Adult obesity treatment and prevention: A trans-agency commentary on the research landscape, gaps, and future opportunities.

Given the high and growing prevalence of obesity among adults in the United States, obesity treatment and prevention are important topics in biomedical and public health research. Although researchers recognize the significance of this problem, much remains unknown about safe and effective prevention and treatment of obesity in adults. In response to the worsening obesity epidemic and the many unknowns regarding the disease, a group of key scientific and program staff members of the National Institutes of Health (NIH) and other federal and non-government agencies gathered virtually in September 2021 to discuss the current state of obesity research, research gaps, and opportunities for future research in adult obesity prevention and treatment. The current article synthesizes presentations given by attendees and shares their organizations' current initiatives and identified gaps and opportunities. By integrating the information discussed in the meeting and current initiatives, we identify potential targets and overlapping priorities for future research, including health equity and disparities in obesity, the heterogeneity of obesity, and the use of technological and innovative approaches in interventions.

Identifying the role of array electrodes in improving the compost quality of food waste during electric field-assisted aerobic composting.

Low composting temperature and long maturation periods are two major problems during food waste composting. In this study, a novel array-based electric field-assisted aerobic composting (Pin-EAC) process was tested on food waste compost. Pin-EAC increase the composting temperature to 69.3 °C, and improved the germination index by 15%. The Pin-EAC took at least 40% less time to reach the standard compost maturity. The fluorescent spectroscopy results showed that Pin-EAC could increase humic acid and fulvic acid by 33% and 37%, respectively. Pin-EAC could increase the diversity of thermophilic bacteria during composting. The co-occurrence network shown that Pin-EAC are more closely related to oxygen and temperature. This work has initially shown that the use of an electric field could improve food waste composting quality, suggesting that the Pin-EAC process is an effective strategy for high-water and high-oil organic solid waste aerobic composting.

Intracellular CD24 disrupts the ARF-NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation.

CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.

Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction.

Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction [corrected] of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid-based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid-based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.

B7 blockade alters the balance between regulatory T cells and tumor-reactive T cells for immunotherapy of cancer.

PURPOSE: In prostate cancer-bearing host, regulatory T (Treg) cells restrain activity of tumor antigen-specific T cells. Because B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers. EXPERIMENTAL DESIGN: The anti-B7-1/B7-2 monoclonal antibodies were administered to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice. RESULTS: Here, we showed that short-term administration of anti-B7-1/B7-2 monoclonal antibodies in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed T antigen-specific CTL effectors in anti-B7-treated but not control IgG-treated TRAMP mice. CONCLUSIONS: Transient blockade of B7-1/B7-2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.

Analysis of Recombinant CD24 Glycans by MALDI-TOF-MS Reveals Prevalence of Sialyl-T Antigen.

CD24 is a glycosyl-phosphatidyl-inositol linked glycoprotein expressed in a broad range of cell types and is heavily glycosylated. It has been found to be over expressed in cancers and tumors and is also a costimulatory molecule. Therefore, this study was carried out to define the structures of the carbohydrates associated with the CD24 recombinant protein. The CD24 glycoprotein's oligosaccharides were released by chemical and enzymatic means prior to being analyzed by MALDI-TOF-MS. The results obtained showed that CD24 is both N- and O-glycosylated. The major oligosaccharides were found to be Neu5Acα-2,3/6Galß-1,3GalNAc, NeuAc(2)Gal ß-1,3GalNAc(1) (O-glycans), GalNAc(2)GlcNAc(2)Man(3)Fuc(1), Gal(1)GalNAc(2)GlcNAc(2)Man(3)Fuc(1), and Gal(2)GalNAc(2)GlcNAc(2)Man(3)Fuc(1) (N-glycans). The results showed that Neu5Acα-2,3/6Galß-1,3GalNAc (sialyl-tumor antigen, sT), a cancer-associated carbohydrate, was the most abundant glycan associated with CD24. This result raised the intriguing possibility that CD24 may be a major carrier of the sialyl-T abundantly found in cancer cells.

Modulation of NKT cell development by B7-CD28 interaction: an expanding horizon for costimulation.

