Genetic ablation of IP3 receptor 2 increases cytokines and decreases survival of SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease characterized by the selective death of motor neurons. Disease pathophysiology is complex and not yet fully understood. Higher gene expression of the inositol 1,4,5-trisphosphate receptor 2 gene (ITPR2), encoding the IP3 receptor 2 (IP3R2), was detected in sporadic ALS patients. Here, we demonstrate that IP3R2 gene expression was also increased in spinal cords of ALS mice. Moreover, an increase of IP3R2 expression was observed in other models of chronic and acute neurodegeneration. Upregulation of IP3R2 gene expression could be induced by lipopolysaccharide (LPS) in murine astrocytes, murine macrophages and human fibroblasts indicating that it may be a compensatory response to inflammation. Preventing this response by genetic deletion of ITPR2 from SOD1G93A mice had a dose-dependent effect on disease duration, resulting in a significantly shorter lifespan of these mice. In addition, the absence of IP3R2 led to increased innate immunity, which may contribute to the decreased survival of the SOD1G93A mice. Besides systemic inflammation, IP3R2 knockout mice also had increased IFNγ, IL-6 and IL1α expression. Altogether, our data indicate that IP3R2 protects against the negative effects of inflammation, suggesting that the increase in IP3R2 expression in ALS patients is a protective response.

Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening.

Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z'-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10 µM. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low µM activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors.

Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 µg vitamin B9, 1 µg vitamin B12, 10 mg zinc, 50 µg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment.

Multiple determinations of sperm DNA fragmentation show that varicocelectomy is not indicated for infertile patients with subclinical varicocele.

Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient's fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA) were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment). TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele.

Presence and regulation of insulin-regulated aminopeptidase in mouse macrophages.

INTRODUCTION: The insulin-regulated aminopeptidase (IRAP) is expressed in several cell types, where it is mainly located in specialized secretory endosomes that are quickly recruited to the cell surface upon cell type-specific activation. Here we describe for the first time the expression and subcellular distribution of IRAP in macrophages. METHODS: IRAP mRNA expression, protein expression and presence at the cell surface was investigated by real-time polymerase chain reaction (PCR), Western blot and [(3)H]IVDE77 binding, respectively. RESULTS: IRAP mRNA expression was increased by interferon-γ (IFN-γ) and lipopolysaccharide (LPS), but not by anti-inflammatory cytokines (interleukin (IL)-4, IL-10, transforming growth factor ß (TGF-ß)). IFN-γ increased [(3)H]IVDE77 binding steadily over time, while LPS quickly and transiently recruited IRAP to the cell surface. Combined stimulations with IFN-γ and LPS showed the same pattern as LPS alone. Latex particles also induced a transient recruitment of IRAP to the cell surface, but no difference was observed in phagocytic uptake between wild-type and IRAP(-/-) macrophages, suggesting that the enzymatic activity of IRAP is not required for the ingestion of particles. CONCLUSION: IRAP is more highly expressed in pro-inflammatory M1-activated macrophages and its presence at the cell surface is modulated upon exposure to IFN-γ, LPS or exogenous particles.

[3H]IVDE77, a novel radioligand with high affinity and selectivity for the insulin-regulated aminopeptidase.

The hexapeptide angiotensin IV (Ang IV) induces diverse biological effects such as memory enhancement and protection against ischemic stroke. Studies on the mechanism of Ang IV however are hampered by its instability and its lack of selectivity. The high-affinity binding site for Ang IV is the insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), but Ang IV also acts as a weak agonist for the Ang II-receptor (AT1), implying the need for stable and highly selective Ang IV-analogues. Here we present the screening of novel Ang IV-analogues, selected on basis of high affinity for IRAP, high selectivity (compared to aminopeptidase N and the AT1 receptor) and resistance against proteases. The selected compound IVDE77 possesses a number of advantages compared to Ang IV: (i) it has a 40 times higher affinity for IRAP (Ki 1.71 nM), (ii) it does not activate the AT1 receptor, (iii) it is easily radiolabeled with tritium and (iv) it is resistant to proteolysis, even in human plasma. In addition, pre-treatment of intact CHO-K1 cells with IVDE77 led to a virtually complete inhibition of subsequent intracellular accumulation of [(3)H]IVDE77-IRAP complexes. IVDE77 thus represents the first Ang IV-analogue able to abolish IRAP-availability completely at the cell surface in vitro. In summary, IVDE77 is a useful tool for the detection of IRAP under physiological conditions, and may contribute to elucidating the mechanism of Ang IV to ascertain which functions are IRAP-dependent.

Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions.

Macrophages display remarkable plasticity, allowing these cells to adapt to changing microenvironments and perform functions as diverse as tissue development and homeostasis, inflammation, pathogen clearance and wound healing. Macrophage activation can be triggered by Th1 cytokines and pathogen-associated or endogenous danger signals, leading to the formation of classically activated or M1 macrophages. On the other hand, anti-inflammatory mediators, including IL-4, IL-10, TGF-ß and M-CSF, induce diverse anti-inflammatory types of macrophages, known under the generic term M2. In human breast carcinomas, tumor-associated macrophage (TAM) density correlates with poor prognosis. In mouse models of breast cancer, eliminating macrophages from the tumor site, either via genetic or therapeutic means, results in retarded tumor progression. Over the years, multiple signals from the mammary tumor microenvironment have been reported to influence the TAM phenotype and TAM have been propagated as anti-inflammatory M2-like cells. Recent developments point to the existence of at least two distinct TAM subpopulations in mammary tumors, based on a differential expression of markers such as CD206 or MHC II and different in vivo behaviour: perivascular, migratory TAM which are less M2-like, and sessile TAM found at tumor-stroma borders and/or hypoxic regions that resemble more M2-like or "trophic" macrophages. Hence, a further refinement of the molecular and functional heterogeneity of TAM is an avenue for further research, with a potential impact on the usefulness of these cells as therapeutic targets.

Amino triazolo diazepines (Ata) as constrained histidine mimics.

Two synthetic routes for the synthesis of amino-triazolodiazepine (Ata) scaffolds are presented. The scope of both of these proceeding through key intra- and intermolecular Huisgen cycloaddition reactions is discussed. The replacement of the His-Pro dipeptide segment in angiotensin IV by the dipeptide mimetic Ata-Gly and subsequent biological evaluation in two inhibitory enzyme assays validated the use of the Ata moiety as a His mimic given the equipotency of both peptidic analogs.

Mononuclear phagocyte heterogeneity in cancer: different subsets and activation states reaching out at the tumor site.

Mononuclear phagocytes are amongst the most versatile cells of the body, contributing to tissue genesis and homeostasis and safeguarding the balance between pro- and anti-inflammatory reactions. Accordingly, these cells are notoriously heterogeneous, functioning in distinct differentiation forms (monocytes, MDSC, macrophages, DC) and adopting different activation states in response to a changing microenvironment. Accumulating evidence exists that mononuclear phagocytes contribute to all phases of the cancer process. These cells orchestrate the inflammatory events during de novo carcinogenesis, participate in tumor immunosurveillance, and contribute to the progression of established tumors. At the tumor site, cells such as tumor-associated macrophages (TAM) are confronted with different tumor microenvironments, leading to TAM subsets with specialized functions. A better refinement of the molecular and functional heterogeneity of tumor-associated mononuclear phagocytes might pave the way for novel cancer therapies that directly target these tumor-supporting cells.

High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis.

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.