Immune reconstitution prevents metastatic recurrence of murine osteosarcoma

Primary tumors developing in immunocompetent hosts escape immunosurveillance by acquiring immune evasive properties. This raises the prospect that metastases derived from such tumors will also evade immunity. To investigate whether immune surveillance plays a role in preventing metastases, we studied a murine model which mimics the clinical progression of osteosarcoma: primary tumor growth in the lower extremity, amputation, minimal residual disease followed by the development of overt metastases. K7M2 implants readily escaped immune surveillance since normal BALB/c mice, T cell deficient SCID and T/NK cell deficient SCID-bg mice showed no difference in the rate of growth of primary osteosarcomas. However, both SCID and SCID-bg mice had higher rates of metastases than immunocompetent mice. Similarly, immune reconstitution following transfer of naive T cells to SCID or SCID-bg mice did not impact primary tumor growth, but significantly diminished metastatic recurrence. T cells in osteosarcoma bearing mice produced IFNγ in response to tumor and IFNγ production by immune reconstituting T cells was required to prevent metastases. These results demonstrate an important role for T cell based immune surveillance in preventing metastases, even when metastases develop from tumors that adeptly evade immunosurveillance. The results further suggest that T cell depleting cancer therapies may eliminate beneficial immune responses and that immune reconstitution of lymphopenic cancer patients could prevent metastatic recurrence of solid tumors. By acceptance of this article, the publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Animal care was provided in accordance with procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH Pub. No. 86-23, 1996). This project was funded in whole or part with funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.     

Discovery of +(2-{4-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy]phenyl}-cyclopropyl)acetic acid as potent and selective alphavbeta3 inhibitor: design, synthesis, and optimization

The integrin alpha(v)beta(3) is expressed in a number of cell types and is thought to play a major role in several pathological conditions. Various small molecules that inhibit the integrin have been shown to suppress tumor growth and retinal angiogenesis. The tripeptide Arg-Gly-Asp (RGD), a common binding motif in several ligands that bind to alpha(v)beta(3), has been depeptidized and optimized in our efforts toward discovering a small molecule inhibitor. We recently disclosed the synthesis and biological activity of several small molecules that did not contain any peptide bond and mimic the tripeptide RGD. The phenethyl group in one of the lead compounds was successfully replaced with a cyclopropyl moiety. The new lead compound was optimized for potency, selectivity, and for its ADME properties. We describe herein the discovery, synthesis, and optimization of cyclopropyl containing analogs that are potent and selective inhibitors of alpha(v)beta(3).     

Synthesis, DNA binding, and cytotoxicity studies of pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates

A series of pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates have been prepared and evaluated for their DNA binding ability as well as anticancer activity. Some of these molecules have shown significant anticancer activity in a number of cancer cell lines.     

The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation

Metastasis is the primary cause of mortality from cancer, but the mechanisms leading to metastasis are poorly understood. In particular, relatively little is known about metastasis in cancers of mesenchymal origins, which are known as sarcomas. Approximately ten proteins have been characterized as 'metastasis suppressors', but how these proteins function and are regulated is, in general, not well understood. Gp78 (also known as AMFR or RNF45) is a RING finger E3 ubiquitin ligase that is integral to the endoplasmic reticulum (ER) and involved in ER-associated degradation (ERAD) of diverse substrates. Here we report that expression of gp78 has a causal role in the metastasis of an aggressive human sarcoma and that this prometastatic activity requires the E3 activity of gp78. Further, gp78 associates with and targets the transmembrane metastasis suppressor, KAI1 (also known as CD82), for degradation. Suppression of gp78 increases KAI1 abundance and reduces the metastatic potential of tumor cells, an effect that is largely blocked by concomitant suppression of KAI1. An inverse relationship between these proteins was confirmed in a human sarcoma tissue microarray. Whereas most previous efforts have focused on genetic mechanisms for the loss of metastasis suppressor genes, our results provide new evidence for post-translational downregulation of a metastasis suppressor by its ubiquitin ligase, resulting in abrogation of its metastasis-suppressing effects.     

Role of plant growth promoting Bacteria (PGPRs) as biocontrol agents of Meloidogyne incognita through improved plant defense of Lycopersicon esculentum

Plant and Soil - As a major plant-derived soil organic carbon (SOC) component, lignin-derived phenolic compounds show varying biogeochemical characteristics compared to plant-derived lipid...     

