There is an obvious and urgent need for novel approaches to treat infectious diseases. The use of monoclonal antibodies in therapy of infectious diseases is now experiencing renewed interest. During the last 5 years radioimmunotherapy (RIT), a modality previously developed only for cancer treatment, has been successfully adapted for the treatment of experimental fungal, bacterial, and viral infections. As our model organism for studying the efficacy, mechanisms, potential toxicity, and radioresistance to RIT, as well as for comparison of RIT with the existing antimicrobial therapies we have chosen the encapsulated yeast Cryptococcus neoformans (CN). The success of RIT approach in laboratory studies provides encouragement for feasibility of therapeutically targeting microbes with labeled antibodies. In addition, the creation of “panantibodies” for RIT which would recognize antigens shared by the whole class of pathogens such as fungi, for example, would facilitate the introduction of RIT into the clinic.
Radioimmunotherapy is the targeted delivery of cytocidal radiation to cells via specific antibody. Although mature for the treatment of cancer, RIT of infectious diseases is in pre-clinical development. However, as there is an obvious and urgent need for novel approaches to treat infectious diseases, RIT can provide us with a powerful approach to combat serious diseases, including invasive fungal infections. For example, RIT has proven more effective than standard amphotericin B for the treatment of experimental cryptococcosis. This review will discuss the concepts of RIT, its applications for infectious diseases, and the strides made to date to bring RIT of infectious diseases to fruition. Finally, we will discuss the potential of PAN-FUNGAL RIT, the targeting of conserved fungal cell surface antigens by RIT, as a treatment modality for fungi prior to the formal microbiological identification of the specific pathogen. In sum, RIT provides a mechanism for the targeted killing of drug susceptible or resistant fungi irrespective of the host immune status and may dramatically reduce the length of therapy currently required for many invasive fungal diseases.
Histoplasma capsulatum; Cryptococcus neoformans; Candida albicans; antibody; heat shock protein 60; beta-glucan; melanin
RNA interference (RNAi) is critical for the assembly of heterochromatin at fission yeast centromeres. Central to this process is the RNA-induced Initiation of Transcriptional gene Silencing (RITS) complex, which physically anchors small non-coding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which bridges between Chp1 and Ago1. Chp1 is a large protein with, apart from its chromodomain, no recognizable domains. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing Chp1's tight embrace of Tas3. The structure also reveals a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1-Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.
The concept of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work 1,2, and the drugs that emerged remain in use for treating Human African Trypanosomiasis (HAT) 3. Ehrlich recognised the importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies, but these mechanisms have remained largely mysterious. Here, we use all five current HAT drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling identifies both known drug importers 4,5 and the only known pro-drug activator 6, and links more than fifty additional genes to drug action. A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while the AP-1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and multiple putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of anti-trypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented levels of molecular insight into the mode of action of anti-trypanosomal drugs.
DFMO; eflornithine; ISG75; nifurtimox; RNAi
RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.
The need for novel approaches to treat infectious diseases is obvious and urgent. This situation has renewed interest in using monoclonal antibodies (mAbs) in therapy of infectious diseases. During the last 5 years radioimmunotherapy (RIT), a modality developed for cancer treatment, has been successfully adapted for the treatment of experimental fungal (C. neoformans and H. capsulatum), bacterial (S. pneumoniae and B. anthracis) and viral (HIV-1) infections. RIT produced none or only transient hematological toxicity in experimental animals. Investigation of radiobiological mechanisms of RIT of infections showed that microbial cells are killed by both "direct hit" and "cross-fire" radiation. MAbs radiolabeled with either alpha- or beta-emitters stimulated apoptosis-like cell death, while only mAbs radiolabeled with alpha-emitter 213Bi also decreased the metabolic activity of microbial cells. The success of this approach in laboratory studies combined with earlier nuclear medicine experience on pre-clinical and clinical studies utilizing radiolabeled organism-specific antibodies for imaging of infections provides encouragement for feasibility of therapeutically targeting microbes with labeled antibodies. We envision that first the organism-specific mAbs will be radiolabeled with imaging radionuclides such as 99mTc or 111In to localize the sites of infection with SPECT followed by RIT with 188Re- or 90Y-labeled mAb, respectively. Also, immunoPET might be utilized for imaging of infection before treatment if such positron-emitting radionuclides as 86Y (matching pair for 90Y) or 124I (matching pair for 131I) are available. It might be possible to create a so-called “pan-antibody” which would recognize an antigen shared by a particular class of human pathogens such as fungi, for example. The availability of such antibodies would eliminate the necessity of having antibodies specific for each particular microorganism and would enormously enhance the development of RIT of infectious diseases.
