bluuebunny/crossref_metadata_2025 huggingface dataset:
TULIPES: Tn5-mediated ultra low input parallel epigenetic sequencing
Authors: Patterson Dillon , Scharer Christopher , Boss Jeremy M
Published: January 2023

Accumulating evidence reveals that changes in the epigenome regulate gene expression profiles of developing, differentiating, and effector immune cells. Importantly, disturbances in this developmental road map may contribute to aberrant immune responses. The recent development of the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) allows for the epigenetic programming of rare immune cells to be determined. This method utilizes the Tn5 transposase to simultaneously fragment and tag genomic DNA by inserting Illumina sequencing adapters. Unique sequencing indexes are then added to the adapter-modified genomic fragments during the PCR enrichment step of sample preparation. This tagmentation-based approach has emerged as a popular approach to generating sequence-ready libraries. Here, we describe a novel strategy in which we have customized the tagmentation process by using in-house purified Tn5 transposase pre-loaded with indexed adapters. This method allows for a more efficient analysis of samples which increases experimental throughput while significantly reducing sequencing cost.
Tools to simplify panel design for multi-color flow cytometry experiments.
Authors: Bashratyan Roman , Luan Feng-Jun , Widmann Stephanie , Corselli Mirko , Semyonova Maria , Rabenstein Jacob , Stall Alan M
Published: January 2023

While flow cytometry continues to be a critical tool for immunologists, one of the more difficult aspects of this practice is panel design. Fully resolving dim populations of interest can be challenging. Low antigen density, fluorescence spillover, available fluorochromes and limited catalog offerings are just a few of the factors that make panel design difficult often leading to sub-optimal resolution. Previously, we have shown that following a basic set of principles can reduce the number of iterations needed to maximize the resolution for populations of interest. Here we show how recent developments, including the BD Horizon™ Guided Panel Solution (GPS) and BD OptiBuild™ reagents, help simplify panel design even further. The BD GPS was designed with the best practices of panel design at its core. By guiding the user through critical steps in this process and flagging potential problem areas in a proposed panel, the GPS minimizes the need for early panel iteration. BD OptiBuild reagents vastly improves the catalog offering of fluorochrome/specificity combinations, thus reducing the need for panel redesign due to limited reagent availability. To demonstrate the utility of these new tools, we walk through panel design for an 8 color assay on both 3 laser (8 parameter) and 5 laser (18 parameter) systems. The analysis of these panels shows that by using these tools, the chances of obtaining a successful panel increase. Additionally, panel design can be made easier and population resolution improved when utilizing a 5 laser system where impacts of spillover and spread can be minimized. Together, using these tools and approaches the goal is to minimize the number of iterations required to develop panels that meet the needs of the investigator.
Umbilical cord derived monocytes and platelet rich plasma for diabetic wound healing
Authors: Greene-Roos Jennifer A , Laughlin Mary
Published: January 2023

Chronic non-healing ulcers are a significant complication present in approximately 15% of diabetic patients. Despite standard clinical treatment including wound dressing, debridement of necrotic tissue, and offloading, 15–27% of patients with diabetic ulcer will require amputation. Deep infection is a risk factor for amputation. Platelet rich plasma (PRP) and stem cells have been tested for potential application to treat diabetic ulcers and have been shown to improve wound healing. Umbilical cord blood provides robust cellular and secreted products with therapeutic potential, but remains yet to be fully exploited as a resource for wound healing strategies. Our initial proof of concept studies indicate that umbilical cord blood derived monocytes and platelet rich plasma enhance wound healing processes measured by in vitro assays. When compared to monocytes or platelet rich plasma alone, monocytes and platelet rich plasma significantly enhanced neovascularization as measured by the matrigel angiogenesis assay. Monocytes and platelet rich plasma enhanced fibroblast migration in a wound scratch assay. Additionally, monocytes and platelet rich plasma enhance proliferation of endothelial cells and fibroblasts. Furthermore, these monocytes have potent antibacterial effects as measured by their ability to phagocytose P. aeruginosa in vitro. Collectively, this in vitro data indicates that this product could heal wounds by a mechanism involving enhancing neovascularization, proliferation, migration, and antibacterial activities, all processes which are dysregulated in diabetic wound healing.
Combining gene expression data from inflamed tissue and machine learning for blood biomarker discovery
Authors: Milanez-Almeida Pedro , Martins Andrew J , Narayanan Manikandan , Torabi-Parizi Parizad , Franco Luis , Tsang John S , Germain Ronald N
Published: January 2023

Inflammation is common in virtually all diseases. Since immune cells travel through the blood to inflamed tissues, peripheral blood reflects inflammation at distant sites. However, blood biomarkers are hard to detect in global gene expression data since blood is not the site of perturbation and statistical corrections for multiple testing reduce sensitivity. We hypothesized that combining transcriptional signatures from inflamed tissue and machine learning would be useful to derive blood biomarkers. We first tested this in the mouse model of influenza infection. We identified a set of early genes in the lungs associated with lethality after infection. We then tested their differential abundance early in the blood of mice infected with a range of influenza doses. We applied elastic net to build a biomarker able to predict disease outcome (4 classes: early lethal, late lethal, non-lethal vs. non-infected; accuracy on training and validation sets: 91% and 79%). We next confirmed our hypothesis in lung cancer and breast cancer in humans. We identified sets of genes associated with tumors but not with tumor-free tissues and tested their differential abundance in the blood of patients compared to healthy controls. Using elastic net, we built two blood biomarkers to classify subjects according to disease status (case vs. control; accuracy on training and validation sets in breast cancer: 92% and 67%; in lung cancer: 89% and 78%). In conclusion, combining inflamed tissue signatures and machine learning, we were able to derive blood biomarkers for three different diseases in two different species. We expect that this novel strategy can be applied in other diseases as well.

