Neutral or Detrimental Effects of TREM2 Agonist Antibodies in Preclinical Models of Alzheimer's Disease and Multiple Sclerosis.

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining.

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Associations of specific types of physical activities with 10-year risk of cardiovascular disease among adults: Data from the national health and nutrition examination survey 1999-2006.

Background: Physical activity plays a key role in the prevention of cardiovascular disease (CVD). However, previous studies focused predominantly on the associations of the total amount of physical activity with CVD. There were few evidences on the associations of specific sport disciplines with CVD. Furthermore, little was known on the interactions between the different types of sports on CVD risk. Therefore, this study aimed to examine the independent associations of specific types of physical activities with the 10-year risk of CVD, and further evaluate the interactions between specific types of physical activities on the 10-year risk of CVD in US adults. Methods: This study used the data of the National Health and Nutrition Examination Survey (NHANES) 1999-2006. Participants aged ≥ 30 years and with free of CVD were eligible. The physical activity questionnaire is used to collect general information on leisure-time activities in the past 30 days, including the frequency, duration, and intensity of participation in each activity. The exposures of interest included cycling, swimming, aerobics, running, American Football, basketball, and racquet sports. The Framingham risk score algorithm was used to assess 10-year CVD risk based on age, high density lipoprotein, total cholesterol, systolic blood pressure, smoking status, and diabetes. A higher total score reflects a greater risk of CVD. Results: This study included 10829 participants. Compared to no participation, participation in cycling (ß = -0.890, 95% CI:-1.278,-0.502, P < 0.001), running (ß = -1.466, 95% CI:-1.837,-1.095, P < 0.001), American Football (ß = -2.934, 95% CI:-3.750,-2.119, P < 0.001), basketball (ß = -1.968, 95% CI:-2.645,-1.291, P < 0.001), and aerobics (ß = -0.980, 95% CI:-1.352,-0.608, P < 0.001) was associated with a lower CVD risk. Furthermore, cycling was antagonistic with basketball and racquet sports in the associations with CVD risk. An antagonistic action between swimming and aerobics was also observed. Nevertheless, running was synergistic with cycling, aerobics, and racquet sports in the associations with CVD risk. Conclusions: There were inverse associations of specific types of physical activities with CVD risk. Furthermore, there might be synergistic and antagonistic associations of multiple types of physical activities with CVD risk.

Spatial Distribution and Migration Characteristics of Heavy Metals in Grassland Open-Pit Coal Mine Dump Soil Interface.

The open-pit coal mine dump in the study area contains many low-concentration heavy metal pollutants, which may cause pollution to the soil interface. Firstly, statistical analysis and geostatistical spatial interpolation methods described heavy metal pollution's spatial distribution. The mine dump heavy metal pollution distribution is strongly random due to disorderly piles, but it is closely related to slope soil erosion. Furthermore, the soil deposition area is where pollutants accumulate. For example, all heavy metal elements converge at the bottom of the dump. Usually, the pollution in the lower part is higher than that in the upper part; the pollution in the lower step is higher than the upper step; the pollution in the soil deposition locations such as flat plate and slope bottom is higher than the soil erosion locations such as slope tip and middle slope. Finally, the hyperspectral remote sensing method described heavy metals pollution's migration characteristics, that the pollutants could affect the soil interface by at least 1 km. This study provides a basis for preventing and controlling critical parts of mine dump heavy metal pollution and pollution path control.

Specific Types of Physical Exercises, Dietary Preferences, and Obesity Patterns With the Incidence of Hypertension: A 26-years Cohort Study.

Objectives: To examine the associations of specific types of physical exercises, dietary preferences, and obesity patterns with incident hypertension. Methods: In this cohort study, obesity patterns were defined using general and abdominal obesity as G-/A-, G+/A- or G-/A+, and G+/A+. The type of physical exercises and dietary preferences were collected using a validated questionnaire. Participants with systemic blood pressure/diastolic blood pressure ≥140 mmHg/90 mmHg, use of antihypertensive medications, or a self-reported diagnosis were identified as hypertension. Results: There were 10,713 participants in this study. Martial arts, gymnastics, and ping pong could decrease the risk of hypertension (HR: 0.792, 0.884, and 0.855; and 95% CI: 0.743-0.845, 0.825-0.948, and 0.767-0.953, respectively). However, TV or computer usage, and consumption of fast food, soft/sugared drinks, and salty snack food could increase incident hypertension (HR: 1.418, 1.381, 1.233, and 1.225; and 95% CI: 1.315-1.529, 1.269-1.504, 1.157-1.314, and 1.139-1.316, respectively). Obese subjects had an increased risk of hypertension. Conclusion: The type of physical exercises, dietary preferences, and obesity patterns were associated with incident hypertension. More attention should be paid to these lifestyles to benefit health outcomes.