Recombinant immunotoxins (RIT) are targeted anti-cancer agents that are composed of a targeting antibody fragment and a protein toxin fragment. SS1P is a RIT that targets mesothelin on the surface of cancer cells and is being evaluated in patients with mesothelioma. Mesothelin, like many other target antigens, is shed from the cell surface. However, whether antigen shedding positively or negatively affects the delivery of RIT remains unknown. In this study, we used experimental data with SS1P to develop a mathematical model that describes the relationship between tumor volume changes and the dose level of the administered RIT, while accounting for the potential effects of antigen shedding. We found that antigen shedding is a favorable biological process for targeted therapy of solid tumors. Shed antigens acted as a protective reservoir of RIT and buffered against the well-known binding site barrier effect, promoting a more uniform distribution of RIT in the tumor.
In addition, our model reproduced the decrease in tumor size upon RIT treatment in animal experiments. Our findings therefore can be used to study the delivery efficacy of RITs and also antibody drug conjugates currently in clinical trials.
mathematical model; antigen shedding; immunoconjugate delivery
Bacterial Rho-independent terminators (RITs) are important genomic landmarks involved in gene regulation and terminating gene expression. In this investigation we present RNIE, a probabilistic approach for predicting RITs. The method is based upon covariance models which have been known for many years to be the most accurate computational tools for predicting homology in structural non-coding RNAs. We show that RNIE has superior performance in model species from a spectrum of bacterial phyla. Further analysis of species where a low number of RITs were predicted revealed a highly conserved structural sequence motif enriched near the genic termini of the pathogenic Actinobacteria, Mycobacterium tuberculosis. This motif, together with classical RITs, account for up to 90% of all the significantly structured regions from the termini of M. tuberculosis genic elements. The software, predictions and alignments described below are available from http://github.com/ppgardne/RNIE.
Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery. In this study, a series of Rit effector loop mutants was generated to test the importance of these cellular targets to Rit-mediated neuronal differentiation. We find that Rit-mediated neuritogenesis is dependent upon MEK/ERK MAP kinase signaling but independent of RalGEF activation. In addition, in vivo binding studies identified a novel mechanism of Par6 interaction, suggesting that the cell polarity machinery may serve to spatially restrict Rit signaling.
Neuronal differentiation; PC12 cell; Rit; GTPase; Ras; ERK MAP kinase; Par6
Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1''-(beta-O-ribofuranosyl-5''-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.
Radioimmunotherapy (RIT) combines the mechanism of action and targeting capability of monoclonal antibodies with the tumoricidal effect of radiation and has shown promising results in the treatment of various hematologic malignancies. Based on RIT’s efficacy and safety profile, many investigators have evaluated its use in transplant conditioning regimens with the goal of improving long-term disease control with limited toxicity. In lymphoma, two basic transplant approaches targeting CD20 have emerged: 1. Myeloablative doses of RIT with or without chemotherapy, and 2. Standard non-myeloablative doses of RIT combined with high-dose chemotherapy. Myeloablative RIT has been shown to be feasible and efficacious using escalated doses of I-131-Tositumomab (Bexxar), Y-90-ibritumomab tiuxetan (Zevalin), and I-131-rituximab with or without chemotherapy followed by autologous stem cell transplant (ASCT). The second approach predominantly has used standard doses of Y-90-ibritumomab tiuxetan or I-131 Tositumomab plus BEAM chemotherapy followed ASCT. RIT targeting CD-45, CD-33 and CD-66 prior to allogeneic transplantation has also been evaluated for the treatment of acute leukemia. Overall RIT-based transplant conditioning for lymphoma and leukemia has been shown to be safe, effective, and feasible with ongoing randomized trials currently underway to definitively establish its place in the treatment of hematologic malignancies.
Radioimmunotherapy; stem cell transplantation; CD20; CD45; I-131; Y-90
Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent “priRNAs.” The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3WG) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi–independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4+ in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi–independent factors in the assembly of heterochromatin.
