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Granzymes are a family of serine proteases mainly expressed by CD8 + T cells, natural killer cells, and innate-like lymphocytes 1,2 . Although their major role is thought to be the induction of cell death in virally infected and tumor cells, accumulating evidence suggests some granzymes can regulate inflammation by acting on extracellular substrates 2 . Recently, we found that the majority of tissue CD8 + T cells in rheumatoid arthritis (RA) synovium, inflammatory bowel disease and other inflamed organs express granzyme K (GZMK) 3 , a tryptase-like protease with poorly defined function. Here, we show that GZMK can activate the complement cascade by cleaving C2 and C4. The nascent C4b and C2a fragments form a C3 convertase that cleaves C3, allowing further assembly of a C5 convertase that cleaves C5. The resulting convertases trigger every major event in the complement cascade, generating the anaphylatoxins C3a and C5a, the opsonins C4b and C3b, and the membrane attack complex. In RA synovium, GZMK is enriched in areas with abundant complement activation, and fibroblasts are the major producers of complement C2, C3, and C4 that serve as targets for GZMK-mediated complement activation. Our findings describe a previously unidentified pathway of complement activation that is entirely driven by lymphocyte-derived GZMK and proceeds independently of the classical, lectin, or alternative pathways. Given the widespread abundance of GZMK -expressing T cells in tissues in chronic inflammatory diseases and infection, GZMK-mediated complement activation is likely to be an important contributor to tissue inflammation in multiple disease contexts.

Many adult neurons are dynamically remodeled across timescales ranging from the rapid addition and removal of specific synaptic connections, to largescale structural plasticity events that reconfigure circuits over hours, days, and months. Membrane lipids, including brain-enriched sphingolipids, play crucial roles in these processes. In this review, we summarize progress at the intersection of neuronal activity, lipids, and structural remodeling. We highlight how brain activity modulates lipid metabolism to enable adaptive structural plasticity, and showcase glia as key players in membrane remodeling. These studies reveal that lipids act as critical signaling molecules that instruct the dynamic architecture of the brain., Competing Interests: Conflict of interest statement Nothing declared, (Copyright © 2023. Published by Elsevier Ltd.)

Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Competing Interests: None disclosed.

Background: Hypergammaglobulinemic purpura of Waldenström (HGPW), a rare cutaneous eruption characterized by the triad of recurrent episodes of lower extremity petechiae, symptoms of stinging and burning, and lower extremity edema, is poorly described in children. Some children have been reported to follow a benign course, while others are eventually diagnosed with fulminant rheumatologic disease., Objectives: To determine the distinguishing features of HGPW including the spectrum of disease manifestations and clinical outcomes., Methods: This is a multicenter, retrospective case series of six children with HGPW combined with a literature review of 45 previously published pediatric cases., Results: Most children were eventually diagnosed with systemic disease (63%) or developed autoantibody accumulation suggestive of evolving disease (71%). The most common diagnoses were Sjogren's syndrome and systemic lupus erythematosus. The mean duration between onset of cutaneous eruption and diagnosis of systemic disease was 5.6 years, underscoring that HPGW patients often present with a rash that precedes the development of systemic symptoms., Conclusions: Diagnosis of HGPW should prompt initial screening for rheumatologic disease with long-term rheumatology follow-up, as the majority of patients present with evolving manifestations of systemic disease., (© 2020 Wiley Periodicals, Inc.)

The roots of psychopathology frequently take shape during infancy in the context of parent-infant interactions and adversity. Yet, neurobiological mechanisms linking these processes during infancy remain elusive. Here, using responses to attachment figures among infants who experienced adversity as a benchmark, we assessed rat pup cortical local field potentials (LFPs) and behaviors exposed to adversity in response to maternal rough and nurturing handling by examining its impact on pup separation-reunion with the mother. We show that during adversity, pup cortical LFP dynamic range decreased during nurturing maternal behaviors, but was minimally impacted by rough handling. During reunion, adversity-experiencing pups showed aberrant interactions with mother and blunted cortical LFP. Blocking pup stress hormone during either adversity or reunion restored typical behavior, LFP power, and cross-frequency coupling. This translational approach suggests adversity-rearing produces a stress-induced aberrant neurobehavioral processing of the mother, which can be used as an early biomarker of later-life pathology.

