Possible temporal relationship between SARS-CoV-2 infection and anti-NMDA receptor encephalitis: a meta-analysis.

The global impact of SARS-CoV-2 infection has raised concerns about secondary diseases beyond acute illness. This review explores the significance and potential underlying mechanisms of how SARS-CoV-2 infection might elicit an immune response targeting N-methyl-D-aspartate (NMDA) receptors, and its implications for autoimmune-driven neuropsychiatric manifestations. We identified 19 published case reports of NMDA receptor encephalitis associated with SARS-CoV-2 infection or vaccination by a systematic literature search. The significance of these reports was limited since it is not clear if a coincidental or causal relationship exists between SARS-CoV-2 infection or vaccination and manifestation of NMDA receptor encephalitis. The included studies were hampered by difficulties in establishing if these patients had pre-existing NMDA receptor antibodies which entered the brain by infection- or vaccination-associated transient blood-brain barrier leakage. In addition, four cases had comorbid ovarian teratoma, which is a known trigger for development of NMDA receptor encephalitis. Considering that billions of people have contracted COVID-19 or have been vaccinated against this virus, the publication of only 19 case reports with a possible link to NMDA receptor encephalitis, indicates that it is rare. In conclusion, these findings do not support the case that SARS-CoV-2 infection or vaccination led to an increase of existing or de novo encephalitis mediated by an autoimmune response targeting NMDA receptor function. Nevertheless, this work underscores the importance of ongoing vigilance in monitoring viral outbreaks and their potential impact on the central nervous system through basic, epidemiological and translational research.

Glycosylation of bacterial antigens changes epitope patterns.

Introduction: Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines. Methods: Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level. Results: The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination. Discussion: This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

Fast impedimetric immunosensing of IgGs associated with peanut and hazelnut allergens.

Food allergies trigger a variety of clinical adverse symptoms and clinical evidence suggests that the presence of food allergy-related IgG can be helpful in the diagnosis when analyzed at the peptide-epitope level. To validate and select the peptides based on their specificity toward hazelnut or peanut epitopes, the authors of this study developed a silicon-based microchip coupled with click-chemistry bound peptides identified by the Fraunhofer Institute for Cell Therapy and Immunology. Peptides related to hazelnut and peanut allergies were identified and used to develop a silicon-based microchip. Peptides were coupled with click-chemistry to the sensor surface. The immunosensor was developed by electrografting diazotized amino phenylacetic acid and subsequently, dibenzocyclooctyne-amine (DBCO-NH2) was used as click-chemistry to allow coupling of the peptides with a C-terminal linker and azide structure. Energy-dispersive X-ray spectroscopy, electrochemical impedance spectroscopy (EIS), and fluorescence microscopy techniques have been used to analyze the bio-functionalization of the developed electrode. The peptide-epitope recognition was studied for seven allergen-derived peptides. The electrochemical responses were studied with sera from rabbits immunized with hazelnut and peanut powder. The microchips functionalized with the chosen peptides (peanut peptides T12 and EO13 and hazelnut peptides S4 and EO14 with an RSD of 4%, 3%, 9%, and 1% respectively) demonstrated their ability to specifically detect prevalent anti-nut related IgGs in rabbit sera in a range of dilutions from 1:500000 (0.0002%) until 1:50000 (0.002%). In addition, the other peptides showed promising differentiation abilities which can be further studied to perform multivariable detection fingerprint of anti-allergens in blood sera.

Detection of Antibodies against Endemic and SARS-CoV-2 Coronaviruses with Short Peptide Epitopes.

(1) Background: Coronavirus proteins are quite conserved amongst endemic strains (eCoV) and SARS-CoV-2. We aimed to evaluate whether peptide epitopes might serve as useful diagnostic biomarkers to stratify previous infections and COVID-19. (2) Methods: Peptide epitopes were identified at an amino acid resolution that applied a novel statistical approach to generate data sets of potential antibody binding peptides. (3) Results: Data sets from more than 120 COVID-19 or eCoV-infected patients, as well as vaccinated persons, have been used to generate data sets that have been used to search in silico for potential epitopes in proteins of SARS-CoV-2 and eCoV. Peptide epitopes were validated with >300 serum samples in synthetic peptide micro arrays and epitopes specific for different viruses, in addition to the identified cross reactive epitopes. (4) Conclusions: Most patients develop antibodies against non-structural proteins, which are useful general markers for recent infections. However, there are differences in the epitope patterns of COVID-19, and eCoV, and the S-protein vaccine, which can only be explained by a high degree of cross-reactivity between the viruses, a pre-existing immune response against some epitopes, and even an alternate processing of the vaccine proteins.