This work was supported by the Intramural Research Program of NIAID, NIH.
IFN-γ-deficient transplant induces acute gut GVHD via dysregulation of RagIIIγ and intestinal microbiota
Authors: Song Qingxiao , zeng Veronica , Wu Xiwei , Riggs Arthur D. , Zeng Defu
Published: January 2023

Gut GVHD is critical for determining the outcome of allogenic hematopoietic cell transplantation (HCT). Damage of Paneth cells, loss of defensins, and increase of anti-bacterial lectin regenerating islet-derived IIIγ (RagIIIγ) can lead to dysregulation of intestinal microbiota and gut GVHD pathogenesis. RagIIIγ can be produced by Paneth cells and intestinal epithelial cells and its secretion is augmented by IL-22 and MyD88-mediated signaling. However, the circumstances leading to its dysregulation remains largely unknown. Here we show that, in a MHC-mismatched model of C57BL/6 donor to BALB/c recipient, one injection of depleting anti-CD4 effectively prevents acute gut GVHD in recipients given wild-type (WT) transplants but has no effect in recipients given IFN-γ−/− transplants. Most of latter recipients died with diarrhea and severe colitis by two weeks after HCT. As compared to recipients given WT transplants, recipients given IFN-γ−/− transplants showed expansion of gut tissue infiltrating CD8+ Tc17 cells that produced both IL-17 and IL-22, although no damage of Paneth cells. Recipients given IFN-γ−/− transplants had markedly increased mRNA levels of RagIIIγ in the intestinal tissue and markedly increased protein levels in the serum as well as significant reduction of anaerobic bacteria Barnesiella in feces. Co-housing recipients given IFN-γ−/− transplants with recipients given WT transplants significantly increased the survival of the former recipients. These results indicate that IL-22-producing Tc17 cells can augment intestinal tissue secretion of RagIIIγ, leading to imbalance of microbiota and gut GVHD pathogenesis, even in the absence of Paneth cell damage.
Mucosal T-Bet+CD8+T cells are implicated in the pathogenesis of human chronic Graftversus- Host Disease
Authors: da Silva Ana C Costa , Dodge Joshua T , Rose Jeremy J , Dhamala Susan , Hakim Frances T , Pavletic Steven Z , Mays Jacqueline
Published: January 2023

Allogeneic hematopoietic stem cell transplantation is a curative cancer immunotherapy whose use is limited by the high incidence of post-transplant chronic graft-versus-host disease (cGVHD), an alloimmune disease that manifests in multiple organ systems including the oral cavity where buccal mucosa (BM) and minor salivary glands (MSG) are affected. As little is known about the immune network that drives pathogenesis of cGVHD, therapeutic agents are limited, and this study used a multiparameter approach to query the relationship between salivary gland gene expression, salivary proteins and the immune cell network within oral tissues with regard to oral cGVHD severity. Microarray analysis of labial MSG from oral cGVHD patients revealed significant up-regulation in gene expression pathways involving immune/inflammatory responses (IL-1 and IL-6 signaling, IL-32), response to oxidative stress (superoxide dismutase 2) and wounding/fibrosis (TIMP-1, TGF-β) in patients versus non-sicca controls. Immunohistochemistry revealed that MSG of these patients had increased numbers of activated T-bet+ CD8+ T cells which were directly associated with acinar structure alteration and glandular infiltrates. Flow cytometric analysis also demonstrated high frequency of CXCR3+ cells in cGVHD-affected MSG, consistent with increased levels of CXCL9 and CXCL10 detectable in the whole saliva of oral cGVHD patients. Parallel flow cytometric analyses in a mouse model of cGVHD in the submandibular salivary gland demonstrated an increased percentage of CD103+ DCs, which can cross-present antigens to activate CD8+ T cells. These studies are ongoing, but implicate activation of T cytotoxic (Tc1) cells in the pathogenesis of oral cGVHD.
Alloreactive T cell proliferation is suppressed by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) independent of AMP-activated protein kinase
Authors: Beezhold Kevin J , Byersdorfer Craig A
Published: January 2023

