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Protective signaling from the leukemia microenvironment leads to leukemia cell persistence, development of resistance, and disease relapse. Here, we demonstrate that fibroblast growth factor 2 (FGF2) from bone marrow stromal cells is secreted in exosomes, which are subsequently endocytosed by leukemia cells, and protect leukemia cells from tyrosine kinase inhibitors (TKIs). Expression of FGF2 and its receptor, FGFR1, are both increased in a subset of stromal cell lines and primary AML stroma; and increased FGF2/FGFR1 signaling is associated with increased exosome secretion. FGFR inhibition (or gene silencing) interrupts stromal autocrine growth and significantly decreases secretion of FGF2-containing exosomes, resulting in less stromal protection of leukemia cells. Likewise, Fgf2 -/- mice transplanted with retroviral BCR-ABL leukemia survive significantly longer than their +/+ counterparts when treated with TKI. Thus, inhibition of FGFR can modulate stromal function, reduce exosome secretion, and may be a therapeutic option to overcome resistance to TKIs. This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Latently infected CD4+ T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4+ T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription. In addition, we measured the degree to which these transcriptional blocks could be reversed ex vivo by T cell activation (using anti-CD3/CD28 antibodies) or latency-reversing agents. We found that the main reversible block to HIV RNA transcription was not inhibition of transcriptional initiation but rather a series of blocks to proximal elongation, distal transcription/polyadenylation (completion), and multiple splicing. Cell dilution experiments suggested that these mechanisms operated in most of the HIV-infected CD4+ T cells examined. Latency-reversing agents exerted differential effects on the three blocks to HIV transcription, suggesting that these blocks may be governed by different mechanisms.

Despite intensive study, it is unclear which mechanisms are responsible for latent HIV infection in vivo. One potential mechanism is inhibition of HIV transcriptional elongation, which results in short abortive transcripts containing the trans-activation response (TAR) region. Because the relative levels of total (including short) and processive transcripts provide measures of HIV transcriptional initiation and elongation, there is a compelling need for techniques that accurately measure both. Nonetheless, prior assays for total transcripts have been semi-quantitative and have seen limited application to patient samples. This manuscript reports the validation of quantitative reverse transcription (RT) droplet digital PCR assays for measurement of total (TAR) and processive (R-U5/gag) HIV transcripts. Traditional RT priming strategies can efficiently detect the TAR region on long HIV transcripts but detect 10-fold higher than elongated transcripts, implying a substantial block to transcriptional elongation in vivo. This approach may be applied to other difficult-to-prime RNA targets.

BackgroundIn HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels.MethodsThirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition.ResultsThe addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling.ConclusionsTreatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels.

AIMS:We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(–/–) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(–/–) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS:HypoE/SR-BI(–/–) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS:Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

BackgroundMuscle atrophy, fatty infiltration, and fibrosis of the muscle have been described as important factors governing outcome after rotator cuff injury and repair. Muscle fibrosis is also thought to have a role in determining muscle compliance at the time of surgery. The transforming growth factor-β (TGF-β) pathways are highly conserved pathways that exert a potent level of control over muscle gene expression and are critical regulators of fibrosis in multiple organ systems. It has been shown that TGF-β can regulate important pathways of muscle atrophy, including the Akt/mammalian target of rapamycin pathway. The purpose of this study was to evaluate the expression of TGF-β and its downstream effectors of fibrosis after a massive rotator cuff tear (RCT) in a previously established rat model.MethodsTo simulate a massive RCT, infraspinatus and supraspinatus tenotomy and suprascapular nerve transection were performed on Sprague-Dawley rats with use of a validated model. Two and 6 weeks after surgery, supraspinatus muscles were harvested to study alterations in TGF-β signaling by Western blotting, quantitative polymerase chain reaction, and histologic analysis.ResultsThere was a significant increase in fibrosis in the rotator cuff muscle after RCT in our animal model. There was a concomitant increase in TGF-β gene and protein expression at both 2 and 6 weeks after RCT. Evaluation of the TGF-β signaling pathway revealed an increase in SMAD2 activation but not in SMAD3. There was an increase in profibrotic markers collagen I, collagen III, and α-smooth muscle actin.ConclusionsTGF-β signaling is significantly upregulated in rat supraspinatus muscles after RCTs.