Centromeres are the chromosomal regions that promote chromosome movement during cell division. They consist of repetitive DNA sequences that are packaged into heterochromatin. Disruption of centromeric heterochromatin leads to chromosome loss that can result in miscarriages and genetic disorders. We have sought to define the precise steps leading to heterochromatin assembly using fission yeast as the model system. To accomplish this we employed our novel Tas3WG mutant strain that can propagate preassembled heterochromatin but cannot support its de novo establishment. Current models suggest that small RNAs initiate heterochromatin assembly by targeting the RNAi machinery and subsequently the Clr-C chromatin-modifying complex to the centromere. Here, we demonstrate that transient depletion of components of the RNAi pathway that generate or bind small RNAs does not perturb heterochromatin assembly in our Tas3WG strain. Instead, transient depletion of the Clr-C complex blocks heterochromatin assembly, suggesting a critical role for continuous Clr-C activity during heterochromatin assembly in Tas3WG cells. We have directly tested whether Clr-C can target centromeres when expressed in cells deficient for RNAi and Clr-C. We find that RNAi–independent recruitment of Clr-C can occur and likely contributes to the critical initiating mechanisms of heterochromatin assembly.
RitR (formerly RR489) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that has been shown to be required for lung pathogenicity. In the present study, by using the rough strain R800, inactivation of the orphan response regulator gene ritR by allele replacement reduced pathogenicity in a cyclophosphamide-treated mouse lung model but not in a thigh model, suggesting a role for RitR in regulation of tissue-specific virulence factors. Analysis of changes in genome-wide transcript mRNA levels associated with the inactivation of ritR compared to wild-type cells was performed by the use of high-density DNA microarrays. Genes with a change in transcript abundance associated with inactivation of ritR included piuB, encoding an Fe permease subunit, and piuA, encoding an Fe carrier-binding protein. In addition, a dpr ortholog, encoding an H2O2 resistance protein that has been shown to reduce synthesis of reactive oxygen intermediates, was activated in the wild-type (ritR+) strain. Microarray experiments suggested that RitR represses Fe uptake in vitro by negatively regulating the Piu hemin-iron transport system. Footprinting experiments confirmed site-specific DNA-binding activity for RitR and identified three binding sites that partly overlap the +1 site for transcription initiation upstream of piuB. Transcripts belonging to other gene categories found to be differentially expressed in our array studies include those associated with (i) H2O2 resistance, (ii) repair of DNA damage, (iii) sugar transport and capsule biosynthesis, and (iv) two-component signal transduction elements. These observations suggest that RitR is an important response regulator whose primary role is to maintain iron homeostasis in S. pneumoniae. The name ritR (repressor of iron transport) for the orphan response regulator gene, rr489, is proposed.
Rit knockout mice and D-Ric null Drosophila were used to identify the Rit/RIC subfamily of Ras-related GTPases as regulators of an evolutionarily conserved, p38-dependent signaling cascade that functions as a survival mechanism for cells in response to reactive oxygen species exposure.
Ras-related small GTP-binding proteins control a wide range of cellular processes by regulating a variety of effector pathways, including prominent roles in the control of mitogen-activated protein kinase (MAPK) cascades. Although the regulatory role(s) for many Ras family GTPases are well established, the physiological function for the Rit/Rin subfamily has been lacking. Here, using both knockout mice and Drosophila models, we demonstrate an evolutionarily conserved role for Rit subfamily GTPases (mammalian Rit and Rin, and the Drosophila RIC homologue) in governing survival in response to oxidative stress. Primary embryonic fibroblasts derived from Rit knockout mice display increased apoptosis and selective disruption of MAPK signaling following reactive oxygen species (ROS) exposure but not in response to endoplasmic reticulum stress or DNA damage. These deficits include a reduction in ROS-mediated stimulation of a p38-MK2-HSP27 signaling cascade that controls Akt activation, directing Bad phosphorylation to promote cell survival. Furthermore, D-RIC null flies display increased susceptibility to environmental stresses and reduced stress-dependent p38 signaling, extending the Rit-p38 survival pathway to Drosophila. Together, our studies establish the Rit GTPases as critical regulators of an evolutionarily conserved, p38 MAPK–dependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress.