Peptide epitopes as biomarkers of soya sensitization in rBet v 1 immunotherapy of birch-related soya allergy.

BACKGROUND: There are no diagnostic and/or prognostic markers of the treatment outcome in patients receiving allergen immunotherapy (AIT). Although numerous allergen epitopes are known, their value in this context has not been investigated. This paper deals with re-evaluation of sera from patients who underwent AIT against rBet v 1 for treatment of their soya allergy (BASALIT trial). OBJECTIVE: To evaluate the diagnostic and/or prognostic potential of allergen epitopes recognition by antibodies from patients with birch-related soya allergy before and after rBet v 1-immunotherapy. METHODS: PR-10 epitope-binding profiles from 34 patients were identified in silico using a statistical peptide phage display at start and at end of AIT. IgE- and IgG-binding to these peptide epitopes was measured in peptide microarrays. Clinical relevance of epitopes was evaluated by comparing these measurements to a number of treatment outcome measures recorded during double-blind placebo-controlled food challenge at start and end of AIT. RESULTS: We showed that IgG- and IgE-recognition of peptide epitopes after AIT were surrogate markers of 5 out of 12 analysed treatment outcome measures using this patient cohort. Seven epitopes were identified from multiple PR-10 allergen sequences. Twenty-six peptide epitopes were used for IgG and IgE measurements. IgE-binding to one of the epitopes was associated with stronger intensity of oral tingling/itching after ingesting soya at start of AIT. IgG recognizing two other epitopes at start of AIT could predict decreased Cor a 1-specific IgE concentration (p = .043) and decreased lip swelling intensity (p = .016) after AIT. Tolerance to increasing amounts of soy at food challenge correlated with IgG-binding to another epitope at start of AIT (p = .046). CONCLUSION: IgG- and IgE-binding to peptide epitopes in PR-10 is a potential indicator of the outcome and clinical course of AIT of soya-sensitized patients with rBet v 1.

Enzymatic Hydrolysis and Fermentation of Pea Protein Isolate and Its Effects on Antigenic Proteins, Functional Properties, and Sensory Profile.

Combinations of enzymatic hydrolysis using different proteolytic enzymes (papain, Esperase®, trypsin) and lactic fermentation with Lactobacillus plantarum were used to alter potential pea allergens, the functional properties and sensory profile of pea protein isolate (PPI). The order in which the treatments were performed had a major impact on the changes in the properties of the pea protein isolate; the highest changes were seen with the combination of fermentation followed by enzymatic hydrolysis. SDS-PAGE, gel filtration, and ELISA results showed changes in the protein molecular weight and a reduced immunogenicity of treated samples. Treated samples showed significantly increased protein solubility at pH 4.5 (31.19-66.55%) and at pH 7.0 (47.37-74.95%), compared to the untreated PPI (6.98% and 40.26%, respectively). The foaming capacity was significantly increased (1190-2575%) compared to the untreated PPI (840%). The treated PPI showed reduced pea characteristic off-flavors, where only the treatment with Esperase® significantly increased the bitterness. The results from this study suggest that the combination of enzymatic hydrolysis and lactic fermentation is a promising method to be used in the food industry to produce pea protein ingredients with higher functionality and a highly neutral taste. A reduced detection signal of polyclonal rabbit anti-pea-antibodies against the processed protein preparations in ELISA furthermore might indicate a decreased immunological reaction after consumption.

Microlyse: a thrombolytic agent that targets VWF for clearance of microvascular thrombosis.

Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.

Identification of Seasonal Variations of Antibodies against PR-10-Specific Epitopes Can Be Improved Using Peptide-Phage Display.

BACKGROUND: In pollinosis patients, allergen-specific antibody titers show seasonal variations. Little is known about these variations at the epitope level. OBJECTIVES: We aimed at investigating seasonal variations on the level of allergen epitope recognition in patients with Bet v 1-related food allergy using a peptide phage display approach. METHODS: Serum samples collected over 1 year from 4 patients of the placebo arm of the birch-associated soya allergy immunotherapy trial were included. To identify epitopes from Bet v 1-related food allergens, patient sera were used in peptide phage display experiments. In silico analysis of enriched allergen-related motifs was performed. RESULTS: We identified epitope motifs related to Bet v 1 and its homologs in soya and hazelnut (Gly m 4 and Cor a 1, respectively) that were enriched in accordance with birch and hazel pollen exposure. Within several weeks after the birch pollen season peak, the pattern of identified epitope motifs differed considerably among patients. Data for amino acid preferences in homologous Bet v 1 and Cor a 1 epitope motifs identified for one of the investigated patients suggest changes in concentration or specificity of serum antibodies for the Cor a 1 epitope motif. CONCLUSIONS: Peptide phage display data suggest an impact of birch and hazel pollen exposure on the recognition pattern of Bet v 1-like allergen epitopes. Epitope-oriented analyses could provide deeper, personalized details regarding the allergen epitope recognition influenced by pollen exposure beyond the capability of current methods.