Backgroundextracellular matrix (ECM) components are instrumental in maintaining homeostasis and muscle fiber functional integrity. Skeletal muscle hypertrophy is associated with ECM remodeling. Specifically, recent studies have reported the involvement of matrix metalloproteinases (MMPs) in muscle ECM remodeling. However, the functional role of MMPs in muscle hypertrophy remains largely unknown.Methodsin this study, we examined the role of MMP-2 in skeletal muscle hypertrophy using a previously validated method where the plantaris muscle of mice were subjected to mechanical overload due to the surgical removal of synergist muscles (gastrocnemius and soleus).Resultsfollowing two weeks of overload, we observed a significant increase in MMP-2 activity and up-regulation of ECM components and remodeling enzymes in the plantaris muscles of wild-type mice. However, MMP-2 knockout mice developed significantly less hypertrophy and ECM remodeling in response to overload compared to their wild-type littermates. Investigation of protein synthesis rate and Akt/mTOR signaling revealed no difference between wild-type and MMP-2 knockout mice, suggesting that a difference in hypertrophy was independent of protein synthesis.Conclusiontaken together, our results suggest that MMP-2 is a key mediator of ECM remodeling in the setting of skeletal muscle hypertrophy.

Previous studies have evaluated role of Akt/mTOR signaling in rotator cuff muscle atrophy and determined that there was differential in signaling following tendon transection (TT) and suprascapular nerve (SSN) denervation (DN), suggesting that atrophy following TT and DN was modulated by different protein degradation pathways. In this study, two muscle proteolytic systems that have been shown to be potent regulators of muscle atrophy in other injury models, the ubiquitin-proteasome pathway and autophagy, were evaluated following TT and DN. In addition to examining protein degradation, this study assessed protein synthesis rate following these two surgical models to understand how the balance between protein degradation and synthesis results in atrophy following rotator cuff injury. In contrast to the traditional theory that protein synthesis is decreased during muscle atrophy, this study suggests that protein synthesis is up-regulated in rotator cuff muscle atrophy following both surgical models. While the ubiquitin-proteasome pathway was a major contributor to the atrophy seen following DN, autophagy was a major contributor following TT. The findings of this study suggest that protein degradation is the primary factor contributing to atrophy following rotator cuff injury. However, different proteolytic pathways are activated if SSN injury is involved.

Repair of peripheral nerve defects with current synthetic, tubular nerve conduits generally shows inferior recovery when compared with using nerve autografts, the current gold standard. We tested the ability of composite collagen and hyaluronan hydrogels, with and without the nerve growth factor (NGF), to stimulate neurite extension on a promising aligned, nanofiber poly-L-lactide-co-caprolactone (PLCL) scaffold. In vitro, the hydrogels significantly increased neurite extension from dorsal root ganglia explants. Consistent with these results, the addition of hydrogels as luminal fillers within aligned, nanofiber tubular PLCL conduits led to improved sensory function compared to autograft repair in a critical-size defect in the sciatic nerve in a rat model. Sensory recovery was assessed 3 and 12 weeks after repair using a withdrawal assay from thermal stimulation. The addition of hydrogel did not enhance recovery of motor function in the rat model. The NGF led to dose-dependent improvements in neurite out-growth in vitro, but did not have a significant effect in vivo. In summary, composite collagen/hyaluronan hydrogels enhanced sensory neurite outgrowth in vitro and sensory recovery in vivo. The use of such hydrogels as luminal fillers for tubular nerve conduits may therefore be useful in assisting restoration of protective sensation following peripheral nerve injury.