It has been demonstrated that the development of NKT cells requires CD1d. The contribution of costimulatory molecules in this process has not been studied. Here we show that in mice with targeted mutations of B7-1/2 and CD28, the TCRbeta(+)alpha-Galcer/CD1d(+) (iValpha14 NKT) subset is significantly reduced in the thymus, spleen and liver. This is mainly due to decreased cell proliferation; although increased cell death in the thymi of CD28-deficient mice was also observed. Moreover, in the B7-1/2- and CD28-deficient mice, we found a decreased percentage of the CD4(-)NK1.1(+) subset and a correspondingly increased portion of the CD4(+)NK1.1(-) subset. In addition, the mice with a targeted mutation of either B7 or CD28 had a reduced susceptibility to Con A induced hepatitis, which is known to be mediated by NKT cells. Our results demonstrate that the development, maturation and function of NKT cell are modulated by the costimulatory pathway and thus expand the horizon of costimulation into NKT, which is widely viewed as a bridge between innate and adaptive immunity. As such, costimulation may modulate all major branches of cell-mediated immunity, including T cells, NK cells and NKT cells.

Autoreactive T cells escape clonal deletion in the thymus by a CD24-dependent pathway.

Despite negative selection in the thymus, significant numbers of autoreactive T cells still escape to the periphery and cause autoimmune diseases when immune regulation goes awry. It is largely unknown how these T cells escape clonal deletion. In this study, we report that CD24 deficiency caused deletion of autoreactive T cells that normally escape negative selection. Restoration of CD24 expression on T cells alone did not prevent autoreactive T cells from deletion; bone marrow chimera experiments suggest that CD24 on radio-resistant stromal cells is necessary for preventing deletion of autoreactive T cells. CD24 deficiency abrogated the development of experimental autoimmune encephalomyelitis in transgenic mice with a TCR specific for a pathogenic autoantigen. The role of CD24 in negative selection provides a novel explanation for its control of genetic susceptibility to autoimmune diseases in mice and humans.

Alpha-lactosylceramide as a novel "sugar-capped" CD1d ligand for natural killer T cells: biased cytokine profile and therapeutic activities.

The invariant natural killer T cells (iNKT) cells have emerged as an important regulator of immunity to infection, cancer, and autoimmune diseases. They can be activated by glycolipids that bind to CD1d. The most effective iNKT ligand reported to date is alpha-galactosylceramide (alpha-GalCer), which stimulates iNKT cells to secrete both Th-1 and Th-2 cytokines. Indiscriminate induction of both types of cytokines could limit the therapeutic potential of iNKT ligands, as Th-1 and Th-2 cytokines play different roles under physiological and pathological conditions. Therefore, a ligand with a biased cytokine-release profile would be highly desirable. Here, we report the synthesis and biological activity of alpha-lactosylceramide (alpha-LacCer). Our data demonstrate that alpha-LacCer can stimulate iNKT cells to proliferate and release cytokines, both in vitro and in vivo. Interestingly, while alpha-LacCer is approximately 1000-times less efficient than alpha-GalCer in inducing Th-1 cytokines, it is as potent as alpha-GalCer in the induction of Th-2 cytokines; therefore, alpha-LacCer is a novel compound that induces a biased cytokine release. Processing by beta-glycosidase was critical for alpha-LacCer activity. Moreover, in vivo experiments suggest that alpha-LacCer is at least as potent as alpha-GalCer in the treatment of tumors and experimental autoimmune encephalomyelitis.

A dinucleotide deletion in CD24 confers protection against autoimmune diseases.

It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527 approximately 1528 (P1527(del)) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34-0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22-0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527(del) allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3' UTR of CD24 mRNA conveys significant protection against both MS and SLE.

Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia.

PURPOSE: A high-rate glycolysis is a fundamental property of solid tumors and is associated with an over-expression of glucose transporters and glycolytic enzymes. We hypothesize that over-expression of glucose transporters in tumors prevents apoptosis, promotes cancer cell survival, and confers drug resistance. Inhibition of glucose transporter will preferentially sensitize the anticancer effects of chemotherapeutic drugs to overcome drug resistance in hypoxia. METHODS: Glucose transporter expressions were detected in cancer tissues and NCI 60 cancer cells with immunostaining and DNA microarray. Glucose uptake was measured with 3H-2-deoxy-glucose. Cytotoxicity of daunorubicin (DNR) in combination of glucose inhibitor was detected by MTS assay under hypoxic condition. Early stage apoptosis was monitored with Annexin V-FITC staining. RESULTS: Immunostaining showed that GLUT1 was significantly increased in hypoxic regions of the human colon and breast tumors. The expression profiles of all glucose transporters in NCI 60 cancer cells exhibited distinct expression patterns. Phloretin exhibited more than 60% glucose uptake inhibition. Hypoxia conferred two to fivefold higher drug resistance in SW620 and K562 to DNR. Inhibition of glucose uptake by phloretin sensitized cancer cells to DNR for its anticancer activity and apoptosis to overcome drug resistance only under hypoxia. CONCLUSION: Cancer cells heavily rely on glucose transporters for glucose uptake to facilitate a high-rate glycolysis under hypoxia for their survival and drug resistance. Combination of glucose transporter inhibitors and chemotherapeutic drugs may provide a preferential novel therapeutic strategy to overcome drug resistance in hypoxia.