The ability of neurons to identify correct synaptic partners is fundamental to the proper assembly and function of neural circuits. Relative to other steps in circuit formation such as axon guidance, our knowledge of how synaptic partner selection is regulated is severely limited. Drosophila Dpr and DIP immunoglobulin superfamily (IgSF) cell-surface proteins bind heterophilically and are expressed in a complementary manner between synaptic partners in the visual system. Here, we show that in the lamina, DIP mis-expression is sufficient to promote synapse formation with Dpr-expressing neurons and that disrupting DIP function results in ectopic synapse formation. These findings indicate that DIP proteins promote synapses to form between specific cell types and that in their absence, neurons synapse with alternative partners. We propose that neurons have the capacity to synapse with a broad range of cell types and that synaptic specificity is achieved by establishing a preference for specific partners., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To describe which cannabinoids and terpenes are effective for treating pain., Data Sources: Peer-reviewed articles, book chapters., Conclusion: Cannabis and cannabinoid medicines, as modulators of the endocannabinoid system, offer novel therapeutic options for the treatment of cancer-related pain, not only for patients who do not respond to conventional therapies, but also for patients who prefer to try cannabis as a first treatment option., Implications for Nursing Practice: Understanding the endocannabinoid system, cannabinoids, terpenes, routes of administration, potential drug interactions, clinical implications, and potential side effects ensures nurses can better assist patients who use cannabis for the treatment of cancer pain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Dual degree program MD/PhD candidates typically train extensively in basic science research and in clinical medicine, but often receive little formal experience or mentorship in clinical and translational research., Methods: To address this educational and curricular gap, the University of Wisconsin Medical Scientist Training Program partnered with the University of Wisconsin Institute for Clinical and Translational Research to create a new physician-scientist preceptorship in clinical and translational research. This six-week apprentice-style learning experience-guided by a physician-scientist faculty mentor-integrates both clinical work and a translational research project, providing early exposure and hands-on experience with clinically oriented research and the integrated career of a physician-scientist. Five years following implementation, we retrospectively surveyed students and faculty members to determine the outcomes of this preceptorship., Results: Over five years, 38 students and 36 faculty members participated in the physician-scientist preceptorship. Based on student self-assessments (n = 29, response rate 76%), the course enhanced competency in conducting translational research and understanding regulation of clinical research among other skills. Mentor assessments (n = 17, response rate 47%) supported the value of the preceptorship in these same areas. Based on work during the preceptorship, half of the students produced a peer-reviewed publication or a meeting abstract. At least eleven peer-reviewed manuscripts were generated. The preceptorship also provided a structure for physician-scientist mentorship in the students' clinical specialty of choice., Conclusion: The physician-scientist preceptorship provides a new curricular model to address the gap of clinical research training and provides for mentorship of physician-scientists during medical school. Future work will assess the long-term impact of this course on physician-scientist career trajectories.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and are commonly used for pain relief and fever reduction. NSAIDs are used following childhood vaccinations and cancer immunotherapies; however, how NSAIDs influence the development of immunity following these therapies is unknown. We hypothesized that NSAIDs would modulate the development of an immune response to Listeria monocytogenes -based immunotherapy. Treatment of mice with the nonspecific COX inhibitor indomethacin impaired the generation of cell-mediated immunity. This phenotype was due to inhibition of the inducible COX-2 enzyme, as treatment with the COX-2-selective inhibitor celecoxib similarly inhibited the development of immunity. In contrast, loss of COX-1 activity improved immunity to L. monocytogenes Impairments in immunity were independent of bacterial burden, dendritic cell costimulation, or innate immune cell infiltrate. Instead, we observed that PGE 2 production following L. monocytogenes is critical for the formation of an Ag-specific CD8 + T cell response. Use of the alternative analgesic acetaminophen did not impair immunity. Taken together, our results suggest that COX-2 is necessary for optimal CD8 + T cell responses to L. monocytogenes , whereas COX-1 is detrimental. Use of pharmacotherapies that spare COX-2 activity and the production of PGE 2 like acetaminophen will be critical for the generation of optimal antitumor responses using L. monocytogenes ., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background . Sipuleucel T, an autologous cell-based vaccine targeting prostatic acid phosphatase (PAP), has demonstrated efficacy for the treatment of advanced prostate cancer. DNA vaccines encoding PAP and live attenuated Listeria vaccines have entered clinical trials for patients with prostate cancer, and have advantages in terms of eliciting predominantly Th1-biased immunity. In this study, we investigated whether the immunogenicity and anti-tumor efficacy of a DNA and Listeria vaccine, each encoding PAP, could be enhanced by using them in a heterologous prime/boost approach. Methods . Transgenic mice expressing HLA-A2.01 and HLA-DRB1*0101 were immunized alone or with a heterologous prime/boost strategy. Splenocytes were evaluated for MHC class I and II-restricted, PAP-specific immune responses by IFNγ ELISPOTs. Anti-tumor activity to a syngeneic, PAP-expressing tumor line was evaluated. Results . PAP-specific cellular immunity and anti-tumor activity were elicited in mice after immunization with DNA- or listeria-based vaccines. Greater CD4+ and CD8+ responses, and anti-tumor responses, were elicited when mice were immunized first with DNA and boosted with Listeria , but not when administered in the opposite order. This was found to be dependent on CD4+ T cells elicited with DNA priming, and was not due to inflammatory signals by Listeria itself or due to B cells serving as antigen-presenting cells for DNA during priming. Conclusions . Heterologous prime/boost vaccination using DNA priming with Listeria boosting may provide better anti-tumor immunity, similar to many reports evaluating DNA priming with vaccines targeting foreign microbial antigens. These findings have implications for the design of future clinical trials.