Rotator cuff tears (RCTs) are among the most common injuries seen in orthopedic patients. Chronic tears can result in the development of muscular atrophy and fatty infiltration. Despite the prevalence of RCTs, little is known about the underlying molecular pathways that produce these changes. Recently, we have shown that mammalian target of rapamycin (mTOR) signaling plays an important role in muscle atrophy that results from massive RCTs in a rat model. The purpose of this study was therefore to extend our understanding of mTOR signaling and evaluate its role in fatty infiltration after a combined tendon transection and suprascapular nerve denervation surgery. Akt/mTOR signaling was significantly increased and resulted in the up-regulation of two transcription factors: SREBP-1 and PPARγ. We also saw an increase in expression of adipogenic markers: C/EBP-α and FASN. Upon treatment with rapamycin, an inhibitor of mTOR, we observed a decrease in mTOR signaling, activity of transcription factors, and reduction in fatty infiltration. Therefore, our study suggests that mTOR signaling mediates rotator cuff fatty infiltration via SREBP-1 and PPARγ. Clinically, our finding may alter current treatment methods to address rotator cuff fatty infiltration.

Muscle extracellular matrix (ECM) plays an important role in maintaining muscular integrity and force transduction. However, the role of ECM in skeletal muscle atrophy remains unknown. In this study, we employed two clinically relevant mouse models of Achillotenotomy and hindlimb suspension to simulate Achilles tendon rupture and hindlimb disuse. The gastrocnemius was harvested following two weeks of treatment. We hypothesized that degradation of muscle ECM basement membrane lead to dysfunction of muscle contractility. Our results demonstrated a significant reduction of gastrocnemius single twitch force, isometric tetanic force, and contraction velocity following tendon rupture (p

Proteogenomics refers to the integration of comprehensive genomic, transcriptomic, and proteomic measurements from the same samples with the goal of fully understanding the regulatory processes converting genotypes to phenotypes, often with an emphasis on gaining a deeper understanding of disease processes. Although specific genetic mutations have long been known to drive the development of multiple cancers, gene mutations alone do not always predict prognosis or response to targeted therapy. The benefit of proteogenomics research is that information obtained from proteins and their corresponding pathways provides insight into therapeutic targets that can complement genomic information by providing an additional dimension regarding the underlying mechanisms and pathophysiology of tumors. This review describes the novel insights into tumor biology and drug resistance derived from proteogenomic analysis while highlighting the clinical potential of proteogenomic observations and advances in technique and analysis tools. [ABSTRACT FROM AUTHOR]

Author(s): Qing-Sheng Mi (corresponding author) [1, 2, 3]; Zhong-Bin Deng [1]; Sunil K Joshi [1, 2]; Zai-Zhao Wang [1]; Li Zhou [1, 2]; Sarah Eckenrode [1]; Ratanmani Joshi [1, 2]; [...]

Acute myeloid leukemia (AML) is a clonal hematologic neoplasm characterized by rapid, uncontrolled cell growth of immature myeloid cells (blasts). There are numerous genetic abnormalities in AML, many of which are prognostic, but an increasing number are targets for drug therapy. One of the most common genetic abnormalities in AML are activating mutations in the FMS-like tyrosine kinase 3 receptor (FLT3). As a receptor tyrosine kinase, FLT3 was the first targetable genetic abnormality in AML. The first generation of FLT3 inhibitors were broad-spectrum kinase inhibitors that inhibited FLT3 among other proteins. Although clinically active, first-generation FLT3 inhibitors had limited success as single agents. This led to the development of a second generation of more selective FLT3 inhibitors. This review focuses on quizartinib, a potent second-generation FLT3 inhibitor. We discuss the clinical trial development, mechanisms of resistance, and the recent FDA decision to deny approval for quizartinib as a single agent in relapsed/refractory AML. [ABSTRACT FROM AUTHOR]

