneal cells. LPS-primed Aoah−/− mice were unable to control bacterial growth and therefore were more susceptible to lethal E. coli infection. LPS inactivation by AOAH shortens the duration of tolerance and its associated immunosuppression. A better understanding of how tolerance is terminated may help ameliorate post-infection immunosuppression in humans. Supported by NIAID grant AI18188.

DJ-1 and Parkin Modulate Dopamine-dependent Behavior and Inhibit MPTP-induced Nigral Dopamine Neuron Loss in Mice

Parkin-deficient animals exhibit mitochondrial degeneration and increased oxidative stress vulnerability, and both mice and flies lacking DJ-1 are hypersensitive to environmental toxins associated with Parkinson's disease (PD). We used recombinant adeno-associated virus (AAV) gene transfer to study the influence of DJ-1 and Parkin on the dopaminergic system of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, a model for sporadic PD. After MPTP lesioning, significantly more dopamine neurons survived in the virus-injected substantia nigra of the AAV-DJ-1 and AAV-Parkin mice when compared with AAV-enhanced green fluorescent protein injected controls. Protection at the neuronal level was supported by increased amphetamine-induced contralateral turning behavior. Normal mice expressing DJ-1 showed apomorphine-induced ipsilateral turning, suggesting a hyporesponsiveness of striatal dopamine D1 receptors in the DJ-1-expressing hemisphere. MPTP drastically reduced dopamine to 19% of normal levels and neither DJ-1 nor Parkin protected against MPTP-induced catecholamine loss under these conditions. Our results show that Parkin and DJ-1 inhibit dopamine neuron death and enhance amphetamine-induced dopaminergic function in a mouse model of idiopathic PD. However, DJ-1 overexpression also reduced postsynaptic dopamine receptor responses in normal mice. These results warrant further exploration of DJ-1 and Parkin gene therapy for PD, although a better understanding of their effects on behavior and dopamine neurotransmission is required before these proteins can be safely used.