bluuebunny/crossref_metadata_2025 huggingface dataset:
ERK5 and CCL19 Regulate the Secondary Immune Response in Mice.
Authors: Cervantes Jaisel A , Vines Charlotte M , Knight Colin D , Gehre Mika D , Bill Colin A
Published: January 2023

C-C chemokine receptor 7 (CCR7) binds to its ligand CCL19, which is expressed on the surface of activated dendritic cells, antigen activated B-cells and within lymphoid organs. Previous studies showed that homozygous deletion of CCR7 or its ligands leads to increased production of antibodies. However, it is unclear to what extent CCL19 contributes to this phenotype, since stimulation of naïve T cells with the second CCR7 ligand, CCL21, is sufficient to promote trafficking of T cells to the lymph nodes. We hypothesized that using a CCR7 antagonist during immunization would create a pseudo CCR7(−) state. Previously, we have shown that signaling via CCR7/CCL19 is mediated through ERK5. Therefore, we investigated the effect of the CCL19 antagonist CCL198-83 or blocking downstream signaling through the CCR7/CCL19 axis by using ERK5flox/flox/Lck-CRE mice, on antibody production. We found in the presence of CCL198-83 mice generated significantly higher levels of IgG1, IgG2b, and IgG3 than control mice in response to vaccination with DNP-KLH. In addition, we found that the level of affinity maturation between the mice treated with CCL198-83 was not significantly different from control mice demonstrating that in the absence of CCL19 activity, affinity maturation can occur. To assess the role of ERK5 signaling pathway, we used ERK5flox/flox/Lck-CRE mice to examine the effect of CCR7 downstream signaling on antibody production. We found that these mice produced significantly higher levels of IgG1 and IgG3 following immunization with DNP-KLH than the ERK5flox/flox control mice suggesting that the ERK5 pathway, at least in part, mediates signaling from CCR7/CCL19.

Supported by NIH-SC1GM111172 NIH-2U54MD007592 NSF-HRD-182675 NIH-5UL1GM118970
Myeloperoxidase deficiency impacts the composition of the gut microbiome: a case for variable disease penetrance?
Authors: Neu Savannah , Gurski Cody , Strzepa Anna , Dittel Bonnie N
Published: January 2023

The most abundant proteolytic enzyme found in neutrophilic granules, myeloperoxidase (MPO) is a heme peroxidase with microbicidal activity. While MPO inhibition has therapeutic promise for treatment of inflammatory illness, differential disease penetrance has been observed in MPO-deficient mice during various disease models. In our studies using the DSS-colitis mouse model, MPO-deficient mice under conventional specific pathogen-free (SPF) conditions experienced similar disease penetrance to co-housed, wildtype controls. In contrast, when housed in restricted SPF conditions lacking norovirus and Helicobacter species, MPO-deficient mice experienced more severe colitis while wildtype mice were unchanged. From these data, we hypothesized that an interplay between MPO (microbicidal activity) and the composition of the gut microbiome impacts disease penetrance of colitis. To investigate, we performed 16S rRNA-sequencing on the intestines of MPO-deficient and wildtype mice housed in restricted SPF conditions and found a proportionate increase in Ruminococcus gnavus, which is associated with intestinal dysbiosis. Correspondingly, we found proportionate decreases in the gut beneficial bacteria Akkermansia muciniphilia and Prevotella sp. These data show that MPO does affect the composition of the gut bacteria, and supports an interplay between inflammatory mediators, the microbiome, and altered disease penetrance.
Amniotic fluid metabolites mediate maternal high fat diet expansion of group 3 innate lymphoid cells in offspring.
Authors: Mirpuri Julie
Published: January 2023

Introduction

Previously, we demonstrated that maternal high fat diet (mHFD) exposure results in a unique pattern of microbial colonization in offspring, expansion of IL-17 producing group 3 innate lymphoid cells (ILC3) and increased susceptibility to a model of necrotizing enterocolitis (NEC). We hypothesized that amniotic fluid (AF) metabolites are modified by maternal diet and partially responsible for the phenotype in offspring.

Methods

To assess if AF metabolites in mHFD were unique from regular diet (RD) AF, we collected AF from E15 dams and performed non-targeted global mass spectroscopy. To determine if the mHFD AF is responsible for expansion of ILC3, we gavaged 5-day-old RD germ-free pups with mHFD and RD AF (from conventional dams) every other day and examined them at 2 weeks by flow cytometry for ILC3. mHFD mothers were supplemented with metabolites that were decreased in the mHFD AF and offspring examined for expansion of ILC3 and susceptibility to the LPS/PAF model of NEC.

Results

We identified 1069 metabolites in HFD and RD AF. Principal component analysis revealed that mHFD AF was uniquely clustered from RD AF. We specifically found decreases in indole, short chain fatty acid and furoic acid metabolites in HFD AF compared to RD AF. Gavage of germ-free neonatal pups with mHFD AF resulted in expansion of IL-17 producing ILC3 compared to RD AF at 2 weeks. Supplementation of mHFD dams prenatally with metabolites diminished in HFD AF resulted in decreased expansion of ILC3 and resistance to LPS/PAF model of NEC.

Conclusion

mHFD modifies metabolites present in AF and can expand ILC3 in germ-free offspring. Supplementation of deficient dietary metabolites in mHFD pregnant dams rescues offspring from susceptibility to NEC.