HIV latency may not be governed by a lack of transcriptional initiation, but rather by blocks to RNA elongation, polyadenylation, and splicing. Watching HIV in its hiding place: A barrier to developing a cure for HIV is the problem of viral latency, in which a small population of patient CD4+ T cells harbor inactive virus. These cells remain infected but untargetable by current antiretrovirals, meaning treatment must be lifelong. Now, Yukl et al. have identified and quantified multiple reversible blocks to HIV transcription in latently infected CD4+ T cells taken from infected patients on antiretroviral therapy. They found that viral transcription was stalled not at initiation but rather at downstream stages. These findings may be important for developing therapies to fully eradicate HIV infection. Latently infected CD4+ T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4+ T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription. In addition, we measured the degree to which these transcriptional blocks could be reversed ex vivo by T cell activation (using anti-CD3/CD28 antibodies) or latency-reversing agents. We found that the main reversible block to HIV RNA transcription was not inhibition of transcriptional initiation but rather a series of blocks to proximal elongation, distal transcription/polyadenylation (completion), and multiple splicing. Cell dilution experiments suggested that these mechanisms operated in most of the HIV-infected CD4+ T cells examined. Latency-reversing agents exerted differential effects on the three blocks to HIV transcription, suggesting that these blocks may be governed by different mechanisms. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) remained largely quiescent for nearly six decades after its first appearance in 1947. ZIKV reappeared after 2007, resulting in a declaration of an international "public health emergency" in 2016 by the World Health Organization (WHO). Until this time, ZIKV was considered to induce only mild illness, but it has now been established as the cause of severe clinical manifestations, including fetal anomalies, neurological problems, and autoimmune disorders. Infection during pregnancy can cause congenital brain abnormalities, including microcephaly and neurological degeneration, and in other cases, Guillain-Barré syndrome, making infections with ZIKV a substantial public health concern. Genomic and molecular investigations are underway to investigate ZIKV pathology and its recent enhanced pathogenicity, as well as to design safe and potent vaccines, drugs, and therapeutics. This review describes progress in the design and development of various anti- ZIKV therapeutics, including drugs targeting virus entry into cells and the helicase protein, nucleosides, inhibitors of NS3 protein, small molecules, methyltransferase inhibitors, interferons, repurposed drugs, drugs designed with the aid of computers, neutralizing antibodies, convalescent serum, antibodies that limit antibody-dependent enhancement, and herbal medicines. Additionally, covalent inhibitors of viral protein expression and anti-Toll-like receptor molecules are discussed. To counter ZIKVassociated disease, we need to make rapid progress in developing novel therapies that work effectually to inhibit ZIKV. [ABSTRACT FROM AUTHOR]

In recent years, aquaculture has attained a major economic revolution, however, infectious diseases of bacterial, viral, mycotic and parasitic origin are the most significant restrictive agents in the improvement of intensified aquaculture, which has become a fast blooming seafood industry. For environment-friendly aquaculture and human health concerns owing to the rise in incidences of antimicrobial resistant microbes and food safety hazards, the immunoprophylaxis or vaccination strategies are highly effective and economical in protecting the health of fish and aquaculture animals from various infectious agents. Advancements in science have paved newer avenues in both basic and applied research areas for developing and designing novel and effective vaccines, as well as improving existing vaccines for rendering protection from various types of infectious diseases. Current advances in vaccines and vaccinology offer valuable opportunities to discover new vaccine candidates to combat fish pathogens, including mycotic and parasitic agents, for which vaccines are still lacking. This review focuses on the current knowledge, recent advances and future perspectives of vaccines and vaccination in the aquaculture industry, from traditional inactivated and attenuated vaccines to new generation vaccines comprising of recombinant, subunit, vectored, genetically engineered, DNA and peptide vaccines, reverse vaccinology and plant-based edible vaccines, and nanovaccines. [ABSTRACT FROM AUTHOR]

