A tiny fraction of people immunized with lipid nanoparticle (LNP)-enclosed mRNA (LNP-mRNA) vaccines develop allergic symptoms following their first or subsequent vaccinations, including anaphylaxis. These reactions resemble complement (C) activation-related pseudoallergy (CARPA) to i.v. administered liposomes, for which pigs provide a naturally oversensitive model. Using this model, we injected i.v. the human vaccination dose (HVD) of BNT162b2 (Comirnaty, CMT) or its 2-fold (2x) or 5-fold (5x) amounts and measured the hemodynamic changes and other parameters of CARPA. We observed in 6 of 14 pigs transient pulmonary hypertension along with thromboxane A2 release into the blood and other hemodynamic and blood cell changes, including hypertension, granulocytosis, lymphopenia, and thrombocytopenia. One pig injected with 5x CMT developed an anaphylactic shock requiring resuscitation, while a repeat dose failed to induce the reaction, implying tachyphylaxis. These typical CARPA symptoms could not be linked to animal age, sex, prior immune stimulation with zymosan, immunization of animals with Comirnaty i.v., or i.m. 2 weeks before the vaccine challenge, and anti-PEG IgM levels in Comirnaty-immunized pigs. Nevertheless, IgM binding to the whole vaccine, used as antigen in an ELISA, was significantly higher in reactive animals compared to non-reactive ones. Incubation of Comirnaty with pig serum in vitro showed significant elevations of C3a anaphylatoxin and sC5b-9, the C-terminal complex. These data raise the possibility that C activation plays a causal or contributing role in the rare HSRs to Comirnaty and other vaccines with similar side effects. Further studies are needed to uncover the factors controlling these vaccine reactions in pigs and to understand their translational value to humans.

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In the lifetime of an individual, every single gene will have undergone mutation on about 10¹⁰ separate occasions. Nevertheless, cancer occurs mainly with advancing age. Here, we hypothesize that the evolutionary pressure driving the creation of the T cell receptor (TCR) repertoire was primarily the homeostatic surveillance of the genome. The subtly variable T cells may in fact constitute an evolutionary link between the invariable innate and hypervariable B cell systems. The new model is based on the homeostatic role of T cells, suggesting that molecular complementarity between the positively selected TCR and the self peptide-presenting major histocompatibility complex molecules establishes and regulates homeostasis, strictly limiting variations of its components. Notwithstanding, the ‘homeostatic role of T cells’ model offers a more realistic explanation as to how a naïve clonal immune system can cope with the much faster replicating pathogens, despite a limited repertoire that is capable of facing only a small fraction of the vast antigenic universe at a time.

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BackgroundIntroduction of microbeads into flow‐cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications.MethodsFormaldehyde‐fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow‐cytometric platform for various immunological and biochemical assays.ResultsWe have tested these “biological microbeads” for the simultaneous titration of human α‐fetoprotein (AFP) and human Chorionic Gonadotropin (βhCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers.ConclusionsThe use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow‐cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow‐cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format. © 2005 International Society for Analytical Cytology

Background: Chromatin immunoprecipitation (ChIP) is a widely used technique for the detection of in vivo DNA–protein interactions underlying epigenetic regulation. The standard readout of ChIP is based on semi‐quantitative or quantitative PCR measurements; however, the development of alternative platforms with high throughput potentialities is expected to facilitate the introduction of this method into routine diagnostics. Methods: We have established a flow‐cytometry‐based alternative for the evaluation of ChIP data. The method is based on the capture of the products of a conventional PCR run to low cycle numbers, on microbeads. Results: In vivo histone H4 acetylation and H3 lysine 4 methylation was detected at the promoter of the tissue transglutaminase type 2 gene. These results were confirmed by QPCR measurements. The levels of modifications decreased significantly upon apoptosis and were accompanied by the down‐regulation of TGM2 mRNA expression. Conclusions: This method that we named ChIP‐on‐beads, a combination of flow cytometry and conventional PCR, is a reliable and efficient alternative in the quantitative analysis of ChIP results, especially promising when high throughput monitoring of epigenetic markers of diagnostic importance is required. The method is simple enough to be easily implemented in a routine flow‐cytometric laboratory. © 2006 International Society for Analytical Cytology

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin-and dye labeled reaction products are immobilized on microbeads and the homo-and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero-from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli. ' 2008 International Society for Analytical Cytology Key terms DNA; deletion; insertion; mismatch; nick-translation; flow cytometry; S1 nuclease; chemical cleavage; MLL ENCOURAGED by favorable earlier experience (1-4), we explore the possibilities offered by microbead based flow cytometric technology to perform various forms of molecular biological analysis. Here we demonstrate strategies to detect the frequent double-strand (ds) genetic lesions, insertions and deletions, as well as certain singlestrand (ss) lesions, using a flow cytometric platform. Deletions and insertions can be readily recognized as a result of the changes in the length of PCR products encompassing the particular region. A flow cytometric read-out of such data may offer high throughput (HTP) potential perspective and may also prove useful when small differences are to be recognized; in addition, this approach may be more cost-effective than sequencing in certain cases. Using a model system, the breakpoint-cluster region (bcr) of the mixed lineage leukemia (MLL) gene, we demonstrate that heteroduplices formed after denaturation and reannealing of PCR products of different size are preferentially cleaved by the ss specific nuclease S1, and also by chemical reactions specific for unpaired nucleotides: on the other hand, restriction enzymes are unable to cleave their recognition motives in the overhang area. These reactions lead to significantly altered average fluorescence of the streptavidinated microbeads anchoring the hybrids through their biotinylated ends.

e13102 Background: The problem of the ipilimumab therapy is that a common molecular target (CTLA-4 receptor) is expressed on the targeted and non-targeted T cells, respectively. We suggest that the frequency of immune-related adverse events can be reduced by treating conditions in which the number of the targeted cells is larger than the non-targeted ones and/or by improving the accuracy of the targeting. Methods: The first approach was tested in a phase I clinical trial in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplantation. An ipilimumab blockade was used against CTLA-4 positive allogeneic T cells (responding patients had greater than 80% donor T-cell chimerism). The graft-versus-malignancy (GVM) effects were augmented without a significant impact on graft-versus-host disease (GVHD). A feasible approach could also be pretargeting that has been worked out for radioimmunodetection and radioimmunotherapy. To this end we suggest a testable thought experiment for the treatment of chronic lymphocytic leukemia (CLL). First a streptavidin (StAv) modified anti-CD19 mAb is administered, which is followed by the subsequent delivery of biotin labeled anti-CTLA-4 mAb. The latter endows T cells with the ability to travel to tumor sites without prematurely succumbing to apoptosis, while streptavidin’s ultra-high affinity for biotin (10-15M) ensures a tight bond to the biotin labeled anti-CTLA-4. Results: Using the law of mass action we calculated that above 1 mg/L ipilimumab concentration (i.e. about 5 mg/patient ~70kg) more than 80% of anti-CD19-StAv sensitized B cells will have been bound to anti-CTLA-4-biotin sensitized T cells. Conclusions: This way, the forces of the immune system liberated by the anti-CTLA-4 antibody blockade could be focused with laser sharp accuracy on CLL cells without collateral damage to normal host cells. One should note that 0.3 mg/kg ipilimumab alone already caused mild autoimmunity.