Supported by NIH R01 1K12HD068369
Gene editing at position 71 in DRB1*04:01 blocks collagen sensitization and avoids allorecognition.
Authors: Jha Vibha , Coulombe Marilyne , Rosloniec Edward F , Matsuda Jennifer , Freed Brian M , Roark Christina L
Published: January 2023

DRB1*04:01 is strongly associated with susceptibility to rheumatoid arthritis (RA). Our previous work demonstrated that changing lysine (K) at position 71 in DRB1*04:01 to glutamic acid (E), an amino acid found in resistant alleles, abrogated binding of the immunodominant collagen258-272 peptide. The purpose of this study was to determine if the K71E mutation in DRB1*04:01 would prevent sensitization to collagen and be tolerated by the host immune system in mice. DRB1*04:01 and DRB1*04:01K71E transgenic mice were created on the murine MHC class II knockout background (B6.129S2-H2dlAb1-Ea/J). DRB1*04:01 and DRB1*04:01K71E mice were immunized twice with bovine type II collagen in complete Freund’s adjuvant on days 0 and 21. On day 56, lymph node cells were harvested and stimulated in vitro with collagen258-272 peptide. Introduction of the K71E mutation in DRB1*04:01 resulted in a >90% reduction in proliferation as compared to CD4+ cells from collagen-immunized DRB1*04:01 mice. Next, skin grafts were transplanted from DRB1*04:01K71E mice onto DRB1*04:01 recipients to test for rejection. The skin grafts from DRB1*04:01K71E mice were accepted by the recipient DRB1*04:01 mice with no signs of rejection out to day 70. Our results demonstrate that introducing the K71E mutation in DRB1*04:01 blocks collagen sensitization and avoids allorecognition suggesting that this mutation could safely halt progression of RA by blocking autoimmune T cell responses to collagen.
STAT3 degraders inhibit cellular activation, cytokine production, and Th17 development, resulting in inhibition of autoimmunity in the MOG-EAE model of CNS inflammation.
Authors: Sullivan Jeffrey , Brown Crystal , Mayo Michele , Dixit Vaishali , Enerson Bradley , Rong Haojing , Yang Bin , De Savi Chris , Gollob Jared , Mainolfi Nello , Slavin Anthony , Hubeau Cedric
Published: January 2023

Signal transducer and activator of transcription 3 (STAT3), an “undruggable” transcription factor activated by a variety of receptor- and non-receptor tyrosine kinases, plays a critical role in activation pathways triggered by cytokines, hormones, and growth factors, which makes it an attractive target for the treatment of autoimmune and autoinflammatory disorders. Kymera has developed heterobifunctional molecules that selectively target STAT3 for degradation by the ubiquitin-proteasome pathway. These degraders have broad and potent activity in-vitro against TLR receptor and cytokine-induced activation of immune and stromal cells and attendant mediators release such as MCP-1 (CCL2) and collagen1a1. STAT3 degradation in CD4+ T cells potently inhibited Th17 development, decreasing IL-17, IL-22, IL-8/CXCL8, and TNFa production, with concomitant increase in Treg numbers, that was superior to JAK1/2 kinase inhibition. STAT3 degradation was subsequently evaluated in-vivo in murine models of autoimmune disease. In the Th17-driven inflammatory model of MOG-induced Experimental Autoimmune Encephalomyelitis (EAE), dose-dependent decrease of incidence, disease onset, clinical scores, and histopathology were observed in comparison to a S1P1 inverse agonist or steroid treatment. Ex-vivo MOG-stimulated cytokine release by leukocytes isolated from draining lymph node was also robustly inhibited. These data demonstrate the broad activity of STAT3 degradation in alleviating autoimmune inflammation in systems relevant to human disease.
Tolerogenic Artificial Antigen Presenting Cells for Selective Tolerance in Autoimmune Disease
Authors: Raimondi Giorgio , Rhodes Kelly , Tzeng Stephany , Lozano Marcos Igleasias , VanDyke Derek , Neshat Sarah , Spangler Jamie , Green Jordan
Published: January 2023

Autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes develop when autoreactive immune cells attack healthy cells and tissues. Current treatments are non-curative and leave patients immunocompromised. As an alternative, therapies that expand regulatory T cells (Tregs) are being actively explored to establish antigen-specific tolerance. We recently showed that an IL-2 immunocytokine (IL-2_IC), a fusion of an antibody to IL-2, preferentially engages and expands Tregs over effector T cells. As a more targeted approach to tolerize the immune system towards a specific autoantigen, we explored the design and therapeutic efficacy of a microparticle bearing an IL-2_IC and an MHC II/peptide complex on its surface (TolAPC).

Microparticles composed of a blend of PLGA and PBAE were loaded with rapamycin, and then chemically conjugated with IL-2_IC and MHC II I-A(b) tetramers loaded with a peptide derived from myelin oligodendrocyte glycoprotein (MOG). Characterized TolAPC were tested in a mouse model of MS, Experimental Autoimmune Encephalomyelitis (EAE). When administered prophylactically, TolAPC delayed EAE development and reduced severity compared to unconjugated controls, with 36.4% of mice remaining asymptomatic for the duration of the study. When administered therapeutically, TolAPC reversed symptomatic EAE. Clinical improvements correlated with a nearly two-fold increase in Tregs in the brain and spinal cord of TolAPC-treated mice compared to controls six days after treatment. Furthermore, administration of additional booster doses led to sustained amelioration of the disease.

Overall, TolAPC can selectively expand Tregs and provide both protection and treatment in an autoimmune mouse model.