The benefits of hematopoietic stem cell transplantation, a therapy used to treat a variety of hematologic and immunologic disorders, are often outweighed by the risks of acute graft-versus-host disease (GVHD), where donor T cells attack and destroy host tissues. Our lab has previously shown that targeting lymphocyte metabolism is a promising and novel strategy for the treatment of GVHD. More recently, we have found that AMP-activated protein kinase (AMPK), a major cellular energy sensor, plays a significant role in T-cell proliferation during disease pathogenesis. To explore this relationship further, we treated alloreactive T cells generated during a mixed leukocyte reaction (MLR) with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMP mimetic that strongly activates AMPK. AICAR treatment of MLR cultures strongly repressed T cell proliferation, with a 70% decrease in total cells by 96 hours. Interestingly this decrease in T cell proliferation was independent of AMPK, as similar results were obtained using AMPK−/− cells. Mechanistically AICAR repressed AKT signaling in stimulated T cells, with a 50% decrease in AKT phosphorylation when cells were concurrently treated with 100 μM AICAR. Cytokine driven STAT5 activation was also significantly blunted by simultaneous AICAR exposure. In summary, despite its wide use as an AMPK agonist, we found that AICAR exerts significant inhibitory effects on T cell allo-activation independent of AMPK. This inhibition occurs, at least in part, through decreased phosphorylation of AKT and down-regulation of STAT signaling, making AICAR a potential therapy to combat GVHD and similar inflammatory disorders.
Human mesenchymal stem cells alleviate bleomycin-induced pulmonary fibrosis in humanized mice
Authors: Ni Ke , Liu Ming , Zheng Jian , Wen Liyan , Chen Qingyun , Xiang Zheng , Lam Kowk-Tai , Liu Yinping , Chan Godfrey Chi-Fung , Lau Yu-lung , Tu Wenwei
Published: January 2023

Pulmonary fibrosis is a chronic disease characterized by progressive pulmonary function disruption and activity limitation, of which the pathogenesis and the effective therapy have not been elucidated. Although aberrant immune cell infiltration was found in the lung of patients with pulmonary fibrosis, the contribution of immune dysregulation in pulmonary fibrosis has not been confirmed in human. In order to directly investigate the role human immune response in pulmonary fibrosis in vivo, human periphery blood mononuclear cells (PBMC)-reconstituted humanized mice were used to establish bleomycin-induced pulmonary fibrosis model in this study. In addition, the suppressive efficacy of human bone marrow-derived mesenchymal stem cells (MSC) on pulmonary fibrosis were also evaluated based on this model. Here we found the abnormal activity of the human immune response in the lung prior to pulmonary fibrosis. Human MSC administration at the early stage could significantly modulate pulmonary human immune dysregulation, alleviate pulmonary damage, and improve lung function in humanized mice with pulmonary fibrosis. Furthermore, PD-L1 and indoleamine 2, 3-dioxygenase (IDO) were found to be involved in the immunomodulatory effect of human MSC on pulmonary fibrosis in humanized mice. This study demonstrates the relationship between human immune dysregulation and pulmonary fibrosis in humanized mice. It also suggests the therapeutic efficacy of human MSC for pulmonary fibrosis.
Sialic acid and Galectins mediate Xenogenic Neutrophil-Endothelial Adhesion
Authors: French Beth , Harris Donald , Laird Christopher , Burdorf Lars , Cheng Xiangfei , Feng Chiguang , Cross Alan S , Goldblum Simeon E , Vasta Gerardo R , Pierson Richard N , Azimzadeh Agnes M
Published: January 2023

Sialic acids (Sias) play multifarious roles in immunity, and likely contribute to inflammation seen in xenograft failure. The ‘net’ cellular sialyation state depends on many things including transient loss of Sia moieties with exposure of subterminal galactosyl structures (mediated by sialyltransferases and sialidases [neuraminidases]), which can function as receptors for extracellular lectins, such as galectins. Here, we report the role of Sias in modulating adhesion of human neutrophils (NΦ’s) to pig endothelial cells.

Human (hAECs) and pig (pAECs) aortic endothelial cells were pretreated with Clostridium perfringens neuraminidase (NA, to remove Sias), human tumor necrosis factor (hTNF, 25 ug/ml), or both. CalceinAM-labelled human NΦ’s were treated with rhIL-8, NA, or N-acetyllactosamine (LacNAc, a galectin inhibitor). NΦ adhesion was measured and expressed as percent adhesion.

NA treatment of AECs increased NΦ adhesion to pig (36.3% vs 22.4%) more than to human (24.2% vs 20.7%) AECs. NΦ adhesion was similarly increased by TNF activation of AECs in static and flow conditions. hIL-8 dramatically increased NΦ adhesion to pAECs (76.5%, p<.001) and hAECs (45.3%, p<.001), with the highest adhesion observed with NA-treated pAECs and hIL-8-activated NΦ’s. Pre-exposure of the NΦ’s to LacNAc prevented their galectin-mediated adhesion to galactose moieties exposed on the NA-treated pAECs (13.7%, p<.0001).

In summary, AEC de-sialylation promotes xenogenic NΦ adhesion, an effect completely inhibited by pre-incubation of Nf’s with LacNAc. These results suggest the inhibition of sialidase activity or galectin binding as promising therapeutic approaches to limit inflammation in xenogeneic organ injury.
A Minimum Conditioning Protocol towards Transplantation Tolerance in NOD Mice by Mixed Hematopoietic Chimerism
Authors: Lin Jiaxin , Chan William F. N. , Anderson Colin C.
Published: January 2023

Stable mixed hematopoietic chimerism is a robust method for inducing donor specific tolerance. However, its clinical application is dampened by the toxicity of current recipient conditioning regimens. We previously showed an irradiation-free mixed chimerism protocol in diabetes prone NOD mice is achievable with antibodies to T cells and CD40L together with busulfan and high dose rapamycin. We sought to generate a more clinically feasible chimerism protocol and tested the hypothesis that more efficient recipient T cell depletion would eliminate the need for anti-CD40L (known to cause thromboembolism in humans) and rapamycin.