The immunome of soy bean allergy: Comprehensive identification and characterization of epitopes.

BACKGROUND: The precise mapping of multiple antibody epitopes recognized by patients' sera allows a more detailed and differentiated understanding of immunological diseases. It may lead to the development of novel therapies and diagnostic tools. OBJECTIVE: Mapping soy bean specific epitopes relevant for soy bean allergy patients and persons sensitized to soy bean, and analysis of their IgE/IgG binding spectrum. METHODS: Identification of epitopes using sera, applying an optimized peptide phage display library followed by next-generation sequencing, specially designed in silico data analysis and subsequent peptide microarray analysis. RESULTS: We were able to identify more than 400 potential epitope motifs in soy bean proteins. More than 60% of them have not yet been described as potential epitopes. Eighty-three peptides, representing the 42 most frequently found epitope candidates, were validated by microarray analysis using 50 sera from people who have been tested positive in skin prick test (SPT). Of these peptides, 56 were bound by antibodies, 55 by serum IgE, 43 by serum IgG and 30 by both. Person-specific epitope patterns were found for each individual and protein. CONCLUSIONS: For individuals with clinical symptoms, epitope resolved analyses reveal a high prevalence of IgE binding to a few soy bean specific epitopes. Evaluation of individual immune profiles of patients with soy bean sensitization allows the identification of peptides that do facilitate studying individual IgE/IgG epitope binding patterns. This enables discrimination of sensitization from disease, such assay test has the potential to replace SPT assays.

Exome sequencing reveals DNMT3A and ASXL1 variants associate with progression of chronic myeloid leukemia after tyrosine kinase inhibitor therapy.

OBJECTIVE: The development of tyrosine kinase inhibitors (TKIs) has significantly improved the treatment of chronic myeloid leukemia (CML). However, approximately one third of patients are resistant to TKI and/or progress to advanced disease stages. TKI therapy failure has a well-known association with ABL1 kinase domain (KD) mutations, but only around half of TKI non-responders have detectable ABL1 KD mutations. METHOD: We attempt to identify genetic markers associated with TKI therapy failure in 13 patients (5 resistant, 8 progressed) without ABL1 KD mutations using whole-exome sequencing. RESULTS: In 6 patients, we detected mutations in 6 genes commonly mutated in other myeloid neoplasms: ABL1, ASXL1, DNMT3A, IDH1, SETBP1, and TP63. We then used targeted deep sequencing to validate our finding in an independent cohort consisting of 100 CML patients with varying drug responses (74 responsive, 18 resistant, and 8 progressed patients). Mutations in genes associated with epigenetic regulations such as DNMT3A and ASXL1 seem to play an important role in the pathogenesis of CML progression and TKI-resistance independent of ABL1 KD mutations. CONCLUSION: This study suggests the involvement of other somatic mutations in the development of TKI resistant progression to advanced disease stages in CML, particularly in patients lacking ABL1 KD mutations.

Sympathetic nerve repulsion inhibited by designer molecules in vitro and role in experimental arthritis.