Supported by grants from DoD (W81XWH-18-1-0735; W81XWH-21-1-0892), NIH (R01EB029455), and JDRF (1-INO-2020-923-A-N)
Dysfunction of Dendritic Cells in Tumor-Draining Lymph Nodes
Authors: Bandola-Simon Joanna Marta , Roche Paul A
Published: January 2023

An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II) and CD8 cytotoxic T cells (through MHC-I). Activation of tumor-specific T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes (TDLN) and to identify the mechanisms that lead to defective tumor antigen presentation. We transplanted tumors into the flank of mice and analyzed DCs from TDLN and contralateral non-draining lymph nodes (NDLN). Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN either in vivo or ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLNs was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake by DCs from TDLN. Interestingly, DCs in TDLN also showed poor removal of the Invariant-chain CLIP peptide from the MHC-II peptide binding groove. This is likely to be due to a functional defect of the peptide-editor H2-M. Using Invariant-chain mutant mice and H2-M knock-out mice we confirmed that defective peptide exchange from MHC-II molecules hampered the anti-tumor responses and lead to enhanced tumor growth. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis could allow us to “correct” these DC defects, thereby allowing CD4 T cell priming to provide the necessary help for antitumor CD8 T cells.
Reactive myelopoiesis and FX-expressing monocyte-derived macrophages triggered by chemotherapy promotes cancer lung metastasis
Authors: Ding Chuanlin , Yan Jun
Published: January 2023

Lung metastasis-associated macrophages (MAMs) play a very important role in tumor metastasis. In mouse models of lung metastasis, interstitial macrophages (IMs) markedly accumulate and differentiate into MAMs. Several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis. In this study, we hypothesize that host responses to chemotherapy treatment enhance breast cancer metastasis by modulation of lung macrophage toward a pro-metastatic phenotype. Here, we show that multi-dose chemo treatment promotes breast cancer lung metastasis in the spontaneous breast cancer animal model. Pretreatment with Gemcitabine and Paclitaxel induces a significantly higher number of pulmonary metastases in the experimental metastasis model. Mechanistically, multi-dose of chemotherapy induces reactive myelopoiesis characterized by increase of BM LSK cells and multipotent progenitors in tumor-free mice, which is associated with the increase of mitochondrial ROS. Further, chemokine CCL2 expression is significantly increased in the plasma and lung tissues of chemo-treated tumor-free mice. Consequently, the percentage of BM Ly6C+ monocytes are significantly increased because of increased progenitor monocyte differentiation potential and monocyte proliferation, which results in an increase of lung monocytes and IMs with enhanced expression of coagulation factor X (FX). Inhibition of activated FX (FXa) can inhibit chemotherapy-induced lung metastasis. Together, these studies suggest a novel mechanism for chemotherapy induced metastasis via the modulation of BM-derived monocytes/macrophages and extravascular clotting in lung tissues.

Supported by grants from NIH (P20GM135004)
Determination of CD91 intracellular signaling pathway following HSP stimulation
Authors: Harkness James F , Binder Robert J
Published: January 2023

Cancer immunosurveillance is dependent on the heat shock protein (HSP) receptor, CD91, which is expressed on professional antigen-presenting cells (APCs). While CD91-facilitated cross-presentation of HSP-chaperoned peptides has been well studied, CD91-mediated signaling is not completely defined. The binding of CD91 by immunogenic HSPs leads to production of unique inflammatory cytokine profiles by APCs. This requires the phosphorylation of tyrosine residues on intracellular domains of CD91 and activation of NF-κB. However, adaptor proteins associated with CD91 and molecules involved in signal transmission have not been characterized and their role in cancer immunosurveillance remains untested. Our general strategy to identify adaptor proteins involves crosslinking and co-immunoprecipitation of CD91 and its associated signaling complex following stimulation of CD91 with the immunogenic HSPs gp96, calreticulin, and hsp70. Preliminary studies with gp96 have identified Shc as an adaptor protein and AXL receptor tyrosine kinase and tyrosine-protein kinase Fgr as membrane associated kinases. The impact on their inhibition on cytokine release by APCs is being tested. We have also developed a novel mouse expressing mutant, non-signaling CD91, and its impact on cancer immunosurveillance will be determined. Investigation of these complexes will aid in understanding the signaling pathway that activates production of cytokine profiles within APCs. This project will allow us understand the pathways to priming immune responses responsible for cancer immunosurveillance and also to develop better therapeutic strategies for cancer and autoimmune diseases.

Supported by grants from NIH (RO1 CA233803)
CD155 axis modulation promotes natural killer cell-mediated graft-versus-tumor effects against osteosarcoma
Authors: Cho Monica , Phillips Madison , Song Longzhen , Erbe-Gurel Amy , Capitini Christian M
Published: January 2023