Epm2a suppresses tumor growth in an immunocompromised host by inhibiting Wnt signaling.

The genetic mechanisms responsible for increased incidence of lymphoma in immunocompromised individuals have not been fully elucidated. We show that, in a line of TCR transgenic TG-B mice, an insertional mutation in one allele of the Epm2a locus and epigenetic silencing of another led to a high rate of lymphoma with early onset. Overexpressing Epm2a suppressed the growth of established tumor cells and the development of lymphoma in the TG-B mice, while specific silencing of the locus increased tumorigenesis in the immune-deficient host. Downregulation of Epm2a expression is widespread among mouse and human lymphoma cell lines. Epm2a-encoded laforin is a phosphatase for GSK-3beta and an important repressor in the Wnt signaling pathway. Inactivation of Epm2a resulted in increased Wnt signaling and tumorigenesis.

Discovery of a daunorubicin analogue that exhibits potent antitumor activity and overcomes P-gp-mediated drug resistance.

Anthracyclines are considered to be some of the most effective anticancer drugs for cancer therapy. However, drug resistance and cardiotoxicity of anthracyclines limit their clinical application. We hypothesize that direct modifications of the sugar moiety of anthracyclines avert P-glycoprotein (P-gp) recognition and efflux, increase drug intracellular concentration in cancer cells, and thus overcome P-gp-mediated drug resistance. Daunorubicin (DNR) analogues with sugar modifications were synthesized by directly transforming the amino group of DNR to an azido group or triazole group. Molecular docking showed that the lead compound (3'-azidodaunorubicin, ADNR) averts P-gp binding, while daunorubicin (DNR) extensively interacts with multidrug-resistance (MDR) protein through H-bonds and electrostatic interactions. FACS assay demonstrated that these new compounds abolished P-gp drug efflux and accumulated high intracellular concentration in the drug-resistant leukemia K562/Dox. P-gp inhibition by CsA confirmed that these new analogues are no longer P-gp substrates. ADNR exhibited potent anticancer activity in both drug-sensitive (K562) and drug-resistant leukemia cells (K562/Dox), with a 25-fold lower drug resistance index than DNR. An in vivo xenograft model demonstrated that ADNR showed more than 2.5-fold higher maximum growth inhibition rate against drug-resistant cancers and significant improvement for animal survival rate versus DNR. No significant body weight reduction in mice was observed for ADNR at the maximum tolerable dose, as compared to more than 70% body weight reduction for DNR. These data suggest that sugar modifications of anthracyclines avert P-gp binding, abolish P-gp-mediated drug efflux, increase intracellular drug concentration, and thus overcome P-gp-mediated drug resistance in cancer therapy.

Expression of tissue-specific autoantigens in the hematopoietic cells leads to activation-induced cell death of autoreactive T cells in the secondary lymphoid organs.

Many tissue-specific antigens are expressed in specialized cells called peripheral antigen-expressing cells (PAE) in the thymus and can induce central tolerance. While thymic medullary epithelial cells are the prototypic PAE that express peripheral antigens via an aire-dependent mechanism, some studies also describe bone marrow (BM)-derived dendritic cells (DC) and macrophages as PAE in both the thymus and secondary lymphoid organs. However, the role of these cells in development of tolerance to tissue-specific antigens has not been elucidated. Here we use BM radiation chimeric mice to study the existence of hematopoietic PAE and their contribution to tolerance to tissue-specific antigens. Our results reveal that BM-derived PAE exist in both central and secondary lymphoid organs and that the expression of peripheral antigens in the BM-derived cells does not correlate with aire expression. Using double-transgenic mice expressing TCR specific for a model antigen expressed under the control of a prostate-specific promoter, we show that expression of self antigen in PAE of non-hematopoietic origin is both necessary and sufficient to induce clonal deletion. Surprisingly, while BM-derived PAE fail to induce clonal deletion, they do cause the activation-induced cell death of autoreactive cells in the secondary lymphoid organs. Thus, BM-derived PAE have a distinct function in the maintenance of tolerance to tissue-specific antigens.