MICA immune complex formed with alpha 3 domain-specific antibody activates human NK cells in a Fc-dependent manner.

BACKGROUND: One of the mechanisms by which tumors evade immune surveillance is through shedding of the major histocompatibility complex (MHC) class I chain-related protein A and B (MICA/B) from their cell surface. MICA/B are ligands for the activating receptor NKG2D on NK and CD8 T cells. This shedding reduces cell surface levels of MICA/B and impairs NKG2D recognition. Shed MICA/B can also mask NKG2D receptor and is thought to induce NKG2D internalization, further compromising immune surveillance by NK cells. METHODS: We isolated human primary NK cells from normal donors and tested the suppressive activity of soluble recombinant MICA in vitro. Utilizing a panel of novel anti-MICA antibodies, we further examined the stimulatory activities of anti-MICA antibodies that reversed the suppressive effects of soluble MICA. RESULTS: We show that suppressive effects of soluble MICA (sMICA) on NK cell cytolytic activity was not due to the down-regulation of cell surface NKG2D. In the presence of an α3 domain-specific MICA antibody, which did not obstruct NKG2D binding, sMICA-mediated NK cell suppression was completely reversed. Reversal of NK cell inhibition by sMICA was mediated by immune complex formation that agonized NKG2D signaling. Furthermore, this restorative activity was dependent on antibody Fc effector function as the introduction of Fc mutations to abrogate Fc receptor binding failed to reverse sMICA-mediated NK cell suppression. Furthermore, MICA immune complexes preformed with an α3 domain-specific antibody (containing a wild-type Fc) induced IFN-γ and TNF-α secretion by NK cells in the absence of cancer cells, whereas MICA immune complexes preformed with the Fc effectorless antibody failed to induce IFN-γ and TNF-α secretion. Finally, we demonstrated that MICA immune complexes formed with the α3 domain-specific antibody activates NKG2D on NK cells leading to the release of IFN-γ. CONCLUSIONS: Our results demonstrate that an α3 domain-specific MICA antibody can circumvent sMICA-mediated suppression of NK cell cytolytic activity. Moreover, our data suggest that MICA immune complexes formed with α3-specific antibodies can activate NKG2D receptor and restore NK cell function in a Fc-dependent manner. The clinical utility of α3 domain-specific MICA/B antibodies may hold great promise as a new strategy for cancer immunotherapy.

High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies.

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens.

Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24 h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl2), benzo[a]pyrene (BaP), and perfluorooctanesulphonic acid (PFOS) from 6 hpf to 24 hpf. We observed significant reductions of MMR efficiency in embryos exposed to 0.1 µM CdCl2 (52%) and 0.5 µM BaP (34%), but no effect in embryos exposed to PFOS. Our study for the first time provides a model system for in vivo measurement of DNA MMR activity at the organism level, which has important implications in risk assessment of various environmental carcinogens.

Overexpression of the Notch target genes Hes in vivo induces lymphoid and myeloid alterations.

To examine the effects of Notch signaling on hematopoiesis, we transplanted mice with progenitors transduced with a constitutively active form of Notch1 (Notch1IC) or the Notch1 target genes Hes. Notch1IC-transduced cells induce T cell tumors and cannot generate B lymphocytes in vivo. Hes-transplanted mice remained healthy but cells transduced with Hes1 or Hes5 were partially impaired in their ability to differentiate into B cells. Both Hes1 and Hes5 were upregulated in the BM of Notch1IC mice and their ability to interfere with the transcriptional activity of E2A in a reporter assay was comparable to that of Notch1IC. This suggests that the inhibition of B cell development in the Notch1IC-transduced cells could be mediated by the interference of HES1/HES5 proteins with E2A. Hes1-, Hes5- and Notch1IC-transduced bone marrow cells cultured ex vivo in a colony forming assay in the presence of cytokines that promote myeloid differentiation remained very immature, indicating that the myeloid potential of these bone marrow cells was altered. Thymocytes overexpressing Hes1, Hes5 or Notch1IC matured normally into CD4 and CD8 single positive cells in vivo. Altogether our data suggest that Notch1IC induces T cell tumors independently of Hes genes but that its interference with lymphoid B and myeloid maturation is partly mediated by Hes1 and Hes5. DOI:

Characterization of retinoic acid-induced neurobehavioral effects in developing zebrafish.