Patients with relapsed/refractory osteosarcoma (OSA) have a poor prognosis with few treatment options. Immunotherapy with allogeneic natural killer (NK) cells after allogeneic bone marrow transplant (BMT) is an attractive approach to employ a graft-versus-tumor (GVT) effect against OSA. However, NK cells have had limited success against solid tumors in vivo. CD155 is overexpressed on OSA and interacts with DNAM-1, an activating ligand, and TIGIT, an inhibitory ligand, on NK cells. The impact of blocking CD155 after allogeneic BMT is unknown. IL-15-expanded C57BL/6 (B6, H-2b) and BALB/c (H-2d) murine NK cells were co-cultured with K7M2 murine OSA (H-2d) after CD155 and CD155 ligand blockade, and analyzed by flow cytometry and cytotoxicity assays. BALB/c mice were also transplanted with T cell depleted allogeneic B6 or syngeneic BALB/c bone marrow, challenged with K7M2 and treated with IL-15-expanded NK cells and CD155 blockade. Allogeneic NK cells showed increased degranulation, interferon-gamma production and cytotoxicity against K7M2 OSA in vitro compared to syngeneic NK cells. NK cell activation and cytotoxicity were further enhanced by CD155 and TIGIT blockade, but decreased with DNAM-1 blockade. In vivo, allogeneic NK cell infusion and anti-CD155 treatment decreased tumor growth and increased survival compared to syngeneic NK cell treatment, without induction of graft-versus-host-disease. CD155 blockade augments NK cell activation, cytotoxicity and GVT effects against OSA without toxicity, suggesting TIGIT is the dominant CD155 checkpoint during allogeneic BMT. CD155 blockade with allogeneic NK cell therapy after BMT may be an effective combination immunotherapy platform for treating relapsed/refractory OSA.

Supported by NIH grants (T32 CA009135, R01 CA215461)
Pre-treatment with a novel synthetic TLR4 agonist prior to challenge with mouse-adapted H1N1 and H2N3 influenza strains reduced morbidity and mortality in BALB/c mice
Authors: Whitacre Margaret , Ward Janine , Jackson Konner , Bess Laura , Jayah Rachel , Sands Cherrokee , Arayangkul Hathaipat , Khalaf Juhienah K , Miller Shannon M , Evans Jay T , Bazin Helene G
Published: January 2023

According to the World Health Organization, influenza causes 3–5 million cases of severe illness and 250,000 – 500,000 deaths annually despite the availability of a seasonal vaccine. As an alternative therapy for use in high-risk groups and environments, we evaluated the efficacy of prophylactic treatment with a novel synthetic TLR4 agonist in BALB/c mice. Animals were anesthetized and treated with a TLR4 agonist intranasally (IN) 0 to 28 days prior to a lethal challenge with either H1N1 or H3N2 mouse-adapted human influenza. Animals were monitored daily for weight loss, decreased body temperature, body score, and mortality. Initial dose-response experiments demonstrated that mice treated with 50 μg or 10 μg of our novel TLR4 agonist in an aqueous formulation were similarly protected from H3N2 influenza challenge with respect to weight loss and survival. However, initial weight loss data demonstrated that 50 μg was not as well tolerated as 10 μg. The 10 μg dose was therefore selected as the lead dose in subsequent experiments. In a series of formulation comparison experiments, the lead formulation resulted in marked improvement in both percent weight loss and survival compared to initial TLR4 agonist formulations. Animals treated with all formulations of our novel synthetic TLR4 agonist 0, 7, or 14 days prior to infection resulted in 80–90% survival. The lead formulation also resulted 80–90% survival when given prophylactically 21 or 28 days prior to infection compared to 0–20% survival in untreated mice. Overall, our results show that pre-treatment with our novel synthetic TLR4 agonist prior to exposure to mouse-adapted H1N1 and H2N3 influenza strains reduced morbidity and mortality in infected mice for up to 28 days after treatment.

This work was supported by NIH grant 5R44AI136081-03
Combined CpG and MPL as vaccine adjuvants improve immunity and heterosubtypic protection against lethal viral challenge in single dose and prime/boost regimens
Authors: Brown Deborah M ,   Clarkson Trudeau Biomedical Scholars, Lampe Anna T
Published: January 2023

Current Influenza A virus (IAV) vaccines show poor efficacy, often 40–60%. A strategy to improve effectiveness of IAV vaccines is to include adjuvants to activate innate immunity. We examined the potential of combined pattern recognition receptor agonists CpG and MPL to increase IAV vaccine induced protection. When administered in a single, low dose intramuscular hemagglutinin (HA) protein vaccine, mice receiving MPL + CpG experienced less morbidity after homologous challenge compared to mice receiving single agonists. In experiments analyzing heterosubtypic protection, vaccines containing MPL delivered intramuscularly (i.m.) showed increased protection against weight loss while the addition of CpG enhanced antibody responses but not survival. To determine if MPL and CpG have combinatory effects at different sites, HA and NP were delivered with MPL i.m. while HA and NP were simultaneously delivered with CpG intranasally (i.n.). Data demonstrate that combination MPL i.m. + CpG i.n. abrogated morbidity, but did not improve survival compared to MPL + CpG i.m. Further, mice given MPL + CpG produced IgG antibodies that were cross-reactive to H1N1 virus, yet magnitude of antibody responses did not correlate with protective efficacy. Finally, comparison of MPL i.m. + CpG i.n. in a prime/boost regimen, followed by heterosubtypic challenge, showed that weight loss was significantly lowered in mice that received prime/boost vaccination compared to mice that received prime only. Higher antibody responses induced by a second dose of vaccine did not correlate with protective capacity, suggesting that antibody levels, while indicators of vaccine immunogenicity, do not directly correlate with ability to protect mice from severe disease.