Background: Bricks have been manufactured in Nepal for hundreds of years and are seen as a component of Nepalese sculpture and architecture. Large quantities of hazardous materials including high concentrations of particulate matter are emitted on a daily basis from brick kilns. Exposure to these hazardous materials can lead to adverse consequences on the environment and human health. This study was conducted to estimate the prevalence of respiratory symptoms/illnesses and the magnitude of respirable and total dust exposures among Nepalese brick kiln workers. Methods: Respiratory symptoms/illnesses were evaluated by questionnaire among brickfield workers (n = 400) and a referent group of grocery workers (n = 400) in Kathmandu valley. Work zones (WZs): green brick molding (GBM), green brick stacking/carrying (GBS/C), red brick loading/carrying (RBL/C), coal preparation (CP) and firemen (FM) were the similar exposure groups (SEGs) from where personal air samples and interviews were taken. Among brickfield workers, personal monitoring was conducted across SEGs for total (n = 89) and respirable (n = 72) dust during February-March 2015 and March-April 2016. Applying multi-stage probability proportionate to size sampling technique, 16 kilns and 400 brick workers for interview were selected. Proportions, means, medians and ranges were calculated for the demographics, samples and respiratory symptoms/illnesses. One-way ANOVA was applied to compare the significance differences of the level of particulate matter among SEGs. Bivariate and multivariate logistic regression analysis were performed to evaluate association between respiratory symptoms/illnesses and participants groups, and SEGs among brick kiln workers at 0.05 level. Statistical analyses were performed using IBM SPSS Statistics 21. Results: Chronic cough (14.3%), phlegm (16.6%) and bronchitis (19.0%) were higher (P < 0.05) among brickfield compared with grocery workers (6.8, 5.8 and 10.8%). Mean respirable (5.888 mg/m3) and total (20.657 mg/m3) dust exposures were highest for red brick loading tasks. The prevalence of chronic cough, chronic phlegm, chronic bronchitis, wheezing and asthma were significantly higher for other WZs workers (p < 0.05) compared with CP; for GBM: 22.9, 34.6, 15.0 and 7.5%; for GBS/C: 13.5, 15.8, 10.0, 8.8 and 7.5%; for RBL/C: 11.1, 17.1, 27.4, 19.0 and 11.9%; for FM: 18.4, 12.5, 28.4, 4.9 and 0.0%; and for CP: 4.9, 6.3, 13.3, 9.3 and 4.0% respectively. Conclusion: High dust exposures identified in this study may explain the increased prevalence of respiratory symptoms/ illnesses among Nepalese brickfield workers, warranting action to reduce exposures. [ABSTRACT FROM AUTHOR]

Author(s): Qing-Sheng Mi; Zhong-Bin Deng; Sunil K Joshi; Zai-Zhao Wang; Li Zhou; Sarah Eckenrode; Ratanmani Joshi; Dalam Ly; Bing Yi; Terry L Delovitch; Jin-Xiong She Nature Medicine 12, 624-626 (2006) [...]

Delayed graft function (DGF) is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2), as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2) that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC), in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are associated with tubular injury in DGF and offer novel therapeutic targets for the prevention of this disorder. [ABSTRACT FROM AUTHOR]

Protective immunity against anthrax is inferred from measurement of vaccine antigen-specific neutralizing antibody titers in serum samples. In animal models, in vivo challenges with toxin and/or spores can also be performed. However, neither of these approaches considers toxin-induced damage to specific organ systems. It is therefore important to determine to what extent anthrax vaccines and existing or candidate adjuvants can provide organ-specific protection against intoxication. We therefore compared the ability of Alum, CpG DNA and the CD1d ligand α-galactosylceramide (αGC) to enhance protective antigen-specific antibody titers, to protect mice against challenge with lethal toxin, and to block cardiotoxicity and hepatotoxicity. By measurement of serum cardiac Troponin I (cTnI), and hepatic alanine aminotransferase (ALT), and aspartate aminotransferase (AST), it was apparent that neither vaccine modality prevented hepatic intoxication, despite high Ab titers and ultimate survival of the subject. In contrast, cardiotoxicity was greatly diminished by prior immunization. This shows that a vaccine that confers survival following toxin exposure may still have an associated morbidity. We propose that organ-specific intoxication should be monitored routinely during research into new vaccine modalities. [ABSTRACT FROM AUTHOR]