AIMS: In rheumatoid arthritis and collagen type II arthritis (CIA), sympathetic nerve fibers get lost in inflamed tissue. The process is probably induced by nerve repellent factors like semaphorin 3F (SEMA3F). Repulsion of sympathetic nerve fibers in inflamed tissue has proinflammatory effects due to the loss of anti-inflammatory neurotransmitters. We hypothesized that design molecules like antibodies and specific peptides that inhibit nerve fiber repulsion can ameliorate CIA. MATERIALS AND METHODS: Two blocking antibodies were used and four blocking peptides were generated using the phage display technique with the targets of SEMA3F and plexin-A2. All blocking molecules were tested in vitro using a sympathetic neurite outgrowth assay. CIA was induced by collagen type II in mice. KEY FINDINGS: In the neurite outgrowth assay, the two antibodies against plexin-A2 and neuropilin-2 as well as the four blocking peptides - two SEMA3F analogous peptides (WLFQRDPGDR, QATVKWLFQRDPGDRR) and two plexin A2 analogous peptides (DSSDQFSFDYELEQN, DSSIQFFSFEKDKERI) - were able to block sympathetic nerve fiber repulsion in vitro (at 150-600nmol/l). Administration of the two antibodies prophylactically on day 4 after immunization did not change clinical CIA. Similarly, using the top candidate antibody to plexin-A2 after CIA onset (mild score of 4 points, maximum=52 points), did not ameliorate CIA. The tested blocking peptides were not recovered in peripheral blood after i.v. and i.p. administration. SIGNIFICANCE: While designer molecules blocked nerve fiber repulsion in vitro, therapeutic administration in vivo did not change CIA. Possible strategies to overcome negative effects demonstrated in vivo are discussed.

Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants.

Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents.

Coexistence of JAK2 and CALR mutations and their clinical implications in patients with essential thrombocythemia.

Janus kinase 2 (JAK2) and calreticulin (CALR) constitute the two most frequent mutations in essential thrombocythemia (ET), and both are reported to be mutually exclusive. Hence, we examined a cohort of 123 myeloproliferative neoplasm (MPN) patients without BCR-ABL1 rearrangement and additional ET patients (n=96) for coexistence of JAK2 and CALR mutations. The frequency of CALR mutations was 20.3% in 123 MPN patients; 31.1% in ET (n=74), 25% in primary myelofibrosis (n=4) and 2.2% in polycythemia vera (n=45). JAK2 and CALR mutations coexisted in 7 (4.2%) of 167 ET patients. Clinical characteristics, progression-free survival (PFS), and elapsed time to achieve partial remission across 4 groups (JAK2+/CALR+, JAK2+/CALR-, JAK2-/CALR+, JAK2-/CALR-) were reviewed. The JAK2+/CALR- group had higher leukocyte counts and hemoglobin levels and more frequent thrombotic events than JAK2-/CALR- group. JAK2 mutations have a greater effect on the disease phenotype and the clinical features of MPN patients rather than do CALR mutation. JAK2+ groups showed a tendency of poor PFS than JAK2- groups regardless of CALR mutation. CALR+ was a predictor of late response to the treatment. Our study also showed that thrombosis was more frequent in ET patients with type 2 CALR mutations than in those with type 1 CALR mutations.

RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy.

Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvß3, but weakly bound to αvß3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvß3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

Clinicopathological Implications of Mitochondrial Genome Alterations in Pediatric Acute Myeloid Leukemia.

BACKGROUND: To the best of our knowledge, the association between pediatric AML and mitochondrial aberrations has not been studied. We investigated various mitochondrial aberrations in pediatric AML and evaluated their impact on clinical outcomes. METHODS: Sequencing, mitochondrial DNA (mtDNA) copy number determination, mtDNA 4,977-bp large deletion assessments, and gene scan analyses were performed on the bone marrow mononuclear cells of 55 pediatric AML patients and on the peripheral blood mononuclear cells of 55 normal controls. Changes in the mitochondrial mass, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were also examined. RESULTS: mtDNA copy numbers were about two-fold higher in pediatric AML cells than in controls (P<0.0001). Furthermore, a close relationship was found between mtDNA copy number tertiles and the risk of pediatric AML. Intracellular ROS levels, mitochondrial mass, and mitochondrial membrane potentials were all elevated in pediatric AML. The frequency of the mtDNA 4,977-bp large deletion was significantly higher (P<0.01) in pediatric AML cells, and pediatric AML patients harboring high amount of mtDNA 4,977-bp deletions showed shorter overall survival and event-free survival rates, albeit without statistical significance. CONCLUSIONS: The present findings demonstrate an association between mitochondrial genome alterations and the risk of pediatric AML.

Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1ß.

Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell populations within tumor tissues colonized by E. coli or attenuated Salmonellae. CT26 tumor-bearing mice were treated with two different bacterial strains: S. typhimurium defective in ppGpp synthesis (ΔppGpp Salmonellae) or wild-type E. coli MG1655. Cytokine profiles and immune cell populations in tumor tissue colonized by these two bacterial strains were examined at two time points based on the pattern of tumor growth after ΔppGpp Salmonellae treatment: 1) when tumor growth was suppressed ('suppression stage') and 2) when they began to re-grow ('re-growing stage'). The levels of IL-1ß and TNF-α were markedly increased in tumors colonized by ΔppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1ß and TNF-α returned to normal level when the tumors started to re-grow. To identify the immune cells primarily responsible for Salmonellae-mediated tumor suppression, we examined the major cell types that produce IL-1ß and TNF-α. We found that macrophages and dendritic cells were the main producers of TNF-α and IL-1ß. Inhibiting IL-1ß production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1ß or TNF-α in conjunction with Salmonella therapy. These findings suggested that IL-1ß and TNF-α play important roles in Salmonella-mediated cancer therapy. A better understanding of host immune responses in Salmonella therapy may increase the success of a given drug, particularly when various strategies are combined with bacteriotherapy.

Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BACKGROUND: The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications. METHODS: Single cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients. RESULTS: Single cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFß super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn't support the prognostic value of the ASCs in AML. CONCLUSIONS: ASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.

Mitochondrial DNA aberrations and pathophysiological implications in hematopoietic diseases, chronic inflammatory diseases, and cancers.

Mitochondria are important intracellular organelles that produce energy for cellular development, differentiation, and growth. Mitochondrial DNA (mtDNA) presents a 10- to 20-fold higher susceptibility to genetic mutations owing to the lack of introns and histone proteins. The mtDNA repair system is relatively inefficient, rendering it vulnerable to reactive oxygen species (ROS) produced during ATP synthesis within the mitochondria, which can then target the mtDNA. Under conditions of chronic inflammation and excess stress, increased ROS production can overwhelm the antioxidant system, resulting in mtDNA damage. This paper reviews recent literature describing the pathophysiological implications of oxidative stress, mitochondrial dysfunction, and mitochondrial genome aberrations in aging hematopoietic stem cells, bone marrow failure syndromes, hematological malignancies, solid organ cancers, chronic inflammatory diseases, and other diseases caused by exposure to environmental hazards.

Engineering of bacteria for the visualization of targeted delivery of a cytolytic anticancer agent.

A number of recent reports have demonstrated that attenuated Salmonella typhimurium are capable of targeting both primary and metastatic tumors. The use of bacteria as a vehicle for the delivery of anticancer drugs requires a mechanism that precisely regulates and visualizes gene expression to ensure the appropriate timing and location of drug production. To integrate these functions into bacteria, we used a repressor-regulated tetracycline efflux system, in which the expression of a therapeutic gene and an imaging reporter gene were controlled by divergent promoters (tetAP and tetRP) in response to extracellular tetracycline. Attenuated S. typhimurium was transformed with the expression plasmids encoding cytolysin A, a therapeutic gene, and renilla luciferase variant 8, an imaging reporter gene, and administered intravenously to tumor-bearing mice. The engineered Salmonella successfully localized to tumor tissue and gene expression was dependent on the concentration of inducer, indicating the feasibility of peripheral control of bacterial gene expression. The bioluminescence signal permitted the localization of gene expression from the bacteria. The engineered bacteria significantly suppressed both primary and metastatic tumors and prolonged survival in mice. Therefore, engineered bacteria that carry a therapeutic and an imaging reporter gene for targeted anticancer therapy can be designed as a theranostic agent.

Co-operative regulation of ligand binding to melanocortin receptor subtypes: evidence for interacting binding sites.

This study evaluates the binding the melanocyte stimulating hormone peptide analogue [125I]NDP-MSH to melanocortin receptors MC1, MC3, MC4 and MC5 in insect cell membranes produced by baculovirus expression systems. The presence of Ca2+ was found to be mandatory to achieve specific [125I]NDP-MSH binding to the melanocortin receptors. Although association kinetics of [125I]NDP-MSH followed the regularities of simple bimolecular reactions, the dissociation of [125I]NDP-MSH from the melanocortin receptors was heterogeneous. Eleven linear and cyclic MSH peptides studied displaced the [125I]NDP-MSH binding to the studied melanocortin receptors, with the shapes of their competition curves varying from biphasic or shallow to super-steep (Hill coefficients ranging from 0.4 to 1.5). Notably the same peptide often gave highly different patterns on different melanocortin receptor subtypes; e.g. the MC4 receptor selective antagonist HS131 gave a Hill coefficient of 1.5 on the MC1 receptor but 0.5-0.7 on the MC(3-5) receptors. Adding a mask of one of the peptides to block its high affinity binding did not prevent other competing peptides to yield biphasic competition curves. The data indicate that the binding of MSH peptides to melanocortin receptors are governed by a complex dynamic homotropic co-operative regulations.