Supported by Trudeau Institute and Clarkson University.
Effect of methotrexate on response to Covid-19 mRNA vaccines in patients with Autoimmune Inflammatory Rheumatic Diseases: a longitudinal study
Authors: Rus Violeta , Nguyen Vinh , Shriver Mallory , Degrezia Mary , Alhassan Eaman , Passetti Marcella , Thomas Afton , Rus Horea G
Published: January 2023

Humoral response to Covid-19 mRNA vaccines was assessed in 30 patients with autoimmune inflammatory rheumatic diseases (AIRD) taking methotrexate (MTX) and 22 volunteers at 8 and 24 weeks after the first vaccine. Ten patients were also followed at 2–4 weeks after the booster dose. Concentrations of neutralizing Anti-Spike and anti-RBD Ab (U/ml) were assessed using the V-PLEX Covid-19 ACE2 Neutralization kit.

At 8 weeks after vaccine, median titers of anti-Spike and anti-receptor binding domain (RBD) Ab were significantly lower in patients receiving MTX (6.2, IQR [3.1–19.87] and 5.4 [2.5–15], respectively) vs. controls (25.4, [10–89] and 22.2 [10–88]). At 24 weeks post vaccine, neutralizing antibody titers declined in both groups. Although median anti-Spike and anti-RBD Ab levels remained significantly lower in patients (1.7 [0.0–3.6] and 1.9 [0.6–2.7], respectively) vs. controls (3.8 [1.2–6.6] and 3.5 [1.5–6.3]), the magnitude of the difference decreased from 4 fold at 8 weeks to 2 fold at 24 weeks. Lower titers of anti-Spike and anti-RBD Ab were found in patients taking MTX in combination with biologics (anti-TNFs or anti-Blyss) vs. those on MTX monotherapy, although only the anti-RBD Ab levels were statistically significant (2.7 [1.6–5.8] vs. 7.6 [3.8–17.4], p=0.04). Levels of neutralizing Ab did not differ in patients that held methotrexate around vaccination compared to those who did not. One control but no patients had breakthrough Covid-19 infections. After booster dose, Ab titers increased by a highly variable amount in all patients, ranging between 1.9 – 649 fold for anti-Spike Ab and 2.1 – 29 fold for anti-RBD Ab. Further studies are needed to assess the duration of the booster vaccine protection in this population.

Supported by an ICTR grant from Univ of Maryland Sch of Medicine
RNA origami as a vaccine assembly platform to promote CD8 T Cell mediated anti-tumor immunity
Authors: Yip Theresa , Qi Xiaodong , Kollings Jack , Yan Hao , Chang Yung
Published: January 2023

CD8 T cell mediated immunity is critical for controlling tumor growth. However, constructing synthetic vaccines that can effectively induce this arm of the immunity has been challenging. With the advancement of nanotechnologies, various nanostructures have been explored to link antigens and adjuvants together for increased stability, lymph node delivery, cellular uptake, and activation of innate immunity, all of which may help elicit potent T cell responses against tumors. Here, we explored a self-assembling RNA origami (RNA-OG) nanostructure as a vaccine platform to assemble tumor antigens. In addition to acting as an antigen carrier, RNA-OG is an intrinsic TLR3 agonist and therefore functions as a potent adjuvant. Thus, these RNA-OG-antigen vaccines offer simultaneous delivery of antigens and adjuvants for better initiation of T-cell mediated immunity. We demonstrated enhanced antigen cross-presentation of RNA-OG-linked antigens by dendritic cells and subsequent induction of antigen-specific CD8-T cell responses. Using B16-OVA (ovalalbumin) murine melanoma tumor model, RNA-OG-OVA vaccines induced an anti-tumor immune response that contributed to long-term tumor immunity and increased survival of the animals. Overall, our data demonstrates that RNA-OG can function as a potent anti-tumor vaccine platform.
Diversity Outbred and Collaborative Cross mouse models identify and validate host genetic loci associated with Immune Checkpoint Inhibitor response
Authors: Hackett Justin , Movaheddin Nasrin , Glassbrook James E , Bross Madeline , Muñiz Maria , Dyson Gregory , Gibson Heather
Published: January 2023

Immune checkpoint inhibitors (ICI) for cancer therapy have improved outcomes for a variety of malignancies, however many patients fail to benefit. While tumor-intrinsic mechanisms are involved in therapy resistance, it is unclear to what extent host genetic background plays in response. To investigate this in a pre-clinical setting, we utilized C57BL/6 (B6) F1 crosses of the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. We find a wide variation in response to anti-PD-1/anti-CTLA-4 ICI in B16F0-bearing DOB6F1 mice (n=142) with ~30% showing partial response and ~13% showing complete tumor elimination. Genetic linkage analysis identified several genomic loci associated with therapeutic outcomes. Of the associated loci, the region within chromosome 13 was further validated in CC mice bearing the positive (NZO) versus negative (C57BL/6) driver genotypes. ICI response was detected in 2/3 CCB6F1 predicted responder models, with no response in predicted non-responders. This locus contains the murine prolactin family which is a known immunomodulating cytokine with association to various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Combined ICI with prolactin shows an interaction effect against B16F0, with 5/8 mice exhibiting slowed tumor growth. Analysis of CD8 infiltrates by IHC demonstrate that dual treatment of prolactin and ICI results in a significant increase of CD8 infiltrates over other groups. Collectively, this study shows that host genetics play a role in ICI response and that prolactin may be a positive regulator of ICI efficacy.