Background: extracellular matrix (ECM) components are instrumental in maintaining homeostasis and muscle fiber functional integrity. Skeletal muscle hypertrophy is associated with ECM remodeling. Specifically, recent studies have reported the involvement of matrix metalloproteinases (MMPs) in muscle ECM remodeling. However, the functional role of MMPs in muscle hypertrophy remains largely unknown. Methods: in this study, we examined the role of MMP-2 in skeletal muscle hypertrophy using a previously validated method where the plantaris muscle of mice were subjected to mechanical overload due to the surgical removal of synergist muscles (gastrocnemius and soleus). Results: following two weeks of overload, we observed a significant increase in MMP-2 activity and up-regulation of ECM components and remodeling enzymes in the plantaris muscles of wild-type mice. However, MMP-2 knockout mice developed significantly less hypertrophy and ECM remodeling in response to overload compared to their wild-type littermates. Investigation of protein synthesis rate and Akt/mTOR signaling revealed no difference between wild-type and MMP-2 knockout mice, suggesting that a difference in hypertrophy was independent of protein synthesis. Conclusion: taken together, our results suggest that MMP-2 is a key mediator of ECM remodeling in the setting of skeletal muscle hypertrophy. [ABSTRACT FROM AUTHOR]

CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide–containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell–derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell–derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants. [ABSTRACT FROM AUTHOR]

Muscle extracellular matrix (ECM) plays an important role in maintaining muscular integrity and force transduction. However, the role of ECM in skeletal muscle atrophy remains unknown. In this study, we employed two clinically relevant mouse models of Achillotenotomy and hindlimb suspension to simulate Achilles tendon rupture and hindlimb disuse. The gastrocnemius was harvested following two weeks of treatment. We hypothesized that degradation of muscle ECM basement membrane lead to dysfunction of muscle contractility. Our results demonstrated a significant reduction of gastrocnemius single twitch force, isometric tetanic force, and contraction velocity following tendon rupture (p<0.001), but not disuse. Additionally, up-regulation of matrix metalloproteinase- 2 (MMP-2) was observed only after tendon rupture (p=0.00234). These findings suggest that ECM remodeling and basement membrane degradation due to MMP-2 may be responsible for declined muscle contractibility. Inhibiting ECM degradation enzymes may be a potential treatment strategy for skeletal muscle atrophy after tendon rupture. [ABSTRACT FROM AUTHOR]

Abstract: CD1d-binding glycolipids exert potent adjuvant effects on T-dependent Ab responses. The mechanisms include cognate interaction between CD1d-expressing B cells and TCR-expressing Type I CD1d-restricted natural killer T cells (NKTs). However, the critical NKT-derived factors that stimulate B cells are poorly understood. We tested the hypothesis that CD1d-driven CD40L expression by NKT cells influences humoral immunity. Bone marrow chimeras with CD40L+/+ or CD40L−/− NKT cells were immunized with Ag plus CD1d ligand before measuring Ab responses. CD40L−/− NKT cells stimulated higher endpoint Ab titers than controls expressing CD40L. In contrast, immunization of CD40L−/− mice revealed that CD40L−/− NKT cells could not provide B cell help when Th cells lacked CD40L. We report that CD40L−/− NKT cells can provide help for Ab production and do so cooperatively with CD40L+/+ Th cells. We suggest that the manner in which NKT cells provide B cell help is distinct from that of Th cells. [Copyright &y& Elsevier]

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand &agr;-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFN&ggr; to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4-/-mice and IFNc2/2 mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing &agr;-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4-/one marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFN&ggr;. NKT-derived IL-4 was required for OCHenhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNc also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by &agr;-GC derivatives can be exploited to optimize anthrax vaccination. [ABSTRACT FROM AUTHOR]

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand &agr;-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFN&ggr; to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4-/-mice and IFNc2/2 mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing &agr;-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4-/one marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFN&ggr;. NKT-derived IL-4 was required for OCHenhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNc also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by &agr;-GC derivatives can be exploited to optimize anthrax vaccination. [ABSTRACT FROM AUTHOR]

Exogenous CD1d-binding glycolipid (a-Galactosylceramide, a-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen. [ABSTRACT FROM AUTHOR]