Recombinant immunotoxins (RITs) are anti-cancer agents that combine the Fv of an antibody against cancer cells with a protein toxin from bacteria or plants. Since RITs contain a non-human protein, immunogenicity can be an obstacle in their development. In this study, we have explored the hypothesis that increasing stability can reduce the immunogenicity of a RIT using HA22-LR, which is composed of an anti-CD22 Fv fused to domain III of Pseudomonas exotoxin A. We introduced a disulfide bond into domain III by identifying and mutating two structurally adjacent residues to cysteines at sites suggested by computer modeling. This RIT, HA22-LR-DB, displays a remarkable increase in thermal stability and an enhanced resistance to trypsin degradation. In addition, HA22-LR-DB retains cytotoxic and anti-tumor activity, while exhibiting significantly lower immunogenicity in mice. This study demonstrates that it is possible to design mutations in a protein molecule that will increase the stability of the protein and thereby reduce its immunogenicity.
disulfide bond; immunotoxin; immunogenicity
In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.
► Stc1 is required for RNAi-dependent heterochromatin formation in fission yeast ► Stc1 associates with both RNAi and chromatin modification subunits ► Stc1 localizes to centromeric heterochromatin, dependent on RNAi ► Tethering Stc1 to DNA induces heterochromatin formation independently of RNAi
Cytoreductive surgery (CS) followed by heated intraperitoneal chemotherapy (HIPEC) is considered the standard of care for the treatment of patients with peritoneal carcinomatosis (PC) of colorectal cancer (CRC). These surgical procedures result in a median survival of 2 years at the cost of considerable morbidity and mortality. In preclinical studies, radioimmunotherapy (RIT) improved survival after CS in a model of induced PC of colonic origin.
In the present studies we aimed to compare the efficacy and toxicity of CS followed by adjuvant RIT in experimental PC to the standard of care, HIPEC.
PC was induced by intraperitoneal inoculation of CC-531 colon carcinoma cells in three groups of Wag/Rij rats. Treatment comprised CS only, CS + RIT or CS + HIPEC, immediately after surgery. RIT consisted of intraperitoneal administration of 74 MBq Lutetium-177 labeled MG1. HIPEC was performed by a closed abdomen perfusion technique using mitomycin C (16 mg/L during 60 minutes). The primary endpoint was survival.
CS only or combined with RIT was well tolerated. Rats receiving CS + HIPEC were lethargic, suffered from diarrhea, and lost significantly more weight in the first postoperative week. Median survival of rats treated with CS + RIT was significantly longer than after CS alone (97 and 57 days, respectively, P < .004), whereas survival after CS + HIPEC or CS alone were not significantly different (76 and 57 days, respectively, P = .17).
Survival after CS was significantly improved by RIT with Lutetium-177-MG1 in rats with PC of colorectal origin. Adjuvant HIPEC did not improve survival and was more toxic than adjuvant RIT.
Radioimmunotherapy; Cytoreductive surgery; Heated intraperitoneal chemotherapy; Peritoneal carcinomatosis; Colon cancer; Adjuvant
Panitumumab (ABX-EGF or Vectibix), the first fully human monoclonal antibody targeting epidermal growth factor receptor (EGFR), was approved by the Food and Drug Administration for treatment of patients with metastatic colorectal cancer. Here, we report for the first time the radioimmunotherapy (RIT) of EGFR-positive human head and neck cancer in a nude mouse model using pure β− emitter 90Y-labeled panitumumab. Biodistribution and planar γ-imaging studies were carried out with 111In-DOTA-panitumumab. The RIT efficacy of 90Y-DOTA-panitumumab was evaluated in UM-SCC-22B tumor model. CD31, Ki67, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, and H&E staining were done on UM-SCC-22B tumor sections after treatment. The tumor uptake of 111In-DOTA-panitumumab in UM-SCC-22B tumor-bearing nude mice was 26.10 ± 4.93, 59.11 ± 7.22, 44.57 ± 9.80, 40.38 ± 7.76, and 14.86 ± 7.23 % injected dose per gram of tissue at 4, 24, 72, 120, and 168 hours after injection, respectively. Immunotherapy with cold panitumumab (four doses of 10 mg/kg) did not cause significant antitumor effect. RIT with a single dose of 100 μCi 90Y-DOTA-panitumumab caused significant tumor growth delay and improved the survival in UM-SCC-22B tumor model. A single dose of 200 μCi 90Y-DOTA-panitumumab led to almost complete tumor regression (tumor volumes were 34.83 ± 11.11 mm3 and 56.02 ± 39.95 mm3 on days 0 and 46 after treatment, respectively). Histopathologic analysis of tumors and normal organs further validated the therapeutic efficacy and limited systemic toxicity of 90Y-DOTA-panitumumab. The high tumor uptake and prolonged tumor retention, as well as effective therapy, reveal that 90Y-DOTA-panitumumab may be a promising radioimmunotherapeutic agent to treat EGFR-positive solid tumors.