Supported by grants from NIH (R37 CA220482, T32-CA009531, P30 CA022453), the Pardee Foundation, and the DMC Foundation
Analysis of biomarkers in melanoma patients receiving ICI treatments: pre- vs post in responders and progressors
Authors: Daftarian Pirouz M , Nadine Nadine , Hushur Orkash , Dronca Roxana , Dutta Navnita , Knutson Keith L
Published: January 2023

Markers of immune related adverse events and response in patients treated with immune checkpoint inhibitors (ICI) may shed light into ICI resistance, T cell exhaustion and other combinatorial therapies. The elevated levels of these biomarkers have potential to predict if PD-1 or PD-1/CTLA-4 antagonist combination will be effective in a certain Melanoma patient. Therefore, it may potentially guide healthcare providers to change treatment plans. In this study, the ICI treatments we focused on were PD-1 or combination of PD-1/CTLA-4 blockades. The measurement of the biomarkers was conducted at Mayo Clinic, from serum samples obtained in 2 time points, pre-treatments and 2–3 months after the first ICI treatments. Validated IVD kits were used per manufacture’s instruction. Biomarkers could be measured using stored frozen serum samples according to the instructions on how the biomarkers will be measured. We investigated 13 biomarkers in the sera of 76 melanoma patients who received immune checkpoint therapies. The biomarkers tested were, Human ELAFIN ELISA, Human IL-18 ELISA, Reg3α + Universal kit, Anti-Nuclear Antibody Test System, FLUORO AID-1 Test for anti-mitochondrial antibodies, anti-smooth muscle antibodies and anti-parietal cell antibodies, Anti-MDA5 ELISA Kit, Anti-synthetase ELISA Skin, Antineutrophil cytoplasmic antibodies MPO-ANCA ELISA, Antineutrophil cytoplasmic antibodies PR3-ANCA ELISA, Dsg1, Dsg3 ELISA, BP180, BP230, type-VII collagen. When compared with the samples obtained before treatments, IL-18 levels significantly elevated (p value < 0.0001) in post treatment samples of non-responders. Further studies are needed to confirm if elevated IL-18 contribute to the biology of resistance to ICI therapies.

Supported by MBLI
Cancer immunotherapy with oncolytic enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways
Authors: Wang Kepeng
Published: January 2023

Tumors are adept at escaping the immune response through multiple mechanisms that disrupt key processes. This is observed with immune checkpoint blockade therapies and oncolytic viral therapies that are only effective in limited numbers of cancer patients. Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here we describe the use of virus-like vesicles (VLV) for the delivery of trivalent combination cancer therapy. This high-capacity oncolytic RNA replicon vector was adapted to simultaneously and synergistically stimulate immune cell activation, inhibit an immune checkpoint pathway, and disrupt a pro-tumor pathway by co-delivering interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA), respectively. This trivalent viral vector (designated CARG-2020) was injected intralesionally to subcutaneous MC38 and BNL-T3 tumors, and it eradicated large established tumors in mice. The concurrent modulation of IL-12, IL-17, and PD-L1 pathways by CARG-2020 suppressed tumor recurrence and prolonged overall survival. Treatment of primary tumors by CARG-2020 also resulted in marked inhibition of distal tumors, demonstrating a strong abscopal effect of the agent. Mechanistically, CARG-2020 potently activates Th1-armed immunity in primary tumors and the spleen, and inhibits the expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence in mice and extending their survival makes it a promising candidate for use in human cancer immunotherapy.

This work is supported by a National Institute of Health/National Cancer Institute grant (NIH/NCI R01CA262430).
Studies on the Antigenic Properties of the Ultraviruses
Authors: Schultz E. W. , Gebhardt L. P. , Bullock L. T.
Published: January 2023

Though viricidal antibodies were easily demonstrated in the sera from human and monkey poliomyelitis convalescents, no evidence could be elicited to indicate that these antibodies are related to antibacterial antibodies. The presence of complement fixing and precipitating antibodies specific for the virus could not be demonstrated even in sera from hyperimmunized poliomyelitis convalescent monkeys. On the other hand, evidence was obtained which strongly suggests that the antibodies parallel antitoxins in their behavior. Like neutral toxin-antitoxin, neutral serum-virus mixtures may be rendered infective again by dilution with physiological saline solution. Moreover, while an undiluted serum of sufficient titer may render a given quantity of virus material immediately innocuous when such a serum is diluted a certain minimum of time is required to accomplish the same result.
Chemical and Immunological Studies of the Pneumococcus
Authors: Wadsworth Augustus , Brown Rachel
Published: January 2023

From type I pneumococcus cells a specific substance, apparently carbohydrate in nature, was isolated. Its properties and antigenic activity are described as follows: Precipitation occurred with type I antipneumococcus serum in a dilution of 1:6,000,000; also, in a dilution of 1:600,000 with type I antipneumococcus serum from which the antibodies against the specific carbohydrate of Heidelberger and Avery had been adsorbed.Complement was fixed in the presence of type I antipneumococcus rabbit serum.Protein tests were negative and carbohydrate, positive.Mice immunized with it were protected against type I pneumococcus infection.It caused fatal anaphylactic shock in guinea pigs passively sensitized with type I antipneumococcus rabbit serum; also, in guinea pigs passively sensitized with type I antipneumococcus rabbit serum from which the antibodies against the specific carbohydrate of Heidelberger and Avery had been absorbed.
Factors Conditioning the Development of the Tuberculin Type of Hypersensitivity
Authors: Dienes L.
Published: January 2023