Retinoic signaling plays an important role in cell proliferation and differentiation. Disruption of retinoic signaling via excessive or deficient retinoic acid can cause teratogenic effects on developing embryos. Similar to retinoic acid, many xenobiotic environmental pollutants have been found to disrupt retinoic signaling through binding and eliciting agonistic activity on retinoic acid receptors. Currently, studies of retinoic acid or retinoic acid-like compounds in aquatic organisms have mainly focused on teratogenicity and few studies have explored their neurobehavioral toxicity. In the present study, the authors used retinoic acid as an example to explore the neurobehavioral toxicity associated with developmental exposure of retinoic acid-like compounds in zebrafish. The findings confirmed retinoic acid's teratogenic effects such as bent spine, malformed tail, and pericardial edema in developing zebrafish with a median effective concentration of 2.47 nM. Retinoic acid-induced cell apoptosis at 24 h postfertilization was consistently found in the eye and tail regions of embryos. Spontaneous movement as characterized by tail bend frequency was significantly increased in zebrafish embryos following exposure to 2 nM and 8 nM retinoic acid. Relatively low-dose retinoic acid exposure of 2 nM led to fast locomotion behavior in the dark period and hyperactivity during light-dark photoperiod stimulation. The 2-nM retinoic acid exposure also led to alterations of neurobehavior- and optic nerve-related genes, with the transforming growth factor-ß signal transduction inhibitor noggin (nog) and the spinal cord marker homeobox c3a (hox) being underexpressed and the retinal G protein-coupled receptor a (rgr), the photoreceptor cell marker rhodopsin (rho), and the short wave-sensitive cone pigment opsin 1 (opn1sw1) being overexpressed. Increased expression of opn1sw1 and rho was confirmed by whole-mount in situ hybridization. Whether the misexpression of these genes leads to the neurobehavioral changes merits further study. The findings demonstrated that low-dose retinoic acid exposure perturbed the visual system and optic nerve development and caused hyperactivity in developing zebrafish.

FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma.

Fc receptor-like 5 (FcRL5/FcRH5/IRTA2/CD307) is a surface protein expressed selectively on B cells and plasma cells. We found that FcRL5 was expressed at elevated levels on the surface of plasma cells from the bone marrow of patients diagnosed with multiple myeloma. This prevalence in multiple myeloma and narrow pattern of normal expression indicate that FcRL5 could be a target for antibody-based therapies for multiple myeloma, particularly antibody-drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, where limited expression on normal tissues is a key component to their safety. We found that FcRL5 is internalized upon antibody binding, indicating that ADCs to FcRL5 could be effective. Indeed, we found that FcRL5 ADCs were efficacious in vitro and in vivo but the unconjugated antibody was not. The two most effective consisted of our anti-FcRL5 antibody conjugated through cysteines to monomethylauristatin E (MMAE) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (anti-FcRL5-MC-vcPAB-MMAE) or conjugated via lysines to the maytansinoid DM4 through a disulfide linker (anti-FcRL5-SPDB-DM4). These two ADCs were highly effective in vivo in combination with bortezomib or lenalidomide, drugs in use for the treatment of multiple myeloma. These data show that the FcRL5 ADCs described herein show promise as an effective treatment for multiple myeloma.

CD40 pathway activation status predicts response to CD40 therapy in diffuse large B cell lymphoma.

