Development of an RNase H2 Activity Assay for Clinical Screening.

As the key enzyme mediating ribonucleotide excision repair, RNase H2 is essential for the removal of single ribonucleotides from DNA in order to prevent genome damage. Loss of RNase H2 activity directly contributes to the pathogenesis of autoinflammatory and autoimmune diseases and might further play a role in ageing and neurodegeneration. Moreover, RNase H2 activity is a potential diagnostic and prognostic marker in several types of cancer. Until today, no method for quantification of RNase H2 activity has been validated for the clinical setting. Herein, validation and benchmarks of a FRET-based whole-cell lysate RNase H2 activity assay are presented, including standard conditions and procedures to calculate standardized RNase H2 activity. Spanning a wide working range, the assay is applicable to various human cell or tissue samples with overall methodological assay variability from 8.6% to 16%. Using our assay, we found RNase H2 activity was reduced in lymphocytes of two patients with systemic lupus erythematosus and one with systemic sclerosis carrying heterozygous mutations in one of the RNASEH2 genes. Implementation of larger control groups will help to assess the diagnostic and prognostic value of clinical screening for RNase H2 activity in the future.

A common framework of monocyte-derived macrophage activation.

Macrophages populate every organ during homeostasis and disease, displaying features of tissue imprinting and heterogeneous activation. The disconnected picture of macrophage biology that has emerged from these observations is a barrier for integration across models or with in vitro macrophage activation paradigms. We set out to contextualize macrophage heterogeneity across mouse tissues and inflammatory conditions, specifically aiming to define a common framework of macrophage activation. We built a predictive model with which we mapped the activation of macrophages across 12 tissues and 25 biological conditions, finding a notable commonality and finite number of transcriptional profiles, in particular among infiltrating macrophages, which we modeled as defined stages along four conserved activation paths. These activation paths include a "phagocytic" regulatory path, an "inflammatory" cytokine-producing path, an "oxidative stress" antimicrobial path, or a "remodeling" extracellular matrix deposition path. We verified this model with adoptive cell transfer experiments and identified transient RELMɑ expression as a feature of monocyte-derived macrophage tissue engraftment. We propose that this integrative approach of macrophage classification allows the establishment of a common predictive framework of monocyte-derived macrophage activation in inflammation and homeostasis.

Trace element content in commercial complementary food formulated for infants and toddlers: Health risk assessment.

The study aimed to analyze trace elements content in baby purees and fruit juices and to evaluate the health risk of young children. The average daily dose, hazard quotient, hazard index and total diet hazard quotient were calculated to assess the potential health risk on per capita and consumers only groups of infants and toddlers. There was no significant health risk for studied groups regarding the intake of trace elements via purees and juices consumption. Health risk for lead was not estimated since the oral reference dose for this metal was not yet established and PTWI value was withdrawn. The average daily dose of lead for infants (0.32 - 0.46 µg/kg bw/day) and toddlers (2.01 - 2.29 µg/kg bw/day) were in accordance with the daily lead exposure intervals estimated by EFSA. Applying statistical analysis, the products were classified into three groups according to the content of trace elements.

A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus. FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

Antiproliferative and antimigratory effects of 3-(4-substituted benzyl)-5- isopropyl-5-phenylhydantoin derivatives in human breast cancer cells.

In this study, a series of synthesized 3-(4-substituted benzyl)-5-isopropyl-5-phenylhydantoin derivatives as a potential antiproliferative and antimigratory agents were investigated. The possible antitumor mechanisms of investigated hydantoin derivatives were examined on human breast cancer cell line MDA-MB-231. The cells were treated with different concentrations of compounds (from 0.01 µM to 100 µM) during 24 h and 72 h. The proliferation index, nitric oxide production, apoptosis rate, and migration capacity were measured. The cell invasion potential was examined by measuring the level of MMP-9 and COX-2 gene expression. All tested compounds expressed antiproliferative activity and induced dose- and time-dependent increase in the level of nitrites. The investigated molecules significantly decreased cell survival rate, migration capacity and the expression levels of genes included in the process of tumor invasion. Obtained data suggest that the tested hydantoin derivatives express considerable antitumor activity by reducing cell division rate, elevating apoptosis level, and inhibiting the motility and invasiveness of breast cancer cells. The results obtained in this study indicate that investigated compounds express potential as a novel chemotherapeutic agents against breast cancer growth and progression.

Modulation of functional characteristics of resident and thioglycollate-elicited peritoneal murine macrophages by a recombinant banana lectin.