Summary and Conclusions

Preliminary treatment with egg-white of a series of animals, thus producing an anaphylactic sensitivieness, before infection with tubercle bacilli and subsequent treatment with egg-white according to methods known to produce a tuberculin type of reaction, apparently prevented in the majority of instances the development of tuberculin-type sensitivity. One-fifth to 0.1 mgm. of egg-white is approximately the minimal single dose required for the constant production of strong tuberculin type of sensitivity. An initial injection of a much smaller amount, however, exerts a marked influence on the effect of injections given at later periods. When successive doses are used in sensitizing the animals, or when repeated skin tests are made, either the anaphylactic or the tuberculin type of sensitivity may develop. Among the factors governing which type of sensitiveness will appear are the intervals between injections, the route of injection not only of the initial but of subsequent doses and the condition of the animal dependent on the state and degree of its infection. Repeated injections in short interval favor the development of anaphylactic type of sensitivity. If the interval after the first injection is long enough for the development of a marked grade of tuberculin type of sensitivity, consecutive injections usually increase the strength of this type of sensitivity. During the first week after intravenous infection with the R1 strain of tubercle bacilli conditions as favorable for the production of the tuberculin type of sensitiveness as those afforded by a strong local infection are not produced. If the intravenous infection is a heavy one the effect appears in contrast to be distinctly inhibitory. It seems fair to conclude that the slight generalized infection which follows a local tuberculous infection has little to do with the effects observed in locally infected guinea pigs.
Blood Grouping in Poliomyelitis
Authors: Jungeblut C. W. , Smith L. W.
Published: January 2023

Summary and Conclusions

The incidence of blood groups in cases of paralytic poliomyelitis occurring under five years of age shows no radical deviation from the normal figures. Above that age there is a distinct diminution in B and a slight increase in O. This shift in the distribution of blood groups becomes progressively more noticeable with the advance in the age at onset, coincident with the drop in general morbidity. The largest number of deaths after five years of age occurred in individuals other than B, with a predominance of A in the five to ten year period and of O in the cases over ten years. The prevalence of poliomyelitis in different parts of the world appears, with certain exceptions, to diminish in direct proportion to the increase in group B and the decrease in groups A and O among the human races. This relative racial insusceptibility seems to occur independently of climatic factors. No more than one-third of six human convalescent sera belonging to blood group A nor more than about half of thirteen sera belonging to blood groups O and AB were capable of neutralizing the virus of poliomyelitis in vitro. On the other hand six out of seven B sera were found to possess this property. These results may be useful in pointing the way to a more judicial selection of suitable donors in the collection of convalescent sera form therapeutic purposes.
Experiments in Passive Transference of Urticarial Hypersensitiveness to Fungous Extracts
Authors: Sulzberger Marion B.
Published: January 2023

Reagins which sensitized the skin to extracts of pathogenic fungi of the trichophyton or achorion group, did not sensitize to to oidium (monilia) extracts. This coincides with previous observations which point to the immunologic relationship of the trichophyton, achorion and microsporon fungi and their immunologic differentiation from the monilias. Three of the 7 patients used as test persons did not accept passive sensitization with these reagins. These 3 and only these had been previously infected (experimenti causa) with an achorion fungus and had received injections of trichophytin prior to their employment in my experiments.
The Rate of Antibody Formation in Monkeys Actively Immunized with Poliomyelitis Virus
Authors: Brodie Maurice
Published: January 2023

In a previous report (1) it was shown that active immunity was produced by skin inoculation of virus-serum combinations, or sub-infective doses of virus-containing tissue, but that the latter gave decidedly more immunity. It is proposed, therefore, to trace the onset and subsequent rate of development of immunity following: (a) a single sub-infective dose of virus containing tissue, and (b) two inoculations, administered at different in-intervals.

Although Römer and Joseph (2) claimed that only after twenty-six days were antibodies for poliomyelitis demonstrable in active immunity, Leiner and Wiesner (3) found them after seven, and in one case, two days, and Amoss (4) three and one-half days after the development of the active disease. Likewise in this laboratory (5) antibody was found in monkeys on the second day after the height of paralysis. In human beings, Netter and Levaditi (6), showed antiviral substance on the twelfth day and Flexner and Amoss (7) on the sixth day of the disease.
Agglutinins and the Conditioned Reflex
Authors: Kopeloff L. M. , Kopeloff N. , Posselt E.
Published: January 2023

Within the limitations of the methods and materials under investigation: Rabbits subjected to a conditioning procedure accompanied by injections of Pn III vaccine failed to show a secondary rise in agglutinins as a result of the conditioning stimulus alone. This is contrary to Metalnikov's conclusions.Agglutinins were measured by Heidelberger and Kabat's microchemical nitrogen technic as well as by the usual agglutination method. Both methods demonstrated the failure to induce an increase of agglutinins as a result of the conditioning procedure employed. Because of the greater accuracy and reproductibility of Heidelberger and Kabat's method more reliance is placed upon the results obtained by their technic.
Toxic Substances in Urine and Sweat of Typhoid Fever Patients As Demonstrated by the Shwartzman Phenomenon
Authors: Stolyhwo Nik
Published: January 2023

In 1928 Gregory Shwartzman described for the first time a new phenomenon which consisted in the following: Rabbits received an intradermal injection of 0.25 cc. of undiluted filtrate of five-to seven-day-old broth culture of a given microörganism (preparatory factors) and then twenty-four hours later were injected intravenously with a given amount of the filtrate (reacting factors). Four to five hours after the intravenous injection, there developed severe hemorrhagic and necrotic reactions at the site of the intradermal injection.