We preconditioned NOD mice with donor specific transfusion from fully mismatched mice (d −10), cyclophosphamide (CYP) (d −8), antibodies against CD90 and/or CD4 + CD8 (d −6, −1, 4, 9, 14), busulfan (d −1) and donor bone marrow transplant (d 0). Flow cytometry was used to detect chimerism.

Mixed chimerism was induced in 33/45 NOD mice. Stable chimerism with multilineage donor cells was maintained in 21/33 recipients. Loss of chimerism could be predicted by a lower early level of chimerism at d 4, 9 or 14. Inclusion of αCD90 mAb, busulfan and CYP was critical for chimerism induction. With anti-CD90 17/23 mice became stable chimeras. 5/5 chimeric mice accepted skin from bone marrow donors but rejected skin from MHC-matched and minor antigen mismatched donors. The loss of anti-donor Vβ11+ T cells and anti-recipient Vβ17+ T cells in stable chimeric mice indicated the establishment of chimerism involves clonal deletion.

Conclusion

A protocol causing rapid and robust recipient T cell depletion generated chimerism without the need for anti-CD40L or rapamycin in tolerance induction resistant NOD mice.
Upregulation of IL-33 protects against chronic allograft rejection
Authors: Turnquist Heth Roderick , Liu Quan , Mathews Lisa , Dai Helong , Lucas Anna , Zhang Zhongqiang , Lott Jeremy , Matta Benjamin M , Watkins Simon
Published: January 2023

Background

IL-33 is a broadly expressed, but poorly understood IL-1 cytokine. After lung and muscle injury, immunoregulatory and tissue reparative functions of IL-33 have been suggested. Yet, pro-inflammatory IL-33 properties are known. How endogenous IL-33 shapes outcomes after of solid organs transplant is an important, but unanswered question.

Methods

Mouse heterotopic heart transplant (HTx) models of both acute [BALB/c (H2d) to WT or Il33−/− B6 (H2b)] and chronic rejection [Il33+/+ or Il33−/− bm12 (H2bm12) to WT or Il33−/− B6 mice] were utilized. Cytokines were quantitated in transplant and naïve heart tissues by qRT-PCR and Western blot. HTx were evaluated microscopically following staining with H&E, Trichrome or IHC. IL-33+ cell types within the HTx were identified by confocal. Splenocytes were analyzed by flow cytometry. IL-33 quantities were determined in clinical samples from pediatric HTx (n=39) recipients.

Results

Acute and chronic rejection, and surgery increased HTx IL-33 levels in mice. BALB/c IL-33+HTx cells were predominantly CD45− cTnI− vimentin+ with subsets of α-SMA+ Desmin+ or CD31+ cells. Clinical samples displayed increases in IL-33 during acute rejection. IL-33 was protective during both acute and chronic rejection in mice. At 100 days post HTx, Il33−/− Bm12 grafts displayed dramatically increased chronic rejection-associated fibrosis and vessel disease, especially in Il33−/−recipients. A lack of IL-33 was associated with increased HTx immune infiltrates as well as decreased systemic and local Treg levels.

Conclusion

Our data support an unappreciated, yet critical role for upregulated IL-33 in heart allograft protection, particularly during alloimmune responses causing chronic rejection.
Clonal Deletion and Anergy Play Dominant Role To Achieve Immune Tolerance After Reduced Intensity Unrelated Donor Cord Blood Transplantation (UCBT)
Authors: Chen Xiaohua , Hill Memphis , Szabolcs Paul
Published: January 2023

The mechanism of immune tolerance after successful hematopoietic stem cell transplantation is not fully understood. Both central (clonal deletion) and peripheral (anergy, Treg, Tr1) mechanisms are conceivable and may even coexist. In this study, we set up serial experiments to test for the presence and relative contribution of these mechanisms after HLA-mismatched UCBT in the GvH direction.

Nine patients (age 1m to 9y) were transplanted and enrolled on a clinical trial (NCT01852370). Five immunocompetent patients were off immunosuppression therapy (IST) with no GVHD, while 4 had either limited or recently resolved GvHD and were still on IST when studied. Donor T cell chimerism was a median 97%. Purified T cells responses to host APCs were measured by MLR (3HTdR) and CTL reaction (Europium assay), along with cytokine secretion profiling (Bioplex). Host-reactive T cell clones were tracked with TCRVβ repertoire by ImmunoSEQ® (Adaptive Biotech). In tolerant patients, there was no significant MLR or CTL response towards host APC. Similarly, cytokine profiles revealed non-responsiveness while T cells responded vigorously to 3rd party APC by all assays. Recipient-specific T cell clones that were identified from infused UCB stimulated by host APC pre-UCBT became undetectable. There was no indication of Treg or Tr1 cell involvement in sustaining tolerance as deletion by IL-2 immunotoxin or IL-10R blockade was unable to “break” tolerance. However, low dose IL-2 (5U/mL) was able to restore some proliferation (S.I.=19; p=0.05) and IL-13, IFNγ, TNFα secretion, but in lesser degree in comparison to 3rd party responses (S.I.=112). In summary, clonal deletion and anergy contribute to immune tolerance after UCBT with deletion being dominant.
Prevention of acute graft-versus-host disease by anti-human OX40L monoclonal antibody
Authors: Tripathi Trivendra , Yin Wenjie , Kim Renee , Xue Yaming , Zurawski Sandy , Hanabuchi Shino , Liu Yong-Jun , Oh SangKon , Joo HyeMee
Published: January 2023