The success of immunotoxin therapy of cancer is limited by host production of neutralizing antibodies, which are directed toward the Pseudomonas exotoxin A (PE) component. In this proof-of-principle study using a well-established murine model, we hypothesized that a newly developed immune depletion regimen consisting of pentostatin plus cyclophosphamide would abrogate anti-immunotoxin reactivity.
BALB/c hosts were injected weekly with recombinant immunotoxin (RIT) SS1P, which is an anti-mesothelin Fv antibody fragment genetically fused to a 38 kDa portion of PE, and has been evaluated in clinical trials. Experimental cohorts received induction chemotherapy consisting of pentostatin (P) plus cyclophosphamide (C) prior to initial RIT exposure; some cohorts received further maintenance PC therapy of varying intensity just prior to each weekly RIT challenge. Cohorts were monitored for T, B, and myeloid cell depletion and for total anti-SS1P antibody (Ab) formation.
Controls uniformly developed anti-SS1P Ab after the third RIT exposure. Induction PC therapy reduced the frequency of hosts with anti-SS1P Ab. Abrogation of antibody generation was improved by maintenance PC therapy: nearly 100% of recipients of intensive PC maintenance were free of anti-SS1P Ab after 9 weekly RIT doses. The most effective PC regimen yielded the greatest degree of host B cell depletion, moderate T cell depletion, and minimal myeloid cell depletion.
Induction and maintenance PC chemotherapy safely prevented anti-immunotoxin antibody formation with uniform efficacy. These data suggest that immunotoxin therapy might be used in combination with pentostatin plus cyclophosphamide chemotherapy to improve the targeted therapy of cancer.
adjuvant chemotherapy; combination chemotherapy; pentostatin; cyclophosphamide; immunotoxin
In recent years a bevy of evidence has been unearthed indicating that ‘silent’ heterochromatin is not as transcriptionally inert as once thought. In the unicellular yeast Schizosaccharomyces pombe, processing of transcripts derived from centromeric repeats into homologous small interfering RNA (siRNA) is essential for the formation of centromeric heterochromatin. Deletion of genes required for siRNA biogenesis revealed that core components of the canonical RNAi pathway are essential for centromeric heterochromatin assembly as well as for centromere function. Subsequent purification of the RITS (RNA-induced initiation of transcriptional gene silencing) complex provided the critical link between siRNAs and heterochromatin assembly, with RITS acting as a physical bridge between non-coding RNA scaffolds and chromatin. Here, we review current understanding of how RITS promotes heterochromatin formation and how it participates in transcription coupled silencing.
The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway.
Bismuth-213 (213Bi) (physical half-life 46 min) is a beta-emitter (97%) and an alpha-emitter (3%) which decays to short lived alpha-emitter Polonium-213 and could therefore be used as an in vivo generator of alpha particles with the energy of around 8 MeV. 213Bi has been successfully used during the last decade in both clinical and pre-clinical work for radioimmunotherapy (RIT) of cancer with 213Bi-labeled monoclonal antibodies (mAbs). RIT has been proposed as a novel techonology for treatment of infectious diseases. 213Bi-labeled mAbs have been successfully used for treatment of experimental fungal, bacterial and viral infections with transient or none hematologic toxicity. The mechanisms of RIT of infection with 213Bi-labeled mAbs include “direct” killing of cells and induction of apoptosis. In vivo RIT results in decrease of inflammation in infected organs. Among the delivery vehicles for RIT of infection whole IgG1 mAbs seem to be the most suitable in terms of the highest uptake in the target organs and the lowest - in normal tissues. RIT with alpha-emitter 213Bi involves the application of established technology developed for the treatment of malignancies to infectious diseases. The development of RIT for infectious diseases is potentially easier than its application to tumor therapy given antigenic and tissue perfusion differences between sites of microbial infection and tumor infiltration. Nevertheless, considerable pre-clinical and clinical development work is likely to be required to learn how to use RIT for infection optimally.