A new role for CD28 in the survival of autoreactive T cells in the periphery after chronic exposure to autoantigen.

Recent work demonstrates that costimulatory molecules play a critical role for clonal deletion of autoreactive T cells in the thymus. The role of CD28 in the survival of autoreactive T cells in the periphery, however, has not been reported. Here we demonstrate that while mutation of the CD28 gene consistently increased the burden of autoreactive T cells in the thymus, such an increase was not always found in the periphery, as the CD28(-/-) autoreactive T cells disappeared in the spleen over a period between 4 and 10 weeks. The disappearance of autoreactive T cells associates with a diminished induction of Bcl-2 protein by the self antigen and an increased proportion of apoptotic cells in the periphery. Moreover, the elimination of autoreactive T cells in the periphery requires chronic stimulation by the self antigen, as adoptive transfer analysis revealed no enhancement of apoptosis in CD28(-/-) T cells in antigen-bearing hosts over a 3 day period. Thus, CD28 plays a significant role in both clonal deletion and survival of autoreactive T cells after chronic exposure to autoantigens, resulting in opposite effects on the burden of autoreactive T cells.

CD24 controls expansion and persistence of autoreactive T cells in the central nervous system during experimental autoimmune encephalomyelitis.

In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.

B7-CD28 interaction promotes proliferation and survival but suppresses differentiation of CD4-CD8- T cells in the thymus.

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25(+)CD4(+) regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4(-)CD8(-) (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25(+)CD44(-) (DN3) to CD25(-)CD44(-) (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.

Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors.

In addition to their overexpression in cancer cells, most of the tumor-associated Ags are expressed at low but detectable levels in normal tissues. It is not clear whether the repertoire of T cells specific for unmutated tumor Ags is shaped by negative selection during T cell development. The transgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Although it has been established that T lymphocytes from TRAMP mice are tolerant to SV40 Tag, the mechanism of the tolerance is largely unknown. To examine whether the T cell clonal deletion is responsible for the tolerance, we crossed the TRAMP mice with mice transgenic for a rearranged TCR specific for SV40 Tag presented by the H-2K(k). Double transgenic TRAMP/TCR mice showed profound thymic deletion of SV40 Tag-reactive T cells, including a 6- to 10-fold reduction in the total thymocyte numbers and a >50-fold reduction in phenotypically mature T cells. Consistent with this finding, we observed that the SV40 Tag and endogenous mouse probasin genes are expressed at low levels in the thymus. These results demonstrate that clonal deletion is a major mechanism for tolerance to Ags previously regarded as prostate-specific, and provide direct evidence that the T cell repertoire specific for an unmutated tumor Ag can be shaped by clonal deletion in the thymus.

Perinatal blockade of b7-1 and b7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells.

A number of in vitro studies have suggested that costimulatory molecules B7-1 and B7-2 and their receptor CD28 can promote clonal deletion, and limited in vivo studies have indicated that CD28 is involved in the clonal deletion of some T cells. However, the significance of B7-mediated clonal deletion in preventing autoimmune diseases has not been studied systematically. Here we report that the perinatal blockade of B7-1 and B7-2 substantially inhibits the clonal deletion of T cells in the thymus and leads to an accumulation of T cells capable of inducing fatal multiorgan inflammation. These results reveal a critical role for costimulatory molecules B7-1 and B7-2 in deleting pathogenic autoreactive T cells in the thymus. The critical role of B7-1 and B7-2 in T cell clonal deletion may explain, at least in part, the paradoxical increase of autoimmune disease in mice deficient for this family of costimulatory molecules, such as cytotoxic T lymphocyte associated molecule 4, CD28, and B7-2. The strong pathogenicity of the self-reactive T cells supports a central hypothesis in immunology, which is that clonal deletion plays an important role in preventing autoimmune diseases.