Graft-versus-host disease (GVHD) is one of the major hurdles for the success of solid organ and bone marrow transplantation. Numerous strategies to control alloreactive donor T cell responses have been used in the clinic with limited success. In this study, we investigated the effectiveness of anti-human OX40L monoclonal antibody (ahOX40L) in the prevention and treatment of acute GVHD (aGVHD) in a human xenogeneic model. NOD/SCID/γc−/−NOD.Cg-PrkdcscidIl2rgtm1Sug/Jic mice were transplanted with human PBMCs. Animals were treated with ahOX40L or control antibody either when human PBMCs were inoculated or at the onset of aGVHD. Clinical manifestations and immune profiles of human lymphocytes were monitored over time. ahOX40L treatment significantly alleviated disease severity and prolonged survival in both preventative and therapeutic models. Serological examination of circulating human PBMCs over the course of aGVHD revealed that ahOX40L treatment controls proliferation of CD45+CD3+ T cells, particularly CD8+ T cell expansion, resulting in an increased CD4+/CD8+ T cell ratio. In addition, ahOX40L treatment greatly reduced the infiltration of OX40-expressing CD3+ T cells in the target organs. In line with this, T cells in mice treated with ahOX40L decreased expression of tissue homing receptors. We further observed that ahOX40L treatment significantly down-regulated IL-21 and TNFα but increased IL-10 expression by T cells. Importantly, ahOX40L treatment was more effective in reducing aGVHD-associated mortality compared with neutralizing IL-21, TNFα or IL-21 plus TNFα. Altogether, our data support the clinical development of ahOX40L for the prevention and treatment of aGVHD.
Correct and Incorrect Methods of Performing the “Daily Titrations” for the Wassermann Reaction and Other Forms of Complement Fixation
Authors: Ottenberg Reuben
Published: January 2023

The strength of guinea pigs complement varies greatly. There are in general two different methods employed by different workers to compensate for this varying potency in determining doses of complement and amboceptor for use in the Wassermann reaction as well as in complement fixation with bacterial antigens. These methods are (1) keeping the dose of complement constant and varying the dose of amboceptor from day to day according to the varying strength of the complement, and (2) keeping the dose of amboceptor constant and varying the dose of complement from day to day according to its varying strength. The fact does not seem to have been recognized that these two procedures represent entirely different things and produce different results.

None of the authors gives any reason for adopting the one or the other procedure.
A Study of Two Hundred and Ninety Post Mortem Wassermann Reactions
Authors: Graves Stuart
Published: January 2023

The diagnostic value of the Wassermann test for syphilis performed on sera from human blood taken post mortem has been the subject of investigation and discussion for the last ten years. Practically all published reports on the work have been made in Germany. The first to carry out this research, Much and Fraenkel (1) and Pick and Proskauer (2), concluded that the Wassermann reaction gave reliable results when done on post mortem blood and that the results confirmed the prevailing comprehension of fibrous meso-arteritis, aneurism of the aorta, etc., as syphilitic lesions. Later investigators, on the contrary, arrived at different conclusions. Beside others, Krefting (3), de Besche (4) and Von Bruck (5) decided against the employment of the Wassermann reaction on post mortem blood because they found a very considerable number of positive reactions in cases where not the slightest probability of the existence of syphilis was apparent.
A Comparative Study of Different Methods of Performing the Wassermann Test for Syphilis
Authors: Smith J. Wheeler , MacNeal Ward J.
Published: January 2023

In a group of 110 syphilitic individuals in various stages of the disease, the cholesterin-reinforced antigen with incubation at 37°C. gave 58.2 per cent, the simple antigen at 37°C., 32.2 per cent, and the simple antigen at 8°C., 77.1 per cent of positive tests. In a group of 43 patients, probably syphilitic, the first method yielded 65.9 per cent, the second 36.3 per cent and the third method 75.0 per cent of positive fixations. In a group of 59 patients, probably not syphilitic, the first method yielded 40.0 per cent, the second 1.6 per cent and the third 5.0 per cent of positive tests. In a group of 265 non-syphilitic patients, tests by all methods were negative throughout.
Studies in Anaphylaxis
Authors: Weil Richard
Published: January 2023

It may now be regarded as a well established fact that cellular antibody alone is responsible for the reaction known as acute anaphylactic shock. There are, however, certain other manifestations of anaphylaxis which are with difficulty explained upon that basis, and on this account many conservative workers incline to the belief that under certain circumstances circulating antibody may also play an important part in the production of the symptoms. At first sight it might not appear to be a matter of great consequence whether the interaction between antigen and antibody takes place within the cells, or in the fluids of the body. Such, however, is far from the fact. The humoral theory requires the assumption of an intermediary toxic product, a hypothetical anaphylatoxin, which secondarily affects the cells. The cellular theory does not require such an hypothesis.
Immunochemistry of the Proteins of Horse Dander
Authors: Wodehouse R. P.
Published: January 2023

Summary and Conclusions

From horse dander there can be dissolved out with weak alkali a large amount of protein matter. Part of this can be precipitated by neutralization with HCl, and if this is treated with 95 per cent alcohol the major portion, the “alkali metaprotein,” remains undissolved, while a very much smaller portion, the “alcohol soluble protein,” is dissolved by the alcohol. Of the protein substance that is not precipitated by neutralization by HCl a portion, the “heat coagulated protein,” can be precipitated by boiling, when the reaction is slightly acid. After this is removed there still remains in the solution another protein, the “hair peptone.”