The primary function of B cells, critical components of the adaptive immune response, is to produce antibodies against foreign antigens, as well as to perform isotype class switching, which changes the heavy chain of an antibody so that it can interact with different repertoires of effector cells. CD40 is a member of the tumor necrosis factor superfamily of cell surface receptors that transmits survival signals to B cells. In contrast, in B cell cancers, stimulation of CD40 signaling results in a heterogeneous response in which cells can sometimes undergo cell death in response to treatment, depending on the system studied. We found an association between sensitivity to CD40 stimulation and mutation of the tumor suppressor p53 in a panel of non-Hodgkin's lymphoma cell lines. Consistent with p53's tumor suppressor role, we found that higher levels of intrinsic DNA damage and increased proliferation rates, as well as higher levels of BCL6, a transcriptional repressor proto-oncogene, were associated with sensitivity to CD40 stimulation. In addition, CD40 treatment-resistant cell lines were sensitized to CD40 stimulation after the introduction of DNA-damaging agents. Using gene expression analysis, we also showed that resistant cell lines exhibited a preexisting activated CD40 pathway and that an mRNA expression signature comprising CD40 target genes predicted sensitivity and resistance to CD40-activating agents in cell lines and mouse xenograft models. Finally, the gene signature predicted tumor shrinkage and progression-free survival in patients with diffuse large B cell lymphoma treated with dacetuzumab, a monoclonal antibody with partial CD40 agonist activity. These data show that CD40 pathway activation status may be useful in predicting the antitumor activity of CD40-stimulating therapeutic drugs.

Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma.

Targeting cytotoxic drugs to cancer cells using antibody-drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor, and are attractive targets for the use of ADCs because they are B-cell-specific, expressed in non-Hodgkin lymphomas (NHL), and are trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stable-linker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of nonHodgkin lymphoma cell lines in vitro. Further, these 2 ADCs are equally effective as low doses in xenograft models of follicular, mantle cell, and Burkitt lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, anti-CD79b antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like major histocompatibility complex class II-positive compartment MIIC. These results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.

Expression pattern of the human FcRH/IRTA receptors in normal tissue and in B-chronic lymphocytic leukemia.

A new family of Ig domain receptors referred to as the immune receptor translocation-associated (IRTA) proteins, FcR homologs (FcRHs) or FcR-like that are expressed in lymphoid cells has been recently described. RNA expression analysis suggests that FcRH1-5/IRTA1-5 are expressed exclusively in subsets of the B-cell compartment. We generated mAbs to FcRH1-5/IRTA1-5 and examined their protein expression pattern in normal tissue and in chronic lymphocytic leukemia (CLL) cells. Our data indicated that FcRH1-5/IRTA1-5 were expressed in B-cell sub-populations; however, in some cases, the protein was not expressed in the same B-cell populations as suggested by the RNA expression analysis. FcRH1/IRTA5 was expressed throughout the B-cell lineage starting at the pro-B-cell stage but was down-regulated in plasma cells. FcRH2/IRTA4 was expressed preferentially in memory B cells. FcRH3/IRTA3 was expressed at low levels in naive, germinal center (GC) and memory B cells but was also expressed in NK cells. FcRH4/IRTA1 was expressed in a sub-population of memory B cells associated with mucosal tissue. FcRH5/IRTA2 was expressed in mature B cells and memory B cells and down-regulated in GC cells and, unlike all other B-cell-specific markers, maintained its expression in plasma cells from tonsil, spleen and bone marrow. We examined the expression of FcRH1-5/IRTA1-5 on the surface of CLL cells and found a similar pattern of expression on CLL cells as in the normal mature B cells, except for FcRH3/IRTA3 which was up-regulated in CLL.

Notch1 perturbation of hemopoiesis involves non-cell- autonomous modifications.

To study the effects of Notch on hemopoiesis we used a bone marrow transduction/transplantation model and compared the transduced and nontransduced populations in reconstituted mice. While cells expressing a constitutively active form of murine Notch1 (Notch1IC) completely lacked B cells, a profound suppression of the B lineage was also seen in the nontransduced compartment. Experiments performed with retroviral supernatants of varying titers showed that the perturbations of B cell development among the nontransduced population correlated with the percentage of Notch1IC-transduced cells inoculated into the mice. The myeloid lineage of the Notch1IC-transplanted mice was altered as well, and this also affected the nontransduced population that had features of excessive maturation. To explore the basis of these non-cell-autonomous modifications we prepared conditioned medium from ex vivo cultures of Notch1IC-transplanted mice bone marrow and showed that it inhibited B cell maturation and promoted myeloid differentiation in a dose-dependent manner. Finally, we found that the T cell leukemia/lymphomas that occur in Notch1IC-transplanted mice were accompanied by abnormal maturation of nontransduced T cells in the bone marrow. These findings indicate that modifications of neighboring cells through non-cell-autonomous modifications take part in multiple facets of the activity of Notch on hemopoiesis.