Observations previously reported by me suggested strongly that the Shwartzman phenomenon offered an opportunity to extend our knowledge concerning the hitherto unknown rôle of B. typhosus and B. paratyphosus toxic substances in human infections. In these investigations I attempted first to determine whether substances similar to those necessary for the Shwartzman phenomenon could be discovered in patients suffering from B. typhosus and B. paratyphosus infections.
Observations on Abnormal Isoantibodies Following Transfusions
Authors: Neter Erwin
Published: January 2023

There can be no doubt that all human beings may be classified as belonging to one of the four blood groups of Landsteiner. However, recent investigations have shown that sub-groups exist within these four main blood groups, especially within the group A. Moreover, specific antigens can be demonstrated in red blood cells which are not at all related to the group properties A and B (e.g., factors M, N and P of Landsteiner and Levine). They can be demonstrated only by means of immunization of rabbits with human blood cells containing the respective substances. There are no corresponding normal antibodies present in human serum. Ehrlich and Moregenroth were the first to produce specific isoagglutinins in goats by means of immunization. There are a few reports in the literature concerning specific isoagglutinins in men caused by immunization. Landsteiner, Levine and Janes (1) observed abnormal agglutinins in man after blood transfusions from donors of the same group.
Carbohydrate-Containing Proteins of the Hemolytic Streptococcus
Authors: Heidelberger Michael , Kendall Forrest E.
Published: January 2023

A modification of earlier methods is given for the microdetermination of carbohydrate in protein. Scarlatinal streptococcus protein fractions contain, in general, more carbohydrate than can be accounted for by their nucleic acid content and quite regularly give rise to anticarbohydrate as well as antiprotein when injected into rabbits. Evidence is presented that the non-nucleic acid carbohydrate of the protein fractions is chemically combined “C” substance.
General and Cerebral Anaphylaxis in the Monkey (Macacus Rhesus)
Authors: Kopeloff Nicholas , Davidoff Leo M. , Kopeloff Lenore M.
Published: January 2023

Fatal anaphylactic shock has been produced at will in monkeys following single or multiple preparatory intravenous injections of liberal amounts of fresh undiluted egg-white and in one instance by sensitization through the gastro-intestinal route. Passive transfer of rabbit anti-egg-white serum to a single monkey resulted in anaphylactic death upon injection of antigen. Local cerebral anaphylaxis as evidenced by contralateral focal symptoms and pathologic brain changes including necrosis have been observed in sensitized monkeys upon subsequent injection of the specific antigen. The pathologic picture of fatal anaphylaxis in monkeys is markedly different from that seen in any of the usual laboratory animals.
An Investigation of the League of Nations Standard Requirements for Schick Toxin
Authors: Taylor E. M. , Moloney P. J.
Published: January 2023

In 1931 the Permanent Commission on Biological Standardization of the League of Nations decided, on the basis of coöperative experiments carried out in various countries, to recommend for international use the following regulations for testing a toxin to be used for the Schick test: In choosing a diphtheria toxin for use in the Schick test, the combining power for antitoxin as well as the toxicity of the preparation should be taken into account.The Commission therefore recommends the acceptance of the following definitions of a diphtheria toxin suitable for use in the Schick test, and of the dose for making the test: The toxin shall be matured toxin.
The Antigenic Structure of Shigella Alkalescens (Andrewes) and the Demonstration of Antibodies in the Serum of Patients with Shigella Alkalescens Infections
Authors: Neter Erwin
Published: January 2023

The antigenic structure of Shigella alkalescens was studied, employing eight strains of different sources, and the following results were obtained: Shigella alkalescens, when injected into rabbits, may readily induce the formation of specific agglutinins.Any of the anti-Shigella alkalescens sera reacted with all eight strains of Shigella alkalescens.This agglutination of suspensions of Shigella alkalescens by means of these anti-sera was obtained after short incubation (15 minutes) at 55°C.Antisera for Shigella alkalescens may agglutinate some strains of Shigella paradysenteriae, Flexner indicating a common antigen in the two species. These anti-sera failed, however, to agglutinate Eberthella typhi, Shigella dysenteriae, Shiga or Shigella paradysenteriae, Duval-Sonne.Agglutinin absorptions showed that Shigella alkalescens contains an antigen apparently characteristic of this species and at least one more in common with Shigella paradysenteriae, Flexner.A bacteriophage for Shigella paradysenteriae, Flexner was ineffective toward six strains of Shigella alkalescens tested, but was effective toward seven out of eight strains of Shigella paradysenteriae, Flexner.Patients infected with Shigella alkalescens may develop specific agglutinins.
Photodynamic Action of Methylene Blue on Diphtheric Antitoxin
Authors: Ross Victor
Published: January 2023

Neutralization of diphtheric toxin by antitoxin is inhibited after the exposure of a mixture of antitoxin and methylene blue to light. Flocculation is also inhibited. In our experiments no attempt was made to demonstrate complete inactivation, but judging by the data this should not be difficult. Inactivation does not occur when the mixture of antitoxin and dye is kept in an evacuated vessel, nor when air is admitted and light excluded.
The Neutralization of Pneumococcal Capsular Polysaccharide by the Antibodies of Type-Specific Antisera
Authors: Goodner Kenneth , Horsfall Frank L. , Bauer Johannes H.
Published: January 2023

The filtration of antipneumococcal sera through graded collodion-membranes is greatly facilitated by a considerable dilution of the serum. Dilution also increased the capacity of a given serum to neutralize the type-specific capsular polysaccharide. It is believed that these parallel phenomena are to be explained on the basis of dispersion of antibody-aggregates.