Pure keratin was also prepared from the hair and from this a peptone, “keratin peptone,” was prepared by hydrolysing with HCl.

When tested by means of the skin reaction the four proteins dissolved out from the dander by dilute alkali showed distinctly different anaphylactogenic properties and they are likewise different from the serum.

Pure keratin is inactive towards horse asthmatics; so, also, is a peptone prepared by acid hydrolysis from it.

From this it is concluded that there are, besides keratin, at least four proteins present in horse dander and that these are not decomposition products of keratin.
Immunologic Studies in Typhus Exanthematicus
Authors: Olitsky Peter K.
Published: January 2023

Before discussing the results of immunologic studies made in typhus exanthematicus, the relationship of the B. typhi-exanthematici to this disease will be briefly stated.

Up to the present time, this organism has been recovered from the blood of patients suffering from typhus fever endemic or epidemic in the various parts of the world—the United States, Serbia, Bulgaria, Russia and Mexico. It has also been recovered from the blood and spleen of animals, guinea-pigs and monkeys, injected with infectious typhus-fever blood. It has been isolated from typhus-infected lice. It has not been recovered from numerous nontyphus patients, nor from tissues of normal animals, nor from “normal” (nontyphus) lice.

In regard to positive blood cultures in human and experimental typhus fever in animals, the organism can be recovered during the entire febrile period of the disease. It is also found in greatest numbers during the early stages of the disease, especially the chill, and more often in severe than in mild cases.
Studies in Anaphylaxis
Authors: Weil Richard
Published: January 2023

Experimental observations

A. Neutralization of cellular antibody by antigen

B. Neutralization of cellular antigen by antibody

Theoretical considerations
Rôle of Hepatic Tissues in the Acute Anaphylactic Reaction
Authors: Manwaring W. H. , Crowe Harold E.
Published: January 2023

The liver of a normal guinea-pig, repeatedly perfused with a mixture of foreign protein and defibrinated normal or anaphylactic blood, produces little or no change in the toxicity of the perfusion mixture on subsequent tests with isolated anaphylactic lungs.

The liver of an anaphylactic guinea-pig, similarly perfused, usually renders the perfusion fluid almost completely non-toxic for these lungs.

This reduction in toxicity is not due to the presence of anaphylactic humoral elements, but to a specific functional action of the fixed hepatic cells.

The reduction in toxicity is not due to a removal or destruction of the foreign protein in the perfusion fluid.

Evidence points to the explosive formation or liberation of vaso-dilator and broncho-dilator substances by the sensitized liver cells.
A New Method for Making Wassermann Antigens from Normal Heart Tissue
Authors: Neymann Clarence A. , Gager Leslie T.
Published: January 2023

The antigenic value and anticomplementary and hemolytic properties of the various lipoids of beef heart have been investigated. It was found that lecithin is the only substance of the primary ether extract of dried pulverized beef heart that had any antigenic value, and that the most important substance as regards antigenic value occurs in the secondary alcohol extract and is a diaminomonophosphatide. On this basis of solubilities new methods for making antigens have been described.
Studies on the “Toxins” of Hemolytic Streptococci
Authors: Ando K.
Published: January 2023

One can differentiate between toxins of value x and 2x if satisfactory numbers of volunteers are available. The ordinary Dick toxin, though usually very stable, may deteriorate under some unknown condition, while dried toxin may retain its full specific activity for a long period of time. Nucleoprotein in the ordinary toxin may cause some confusion in the results of the Dick test. For the standardization of the Dick toxin it is not enough to determine its skin units; its nucleoprotein content must also be considered, the unsuitableness of a toxin containing blood element being taken for granted. It is recommended to adopt a purified toxin free from nucleoprotein and reduced to the dry condition as a standard toxin. The fact that a qualitative difference between toxins produced by various strains, while it is not marked, nevertheless surely exists, indicates the need of further careful comparison of various toxins as to the potency of their toxicity and especially their antigenic activity before the selection of a toxin as the international standard.
Comparative Study of the Anaphylactic and Tuberculin Types of Hypersensitiveness
Authors: Dienes L.
Published: January 2023

Tuberculous guinea pigs sensitized with egg white and also with various other antigens often die after intraperitoneal injection in a protracted shock which is similar in many respects to the tuberculin shock. It is of special significance that a strong hemorrhagic reaction develops around the tuberculous lesion. This hemorrhagic reaction is not the result of the special sensitiveness of the lesion, it might be present in any inflammatory area without regard to the origin of the inflammation.

The eggwhite shock differs in two points from the tuberculin shock. The symptoms often begin to develop early after the injection, and in the early phases of the reaction and in the slight reactions usually a drop of temperature is present. These differences are probably connected with the presence of the usual anaphylactic sensitiveness after treatment with eggwhite. When the latter is associated with the tuberculin sensitiveness, the tuberculin shock often presents the same temperature curve and early onset of symptoms as the eggwhite shock.