The influence of cell death on adaptive immunity has been studied for decades. Despite these efforts, the intricacies of how various cell death pathways shape immune responses in the context of infection remain unclear, particularly with regard to more recently discovered pathways such as pyroptosis. The emergence of Listeria monocytogenes as a promising immunotherapeutic platform demands a thorough understanding of how cell death induced in the context of infection influences the generation of CD8 + T-cell-mediated immune responses. To begin to address this question, we designed strains of L. monocytogenes that robustly activate necrosis, apoptosis, or pyroptosis. We hypothesized that proinflammatory cell death such as necrosis would be proimmunogenic while apoptosis would be detrimental, as has previously been reported in the context of sterile cell death. Surprisingly, we found that the activation of any host cell death in the context of L. monocytogenes infection inhibited the generation of protective immunity and specifically the activation of antigen-specific CD8 + T cells. Importantly, the mechanism of attenuation was unique for each type of cell death, ranging from deficits in costimulation in the context of necrosis to a suboptimal inflammatory milieu in the case of pyroptosis. Our results suggest that cell death in the context of infection is different from sterile-environment-induced cell death and that inhibition of cell death or its downstream consequences is necessary for developing effective cell-mediated immune responses using L. monocytogenes-based immunotherapeutic platforms., (Copyright © 2016 American Society for Microbiology.)

The cytokine IL-1β plays a central role in inflammatory responses that are initiated by microbial challenges, as well as in those that are due to endogenous processes (often called sterile inflammation). IL-1β secretion that occurs independently of microbial stimulation is typically associated with the presence of endogenous alarmins, such as extracellular ATP (an indicator of cytopathic damage). In this study, we show that IL-2-activated human invariant NKT (iNKT) cells stimulate the secretion of IL-1β protein by human peripheral blood monocytes in a manner that requires neither the presence of microbial compounds nor signaling through the extracellular ATP receptor P2X7 Monocyte IL-1β production was specifically induced by iNKT cells, because similarly activated polyclonal autologous T cells did not have this effect. Secretion of IL-1β protein occurred rapidly (within 3-4 h) and required cell contact between the iNKT cells and monocytes. Similar to IL-1β production induced by TLR stimulation, the iNKT-induced pathway appeared to entail a two-step process involving NF-κB signaling and IL1B gene transcription, as well as assembly of the NLRP3 inflammasome and activation of caspase-1. However, in contrast to the classical inflammasome-mediated pathway of IL-1β production, activation of monocytes via P2X7 was dispensable for iNKT-induced IL-1β secretion, and potassium efflux was not required. Moreover, the iNKT-induced effect involved caspase-8 activity, yet it induced little monocyte death. These results suggest that IL-2-activated human iNKT cells induce monocytes to produce IL-1β through a distinctive pathway that does not require the presence of microbial danger signals or alarmins associated with cytopathic damage., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Inflammasomes are cytosolic innate immune surveillance systems that recognize a variety of danger signals, including those from pathogens. Listeria monocytogenes is a Gram-positive intracellular bacterium evolved to live within the harsh environment of the host cytosol. Further, L. monocytogenes can activate a robust cell-mediated immune response that is being harnessed as an immunotherapeutic platform. Access to the cytosol is critical for both causing disease and inducing a protective immune response, and it is hypothesized that the cytosolic innate immune system, including the inflammasome, is critical for both host protection and induction of long-term immunity. L. monocytogenes can activate a variety of inflammasomes via its pore-forming toxin listeriolysin-O, flagellin, or DNA released through bacteriolysis; however, inflammasome activation attenuates L. monocytogenes, and as such, L. monocytogenes has evolved a variety of ways to limit inflammasome activation. Surprisingly, inflammasome activation also impairs the host cell-mediated immune response. Thus, understanding how L. monocytogenes activates or avoids detection by the inflammasome is critical to understand the pathogenesis of L. monocytogenes and improve the cell-mediated immune response generated to L. monocytogenes for more effective immunotherapies.

The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP-β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Maternal surfactant protein A (SP-A), a collectin with innate immune system function, is critical to newborn mouse survival preventing bacterial peritonitis associated with a nonhygienic environmental exposure. We hypothesized that SP-A improves newborn survival by optimizing milk immunoprotection., Methods: Regional (lung) and systemic (milk and serum) immunologic responses to a novel antigen, 2,4-dintirophenyl keyhole limpet hemocyanin (DNP-KLH), and to a nonhygienic environment were evaluated in wild-type (WT) and SP-A null murine dams. Cross-fostering pups assessed the impact of milk on newborn survival., Results: Maternal SP-A optimized antigen-specific milk secretory IgA (sIgA) production following the DNP-KLH exposure. Milk total and environment-specific sIgA production was not dependent on maternal SP-A in the nonhygienic exposure. At baseline, SP-A null milk contained physiologically meaningful increases in two proinflammatory cytokines compared with WT milk. The lack of SP-A plus the nonhygienic environmental exposure synergistically increased the number of proinflammatory cytokines contained in milk. Finally, the SP-A null genotype decreased pup survival during a nonhygienic environmental exposure., Conclusion: Maternal SP-A impacts milk sIgA and cytokine content, and is associated with improved newborn health.