Bismuth-213; radioimmunotherapy; fungal infection; bacterial infection; viral infection; apoptosis; radiobiological mechanisms; inflammation
The 3' terminal forty nucleotides of tobraviral RNAs readily fold into a tertiary structure, resembling that of tymo- and tobamoviral RNAs. The latter RNAs possess a tRNA-like structure at their 3' end that is recognized by a number of tRNA-specific enzymes (Rietveld et al. (1984), EMBO J. 3, 2613-2619). Characteristic for their aminoacyl acceptor arm is the presence of a so-called pseudoknot which we now also find in a corresponding position at the 3' terminus of TRV RNA2 (PSG strain). The nucleotide sequences of all tobraviral RNAs analysed so far indicate that they all possess a similar 3' terminal structure. A domain resembling the anticodon arm of canonical tRNA is not readily recognizable. TRV RNA2 can be adenylated with CTP, ATP; tRNA nucleotidyl transferase and ATP. It is unable, however, to accept any of the twenty common amino acids when incubated with ATP and aminoacyl-tRNA synthetases from wheat germ or yeast. We conclude that TRV RNA contains a tRNA-like structure, which, in contrast to the tymo- and tobamoviral tRNA-like structures, cannot be aminoacylated. It is unlikely therefore, that aminoacylation of plant viral RNAs with a tRNA-like structure is a prerequisite for viral RNA replication.
To report our experience on disease control and functional outcome using three modern combined-modality approaches for definitive radiochemotherapy of locally advanced SCCHN with modern radiotherapy techniques: radiochemotherapy (RChT), radioimmunotherapy (RIT) with cetuximab, or induction chemotherapy with docetaxel, cisplatin, and 5-FU (TPF) combined with either RChT or RIT.
Toxicity and outcome was retrospectively analysed in patients receiving definitive RChT, RIT, or induction chemotherapy followed by RChT or RIT between 2006 and 2009. Outcome was estimated using Kaplan-Meier analyses, toxicity was analysed according to CTCAE v 3.0.
Thirty-eight patients were treated with RChT, 38 patients with RIT, 16 patients received TPF followed by either RChT or RIT. Radiotherapy was mostly applied as IMRT (68%). Long-term toxicity was low, only one case of grad III dysphagia requiring oesophageal dilatation, no case of either xerostomia ≥ grade II or cervical plexopathy were observed. Median overall survival (OS) was 25.7 months (RChT) and 27.7 months (RIT), median locoregional progression-free survival (PFS) was not reached yet. Subgroup analysis showed no significant differences between TPF, RChT, and RIT despite higher age and co-morbidities in the RIT group. Results suggested improved OS, distant and overall PFS for the TPF regimen.
Late radiation effects in our cohort are rare. No significant differences in outcome between RChT and RIT were observed. Adding TPF suggests improved progression-free and overall survival, impact of TPF on locoregional PFS was marginal, therefore radiotherapeutic options for intensification of local treatment should be explored.
The proinflammatory cytokine interferon-γ (IFNγ) alters neuronal connectivity via selective regressive effects on dendrites but the signaling pathways that mediate this effect are poorly understood. We recently demonstrated that signaling by Rit, a member of the Ras family of GTPases, modulates dendritic growth in primary cultures of sympathetic and hippocampal neurons. In this study we investigated a role for Rit signaling in IFNγ-induced dendritic retraction. Expression of a dominant negative Rit mutant inhibited IFNγ-induced dendritic retraction in cultured embryonic rat sympathetic and hippocampal neurons. In pheochromacytoma cells and hippocampal neurons, IFNγ caused rapid Rit activation as indicated by increased GTP binding to Rit. Silencing of Rit by RNA interference suppressed IFNγ-elicited activation of p38 MAP kinase in pheochromacytoma cells, and pharmacological inhibition of p38 MAP kinase significantly attenuated the dendrite-inhibiting effects of IFNγ in cultured sympathetic and hippocampal neurons without altering STAT1 activation. These observations identify Rit as a downstream target of IFNγ and suggest that a novel IFNγ-Rit-p38 signaling pathway contributes to dendritic retraction and may, therefore, represent a potential therapeutic target in diseases with a significant neuroinflammatory component.
dendrite retraction; hippocampal neuron; interferon-γ; p38 MAP kinase; Rit; sympathetic neuron