The capacity to react with severe protracted shock develops later after the treatment than either the tuberculin type of skin sensitiveness or anaphylaxis. We observed it only in actively sensitized guinea pigs, and our experiments to transfer it passively remained unsuccessful. The different manifestations of the hypersensitive condition develop in a large measure independently from each other. The independence of the severe protracted shock from the anaphylactic shock is shown also by the observation that the desensitization to the acute anaphylactic shock does not interfere with the development of the severe protracted shock.

The severe protracted eggwhite shock and tuberculin shock are probably analogous processes; but without a more thorough understanding of the physiologic mechanism of the reaction this conclusion must be regarded as only provisory.
Diphtheria Toxoid. Preparation and Dosage
Authors: Povitzky Olga R. , Jackson Erla , Eisner Minnie
Published: January 2023

Under the old nomenclature the term “toxoid” meant a slightly toxic diphtheria toxin which was used instead of toxin-antitoxin in some cases for immunization against diphtheria (1). At present Ramon's “Anatoxin” and “Toxoid” are synonymous terms in this country. Under the regulation of the Hygienic Laboratories of the Public Health Service “toxoid” or “anatoxin” should not possess any toxicity.

Detoxification shall be so complete that five human doses given subcutaneously in guinea pigs weighing 300 grams will cause no signs of diphtheria poisoning, including paralysis at any time during a period of thirty days.

The minimum antigenic requirements are put down as follows:

The original toxin from which the toxoid is made shall have an L + dose of not more than 0.20 cc. or an m.l.d. of not more than 0.0025 cc.

At least ten guinea pigs weighing 270 to 320 grams shall receive subcutaneously the initial human dose.
Remarks Concerning Landsteiner's Discovery of Isoagglutination and the Blood Groups, with Special Reference to a Paper by J. A. Kennedy
Authors: Zinsser Hans , Coca Arthur F.
Published: January 2023

In a preceding issue of this Journal there appeared a paper by Dr. J. A. Kennedy entitled “Isohemagglutination: The Work of Jan Janský with a Critical Analysis.” The paper was accepted by the Journal since it is the purpose of this publication to offer its columns freely for the discussion of controversial opinions in immunology. It was understood, however, that the Editors reserved the privilege of replying to statements which seemed, in their opinions, to be misinterpretations of facts. Since the paper of Dr. Kennedy questions the priority of Karl Landsteiner on the observation of isohemagglutination, the present note is published, the Editors believing that Dr. Kennedy is in error in this assertion.

In 1900, Landsteiner first observed serological, individual differences in human beings. In 1901, he described the three common groups, further laid the foundations for their scientific classification and for the serological recognition of these groups.
Note Concerning Differences Between the Clumping of Pseudoagglutination and that of Isoagglutination
Authors: Coca Arthur F.
Published: January 2023

In a paper by J. A. Kennedy that appeared in the preceding issue of this Journal, illustrations are reproduced from an article by Shattock, which apparently are considered by Kennedy as representing isoagglutination.

Since I disagree with this interpretation, believing the illustrations to be those of pseudoagglutination, and since I have had occasion to see how mistakes in blood-grouping can be made on account of confusing the two phenomena, it seems to be not superfluous to present side by side photographs of these two sorts of reactions (figs. 1 to 4).

One will see from these pictures that the two kinds of clumping differ sufficiently to permit, in general, a differentiation, which, as a rule, can be made easily by the use of the fact, first described by Shattock, that pseudoagglutination is prevented by slight dilution (1:2 or 1:3) of the serum.

Two features stand out in both of figures 1 and 2: first, the clumping, and secondly, the rouleaux, which are prominent—one may say characteristically so—at the periphery of the clumps.
Antigenic Value of Botulinum Toxoids Kept one Year at Ice Box Temperature
Authors: Graham Robert , Thorp Frank
Published: January 2023

An antigenic decline in botulinum toxoids of 30 to 60 per cent over a period of one to twelve months was measured by the increased number of arbitrary units required to protect guinea pigs against lethal amounts of homologous toxins. None of the eleven toxoids that proved non-toxic in 1-unit doses later became toxic in the same amounts, notwithstanding an occasional toxoid inoculated pig died of intercurrent infection. Seven of eleven samples of botulinum toxins A, B, and C containing 0.3 to 0.9 per cent formalin became detoxified at 37° to 42°C. in one month. The remaining four of eleven samples required two to five months for detoxification. One sample (3826 A) retained its original antigenic properties for five months, another (3217 B) for three months, three for two months, and two for one month. Ten of the eleven toxoids were atoxic and antigenic in 2-unit doses at ten to twelve months of age. The rate of detoxification in botulinum toxoids by formalin and heat seemed uncorrelated with the longevity of the antigenic properties. One toxin with 20,000 guinea pig lethal doses per cubic centimeter required a longer period of time for detoxification than other toxins containing 10,000 guinea pig lethal doses per cubic centimeter. However no consistent difference in the rate of antigenic decline was observed between toxoids prepared from toxins containing 10,000 and 20,000 guinea pig lethal doses per cubic centimeter. The pH of eleven botulinum toxoids changed from an original titer of pH 7.6 in the inoculated mediums to an average of pH 6.7 in seven and one-half months.
Food Allergens
Authors: Vaughan Warren T.
Published: January 2023