Prevalence and Causes of Blindness and Visual Impairment and Their Associated Risk Factors,in Three Tribal Areas of Andhra Pradesh,India

Objective

To assess the prevalence of blindness and visual impairment (VI), their associated causes and underlying risk factors in three tribal areas of Andhra Pradesh, India and compare this data in conjunction with data from other countries with low and middle income settings.

Methods

Using a validated Rapid Assessment of Avoidable Blindness methodology, a two stage sampling survey was performed in these areas involving probability proportionate to size sampling and compact segment sampling methods. Blindness, VI and severe visual impairment (SVI) were defined as per the WHO guidelines and Indian definitions.

Results

Based on a prior enumeration, 7281 (97.1%) subjects were enrolled (mean age  = 61.0+/−7.9 years). Based on the presenting visual acuity (PVA), the prevalences of VI, SVI and blindness were 16.9% (95% CI: 15.7–18.1), 2.9% (95% CI: 2.5–3.4), and 2.3% (95% CI: 1.9–2.7), respectively. When based on the Pinhole corrected visual acuity (PCVA), the prevalences were lower in VI (6.2%, 95% CI: 5.4–6.9), SVI (1.5%, 95% CI: 1.2–1.9) and blindness (2.1%, 95% CI: 1.7–2.5). Refractive error was the major cause of VI (71.4%), whereas, cataract was the major cause of SVI and blindness (70.3%). Based on the PVA, the odds ratio (OR) of blindness increased in the age groups of 60–69 years (OR = 3.8, 95% CI: 2.8, 5.1), 70–79 years (OR = 10.6, 95% CI: 7.2, 15.5) and 80 years and above (OR = 30.7, 95% CI: 19.2, 49). The ORs were relatively higher in females (OR = 1.3, 95% CI: 1.0, 1.6) and illiterate subjects (OR = 4.3, 95% CI: 2.2, 8.5), but lower in those wearing glasses (OR = 0.2, 95% CI: 0.1, 0.4).

Conclusions

This is perhaps the first study to assess the prevalence of blindness and VI in these tribal regions and the majority of the causes of blindness and SVI were avoidable (88.5%). These findings may be useful for planning eye care services in these underserved regions.     

Carbamazepine Potentiates the Effectiveness of Morphine in a Rodent Model of Neuropathic Pain

Approximately 60% of morphine is glucuronidated to morphine-3-glucuronide (M3G) which may aggravate preexisting pain conditions. Accumulating evidence indicates that M3G signaling through neuronal Toll-like receptor 4 (TLR4) may be central to this proalgesic signaling event. These events are known to include elevated neuronal excitability, increased voltage-gated sodium (NaV) current, tactile allodynia and decreased opioid analgesic efficacy. Using an in vitro ratiometric-based calcium influx analysis of acutely dissociated small and medium-diameter neurons derived from lumbar dorsal root ganglion (DRG), we observed that M3G-sensitive neurons responded to lipopolysaccharide (LPS) and over 35% of these M3G/LPS-responsive cells exhibited sensitivity to capsaicin. In addition, M3G-exposed sensory neurons significantly increased excitatory activity and potentiated NaV current as measured by current and voltage clamp, when compared to baseline level measurements. The M3G-dependent excitability and potentiation of NaV current in these sensory neurons could be reversed by the addition of carbamazepine (CBZ), a known inhibitor of several NaV currents. We then compared the efficacy between CBZ and morphine as independent agents, to the combined treatment of both drugs simultaneously, in the tibial nerve injury (TNI) model of neuropathic pain. The potent anti-nociceptive effects of morphine (5 mg/kg, i.p.) were observed in TNI rodents at post-injury day (PID) 7–14 and absent at PID21–28, while administration of CBZ (10 mg/kg, i.p.) alone failed to produce anti-nociceptive effects at any time following TNI (PID 7–28). In contrast to either drug alone at PID28, the combination of morphine and CBZ completely attenuated tactile hyperalgesia in the rodent TNI model. The basis for the potentiation of morphine in combination with CBZ may be due to the effects of a latent upregulation of NaV1.7 in the DRG following TNI. Taken together, our observations demonstrate a potential therapeutic use of morphine and CBZ as a combinational treatment for neuropathic pain.     

The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid Alpha-Glucosidase,and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease

Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.     

Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen,EBNA1

Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to therapeutic development through the use of anti-sense strategies or small molecules targeting EBNA1 mRNA structure.