Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an “eat-me” signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1⁺) derived from cancer cells compared to m/lEVs (annexin A1⁺) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1⁺) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Nutritional strategies that improve an animal's resilience to various challenges may improve animal health and welfare. One such nutrient is niacin, which has reduced inflammation in mice, humans, and swine; however, niacin's anti-inflammatory effects have not been investigated in cattle. Our objective was to determine whether rumen-protected niacin (RPN) alters lactating dairy cows' inflammatory response to intramammary LPS challenges, whether RPN resulted in any carryover effects, and whether repeated LPS challenges result in signs of immune tolerance or innate immune training. Twenty healthy, late-lactation Holstein cows (232 ± 65 DIM; 39 ± 5.8 kg/d of milk) were enrolled in a randomized complete block experiment that lasted 70 d. Cows received 26 g/d of RPN or no top-dress (CON) for the first 42 d of the experiment. During the final milking of d 27 and 55, cows were challenged in their rear right (RR) mammary gland with 100 µg of LPS suspended in 5 mL of PBS. Milk yield, milk conductivity, and feed intake were measured daily. Milk composition was measured on d 14, 23, 24, 30, 37, 45, and 52. Blood samples were collected at 0, 8, 12, 24, 48, 72, 96, and 120 h after each LPS challenge, whereas RR quarter milk samples were collected at 0, 8, 16, 24, 48, 72, 96, 120, 144, and 168 h after each LPS challenge. Body temperature was measured continuously during each challenge with an intravaginal thermometer. Linear mixed models with repeated measures were used to analyze the results. Before LPS challenge, RPN did not affect feed intake or milk production, but it reduced SCS (1.24 ± 0.41 [SE] vs. 0.05 ± 0.45). After challenge, RPN did not affect feed intake, milk production, milk composition, SCS, body temperature, plasma glucose, or plasma insulin concentrations. Our results suggest RPN reduced peak plasma haptoglobin and lipopolysaccharide binding protein during the first LPS challenge. Plasma haptoglobin tended to be less after the second challenge for cows previously supplemented RPN, and lipopolysaccharide binding protein was similar for each treatment group after the second challenge. The second LPS challenge resulted in decreased plasma haptoglobin compared with the first LPS challenge, suggestive of tolerance, but it also induced a greater peak SCS than the first LPS challenge. Our results suggest that repeated LPS challenges promote a systemic tolerance but heightened local response to LPS-induced mastitis. Feeding RPN reduced SCS before challenge and reduced plasma acute phase proteins after challenge, suggesting that RPN may reduce systemic inflammation without altering the local inflammatory responses.

Despite the addition of several new agents to the armamentarium for the treatment of multiple myeloma (MM) in the last decade and improvements in outcomes, the refractory and relapsing disease continues to take a great toll, limiting overall survival. Therefore, additional novel approaches are needed to improve outcomes for MM patients. The oncogenic transcription factor MYC drives cell growth, differentiation and tumor development in many cancers. MYC protein levels are tightly regulated by the proteasome and an increase in MYC protein expression is found in more than 70% of all human cancers, including MM. In addition to the ubiquitin-dependent degradation of MYC by the 26S proteasome, MYC levels are also regulated in a ubiquitin-independent manner through the REGγ activation of the 20S proteasome. Here, we demonstrate that a small molecule activator of the 20S proteasome, TCH-165, decreases MYC protein levels, in a manner that parallels REGγ protein-mediated MYC degradation. TCH-165 enhances MYC degradation and reduces cancer cell growth in vitro and in vivo models of multiple myeloma by enhancing apoptotic signaling, as assessed by targeted gene expression analysis of cancer pathways. Furthermore, 20S proteasome enhancement is well tolerated in mice and dogs. These data support the therapeutic potential of small molecule-driven 20S proteasome activation for the treatments of MYC-driven cancers, especially MM.

Women frequently experience longer-lasting pain than men, indicating delayed pain resolution, but the mechanisms underlying this sex difference remain unclear. Here, we show that interleukin-10 ⁺ (IL-10 ⁺ ) monocytes resolve inflammatory pain by signaling to IL-10R1 ⁺ sensory neurons in a mouse model of skin inflammation. Male mice exhibited faster pain resolution than females, which was associated with higher numbers of IL-10 ⁺ monocytes. In both sexes, pain resolution was impaired by deleting Il10 from monocytes or Il10ra from sensory neurons. Androgen signaling promoted IL-10 production by monocytes, driving sex differences in IL-10 ⁺ monocyte abundance. Enhancing IL-10 ⁺ monocytes with resolvin D1 accelerated pain resolution in both sexes. In humans, pain resolved faster in men than in women after traumatic injury and was associated with higher circulating monocytes and IL-10 levels in men. These findings identify a role for peripheral IL-10 ⁺ monocytes in sex-specific pain resolution and highlight immune mechanisms that may prevent chronic pain.

Immune cells have been observed in many tissues of the mammal including the gut, liver, adipose, bone, and neural tissue. Our objective was to demonstrate whether immune cells are present within the stratified rumen epithelium of healthy lactating dairy cattle. We hypothesized that immune cells would be found in the rumen tissue and that the total amount of leukocytes present would increase during a ruminal acidosis challenge. We conducted a 2-phase experiment with 9 rumen-cannulated lactating Holstein cows. During phase 1, cows were fed a diet containing 31% NDF, 24% forage NDF, and 27% starch (CON) for 14 d. The starch sources were corn silage and dry ground corn. During phase 2, cows were fed a diet with 28% NDF, 16% forage NDF, and 32% starch (SARA) for 7 d. The SARA starch sources were corn silage, dry ground corn, ground wheat, and ground barley. During both phases, rumen fluid, fecal, blood, and rumen tissue samples were collected. Feed intake and milk yield were recorded each day and milk samples were collected at each milking during the final 2 d of each period. Data were analyzed with mixed models that included the fixed effect of phase (CON or SARA) and the random effect of cow. The SARA diet reduced rumen pH and increased area under the curve for rumen pH beneath 5.8 and 5.6. Feed intake, milk yield, and milk protein yield increased for SARA. Phase did not affect ruminal immune cell phenotype proportions or total presence. Most immune cells observed were CD3⁺ (T cells) for both CON and SARA. The ruminal immune cells were located within the lamina propria, stratum basale, and stratum spinosum of the rumen papillae. Our data demonstrate that T lymphocytes are prominent in rumen tissue. Understanding the role of ruminal leukocytes may yield new insights into ruminant gut health, function, development, and maintenance.

The loss of major histocompatibility complex class I (MHC-I) molecules has been proposed as a mechanism for cancer immune evasion. Nevertheless, the mechanism is poorly understood. We report here that membrane-associated RING-CH-type finger 8 (MARCHF8), upregulated by human papillomavirus (HPV), ubiquitinates and degrades MHC-I in HPV-positive head and neck cancer (HPV+ HNC). Inhibiting MARCHF8 restores MHC-I levels on HPV+ HNC cells, suppresses tumor growth, and increases the infiltration of natural killer (NK) and T cells in the tumor microenvironment. Furthermore, Marchf8 knockout markedly increases cross talk between cytotoxic NK cells and CD8 ⁺ T cells with macrophages and enhances the tumor-killing activity of CD8 ⁺ T cells. Interestingly, Marchf8 knockout, in combination with anti-PD-1 treatment, further enhances tumor suppression and increases NK and T cell infiltration in mice bearing immune checkpoint inhibitor–refractory tumors. Our findings suggest that MARCHF8 could be a promising target for immunotherapy for HPV+ HNC patients.