While cutaneous testing for allergens has been of great aid in the study of the allergic disease, the tests not infrequently fail to identify the etiologic agent even when the case is manifestly allergic. Foods are less likely to give positive reactions than are pollens. Rowe (1) finds that frequently less than 50 per cent of those sensitive to a certain food give positive skin reaction to that food. Alexander (2) concludes that positive skin reactions are obtained in about 50 per cent of asthmatics. Several writers, including Schloss (3), O'Keefe (4), Spain (5), Eyermann (6), Dale and Thornburg (7), Rowe and Richet (8) have discussed the occurrence of specific food allergy in cases giving negative skin tests to these foods. Conversely it is a common observation that some foods, pollens and other allergens which give clear cut positive scratch or intradermal reactions do not cause allergic symptoms when the patient comes into contact with them through the normal channels.
On the Spontaneous Immunization of Rabbits to Vaccine Virus
Authors: Duran-Reynals F.
Published: January 2023

It has been shown in previous publications (1, 2) that in the testicle, especially, and also to some degree in certain other organs, there exists a factor which is capable of greatly augmenting the lesions produced by viruses and bacteria. In the course of experiments designed to test the possibility of neutralizing this factor by an antitesticular serum, a number of rabbits were given repeated injections of testicle extract subcutaneously, intravenously and intraperitoneally. As a control, others were similarly injected with spleen extract. In the course of testing the effects of the resultant sera on the factor of testicle extract, it was observed that in certain instances not only was the enhancing power of testicle extract neutralized, but the vaccine virus used in the test was inactivated. An examination was made into this unexpected result.
ADAM17-deficient mast cells exhibit decreased IgE-mediated activation due to impaired Lyn phosphorylation.
Authors: Luker Andrea , Conrad Daniel
Published: January 2023

Allergies and asthma are common ailments that are on the rise globally. Mast cells play a direct role in the signs and symptoms commonly displayed by allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAM17 in mast cell activity. Using inhibitor-based and genetic knockout studies, loss of ADAM17 reduced mast cell degranulation. This resulted in decreased cytokine and histamine release. ADAM17MC−/− mice exhibited less severe IgE-mediated passive systemic anaphylaxis, while no difference was seen when initiated by exogenous histamine. We previously showed that ADAM17 forms a protein complex with the signaling molecule Lyn. Upon cross-linking, ADAM17−/− mast cells have reduced Lyn phosphorylation which affects downstream signaling events. ADAM17 may play a role in apical FcɛRI signaling through interactions with Lyn.
MicroRNA-146a is a negative regulator of IL-33 stimulated mouse mast cells.
Authors: Taruselli Marcela T. , Qayum Amina Abdul , Ryan John J.
Published: January 2023

Interleukin 33 (IL-33) is an inflammatory cytokine that promotes allergic disease by activating ILC2, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. The IL-33 receptor, ST2, shares signaling cascades with the TLR family, but homeostatic control of ST2 function is poorly understood. MicroRNA-146a (miR-146a) is induced by LPS and suppresses TLR4 signaling in macrophages. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow derived mast cells (BMMC). Induction required MyD88, Akt, and NFκB, since antagonizing these pathways decreased miR-146 expression. BMMC transfected with a miR-146a antagomir or derived from miR-146a KO mice showed enhanced cytokine expression in response to IL-33, suggesting that mir46a is a negative regulator of IL-33-ST2 signaling. Our data further suggest that miR-146a may act by targeting IRAK proteins, because mir146a KO BMMC have increased IRAK1 and IRAK4 expression. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.
Characterization of the phenotype of the connective tissue mast cell knockout Mcpt5Cre
+
;Rosa(DTA) mice in the lung
Authors: Cohen Jonathan
Published: January 2023

Mast cells are roughly grouped as resident connective tissue mast cells (CTMCs) and mucosal mast cells which are recruited to mucosal epithelia during inflammation. Although mast cells are known to play critical roles in allergic lung inflammation, little is known about the specific function of pulmonary CTMCs under any condition. To investigate their function, we employed Mcpt5Cre+; Rosa(DTA) transgenic mice to eliminate cells that ever expressed Mcpt5, a classic marker of CTMCs. Unexpectedly, we discovered that the lungs of these mice contained an increased number of mast cells, likely mucosal mast cells. In addition, we found spontaneous inflammation characterized by recruitment of neutrophils and eosinophils, elevated il-33 expression, mucus overproduction and excessive collagen in the airway. Inflammation was detected as early as postnatal day 21 but resolved around 2–3 months of age. In contrast, other tissues that normally have abundant CTMCs, such as the skin and intestine, present no baseline phenotypes in Mcpt5Cre+;Rosa(DTA) mice. These findings suggest a tissue-specific role of CTMCs in maintaining tolerance in the lung, potentially by inhibiting the recruitment of pro-inflammatory mucosal mast cells. Further, in conflict with previous findings that the Mcpt5Cre line specifically targets CTMCs, lineage labeling using Mcpt5Cre+;Rosa(TmRed) mice revealed the presence of TmRed reporter in CTMCs and a small subset of other leukocytes in the lung. While these leukocytes are being mapped, the already identified cell types are unlikely to contribute to the baseline phenotype of Mcpt5Cre+;Rosa(TmRed) mice. Our ongoing studies aim to determine the role of CTMCs in establishing immune tolerance.