We demonstrated that a recombinant banana lectin (rBanLec), which structural characteristics and physiological impacts highly resemble those reported for its natural counterparts, binds murine peritoneal macrophages and specifically modulates their functional characteristics. By using rBanLec in concentrations ranging from 1 µg to 10 µg to stimulate resident (RMs) and thioglycollate-elicited (TGMs) peritoneal macrophages from BALB/c and C57BL/6 mice, we have shown that effects of rBanLec stimulation depend on its concentration but also on the functional status of macrophages and their genetic background. rBanLec, in a positive dose-dependent manner, promotes the proliferation of TGMs from both BALB/c and C57BL/6 mice, while its mitogenic influence on RMs is significantly lower (BALB/c mice) or not detectable (C57BL/6 mice). In all peritoneal macrophages, irrespective of their type and genetic background, rBanLec, in a positive dose dependent manner, enhances the secretion of IL-10. rBanLec stimulation of RMs from both BALB/c and C57BL/6 resulted in a positive dose-dependent promotion of proinflammatory phenotype (enhancement of NO production and IL-12 and TNFα secretion, reduction of arginase activity). Positive dose-dependent skewing toward proinflammatory phenotype was also observed in TGMs from C57BL/6 mice. However, the enhancement of rBanLec stimulation promotes skewing of TGMs from BALB/c mice towards anti-inflammatory profile (reduction of NO production and IL-12 secretion, enhancement of arginase activity and TGFß and IL-4 secretion). Moreover, we established that rBanLec binds oligosaccharide structures of TLR2 and CD14 and that blocking of signaling via these receptors significantly impairs the production of TNFα and NO in BALB/c macrophages. Since the outcome of rBanLec stimulation depends on rBanLec concentration as well as on the functional characteristics of its target cells and their genetic background, further studies are needed to investigate its effects under physiological and specific pathological conditions.

Lignin model compound in alginate hydrogel: a strong antimicrobial agent with high potential in wound treatment.

Nowadays bacterial resistance to known antibiotics is a serious health problem. In order to achieve more efficient treatment, lately there is an effort to find new substances, such as certain biomaterials, that are non-toxic to humans with antibiotic potential. Lignins and lignin-derived compounds have been proposed to be good candidates for use in medicine and health maintenance. In this study, the antibacterial activity of the lignin model polymer dehydrogenate polymer (DHP) in alginate hydrogel (Alg) was studied. The obtained results show that DHP-Alg has strong antimicrobial activity against several bacterial strains and biofilms and does not have a toxic effect on human epithelial cells. These results strongly suggest its application as a wound healing agent or as an adjunct substance for wound treatments.

Production, characterization and applications of a tetanus toxin specific monoclonal antibody T-62.

Tetanus neurotoxin (TeNT) represents a potent toxin that binds to its receptors on neurons and inhibits the release of neurotransmitters. Additionally, its fragments are used to transport pharmacological substances to neuronal cell bodies. The main objective of this study was the development of a suitable model system to study internalization of the TeNT. We have produced a monoclonal antibody (MoAb) specific for TeNT by hybridoma technology, after immunization of BALB/c mice with tetanus toxoid, and have named it T-62. The immunochemical characteristics of MoAb T-62 were tested using ELISA, PAGE and immunoblotting. Finally, we have used an immunohistochemical method to detect specific binding of MoAb T-62 to TeNT bound to PC 12 cells. Our results show that MoAb T-62 is highly specific for TeNT, even when it is bound to its receptor, and that it could be of considerable importance in studies regarding fundamental research on TeNT receptors, intracellular transport of TeNT, as well as retrograde transport of pharmaceutical substances and non-invasive delivery of polypeptides through the blood brain barrier. In addition, MoAb T-62 is an invaluable tool in TeNT vaccine production as it can be used for the detection of reverse toxicity, which could drastically reduce the need to use animals in these experiments.

Hexameric immunoglobulin M in humans: desired or unwanted?

Immunoglobulin M (IgM) is the first antibody produced upon infection, and is often suggested as the first line of defense of human immune system. In addition to being present on the surface of naïve B cells as a monomeric molecule, IgM is always secreted as a polymer. The most abundant IgM polymer in humans is pentamer, composed of five monomeric units, joined together by so-called joining or J chain. On the other hand, it is well known that hexameric IgM can be also found in human sera. Its presence is often related to different dissorders (Waldenström's macroglobulinemia, cold agglutinin, and recurrent urinary bacterial infections), although it is believed that small amounts of hexamer are present in normal human sera as well. Unlike pentamer, IgM hexamer contains six monomeric blocks and completely lacks J chain. Although it has been decades since its discovery, the precise function of IgM hexamer is still unknown. Since it was documented that hexamer is very potent in activating complement, it is suggested that its production in humans must be under strict control, and that it is produced in special conditions, when strong activation of complement is absolutely needed. However, the question is whether hexameric IgM is really a secret weapon or just an undesirable molecule in humans. According to structural and known functional characteristics of both pentamers and hexamers of IgM, it can be concluded that hexamers are, in addition to being maybe too reactive to be around, probably not that efficient in protecting us from bacterial and viral infections.

Antigenic specificity and expression of a natural idiotope on human pentameric and hexameric IgM polymers.

Natural antibodies (NAbs) are present in circulation even before the exposure to antigen and they exert various biological functions. They are polyreactive and mainly represented by immunoglobulin M (IgM), which is the first antibody produced in an ongoing immune response to infection and/or immunization. IgM is always secreted as a polymer with predominant pentameric structure, although other polymeric forms such as hexamer can be also formed. The biological functions of hexameric IgM are still not known and it is proposed that its existence as a NAb could be deleterious. However, the nature of IgM hexamers has not been investigated yet. In this paper, we have tested the expression of natural idiotope and antigenic specificities of pentameric and hexameric IgM polymers originating from sera of patients with Waldenström's macroglobulinemia, as well as patients suffering from recurrent urinary bacterial infections. We demonstrate that although pentameric IgM polymers can exist as natural and immune antibodies, IgM hexamers are exclusively immune and do not exist as NAbs.