Cryptic late-onset myocarditis after coronavirus vaccination.

As we enter a new era of mRNA-based therapeutics, evidence on genetic or environmental factors that might predispose to unknown off-target side effects, gains in importance. Among these factors, exercise appears likely to have influenced otherwise cryptic cases of early-onset postvaccination myocarditis. And the existence of a distinct late-onset myocarditis is now being recognized. Here, three case-history reports suggest crypticity (the author's own case), unless provoked by a preexisting cardiac morbidity (one case), or by immune checkpoint blockade to enhance anticancer autoimmunity (several cases). These reports are supported by noninvasive fluorodeoxyglucose-based cardiac scan comparisons of multiple vaccinated and unvaccinated subjects. In pre-pandemic decades, applications for funds by the leading innovator in mRNA-based therapeutics seldom gained peer-review approval. Thus, at the start of the pandemic, the meager data on such side effects could justify only emergency approval. We must do better.

Aggregation-prone peptides from within a non-self-protein homoaggregate are preferred for MHC association: Historical overview.

New technologies assist re-evaluation of hypotheses on generation of immune cell repertoires and distinctions of self from non-self. Findings include positive correlations between peptide propensities to aggregate and their binding to major histocompatibility complex (MHC) proteins. This recalls the hypothesis that foreign proteins may homoaggregate in host cytosols prior to releasing their peptides (p) to form pMHC complexes. Clues to this included aggregation-related phenomena associated with infections (rouleaux formation, pyrexia, certain brain diseases). By virtue of 'promiscuous' gene expression by thymic presenting cells - perhaps adapted from earlier evolving gonadal mechanisms - developing T cells monitor surface pMHC clusterings. This evaluates intracellular concentrations of the corresponding proteins, and hence, following Burnet's two signal principle, degrees of self-reactivity. After positive selection in the thymic cortex for reactivity with 'near-self', high-level pMHC clustering suffices in the medulla for negatively selection. Following Burnet's principle, in the periphery low-level clustering suffices for T cell stimulation and high-level clustering again provokes negative selection (immunological tolerance). For evolving intracellular pathogens, fine-tuned polymorphisms of their host species have limited to 'near-self' some mimicking adaptations. It is proposed that while entire pathogen proteins may have evolved to minimize their aggregability, the greater aggregability of their peptides remains partially hidden within. Two-step proofreading mechanisms in prospective hosts select proteins containing aggregable peptide for the generation of pMHC clusters at the surface of presenting cells. Through mutations, some proteins of pathogens and cancer cells tend to converge towards the host 'near-self' that its T cells have auditioned to address.

Positive selection of immune repertoires: A short further history.

The importance of the negative selection of self-reacting cells from immune repertoires was easily recognized since it would militate against autoimmune disease. However, while the existence of positive selection in the auditioning of newly formed immune cells is now recognized, it is taking longer to understand its role. With the removal or suppression by negative selection of a subset of immune cells that self-react, would the specificities of remaining immune cells range widely to confront the universe of 'non-self' antigens, including some borne by potential microbial pathogens? Alternatively, from among those remaining immune cells, could some be picked out (positively selected) based on 'advanced knowledge' of some character likely to be common to those pathogens? To exploit 'holes' created by negative selection, it was predicted that pathogens would attempt to mimic their hosts by progressive stepwise mutation towards host 'self'-a process entailing passage through a host anti-'near-self-reactivity' arena. Anticipating this, those hosts that over evolutionary time 'learned' to positively select from developing immune repertoires, cells with reactivities against 'near-self', would be advantaged by natural selection. The benefits of this narrowing of the range of host immune reactivities are now clearer and may solve Burnet's paradox, which concerns the ability of an organism's immune cells to attack its own cancer cells. However, while supporting this in the context of T cell immunity, Manczinger and his colleagues now suggest that some wily pathogens may be exploiting their hosts' narrow defence foci by 'seeking', through mutation, dissimilarity from host 'self'.

Neutralism versus selectionism: Chargaff's second parity rule, revisited.

Of Chargaff's four "rules" on DNA base frequencies, the functional interpretation of his second parity rule (PR2) is the most contentious. Thermophile base compositions (GC%) were taken by Galtier and Lobry (1997) as favoring Sueoka's neutral PR2 hypothesis over Forsdyke's selective PR2 hypothesis, namely that mutations improving local within-species recombination efficiency had generated a genome-wide potential for the strands of duplex DNA to separate and initiate recombination through the "kissing" of the tips of stem-loops. However, following Chargaff's GC rule, base composition mainly reflects a species-specific, genome-wide, evolutionary pressure. GC% could not have consistently followed the dictates of temperature, since it plays fundamental roles in both sustaining species integrity and, through primarily neutral genome-wide mutation, fostering speciation. Evidence for a local within-species recombination-initiating role of base order was obtained with a novel technology that masked the contribution of base composition to nucleic acid folding energy. Forsdyke's results were consistent with his PR2 hypothesis, appeared to resolve some root problems in biology and provided a theoretical underpinning for alignment-free taxonomic analyses using relative oligonucleotide frequencies (k-mer analysis). Moreover, consistent with Chargaff's cluster rule, discovery of the thermoadaptive role of the "purine-loading" of open reading frames made less tenable the Galtier-Lobry anti-selectionist arguments.

Metabolic optimization of adoptive T cell transfer cancer immunotherapy: A historical overview.

After prolonged extracorporeal multiplication in physiological culture media, there can be curative infusions of a cancer patient's own cytotoxic T cells (adoptive T cell transfer; ACT), which must achieve efficient activation in potentially adverse tumour microenvironments. With spectacular, yet irregular, success, improvements are needed. Developing lymphoid cells are biologically selected, not only for 'near-self' reactivity (positive selection), but also to avoid self-reactivity (negative selection). Thus, success requires harnessing near-self cells while avoiding extreme autoimmune phenomena. Abrupt metabolic changes accompanying T cell activation to leave the G0 stage and enter the G1 stage of the cell cycle (eg enhanced glycolysis) are accompanied by increased transcription of the G0S9 gene that mediates salvage synthesis of NAD+ from nicotinamide; the latter has recently been shown to increase the efficiency of ACT. Despite theoretical and experimental advances, there has not been parallel progress in simulating in vivo conditions with culture media that were initially formulated for their positive benefits for tumour cell lines (cell survival and proliferation). Yet for lymphoid cells, inhibition or death (ie immunological tolerance) is as important as their activation and proliferation (immunological response). Thus, use of media optimized for the latter may mask the former. The resilience of established culture protocols may have been partly politically driven. However, unphysiological conditions have sometimes yielded fortuitous insights. Optimization of culture media for specific tissues must consider the nature of problems addressed in research settings and the need to avoid mishaps in clinical settings.

Two signal half-century: From negative selection of self-reactivity to positive selection of near-self-reactivity.

With the emergence of clonal selection ideas in the 1950s, the development of immune cell repertoires was seen to require the negative selection of self-reacting cells, with surviving cells exhibiting a broad range of specificities. Thus, confronting a universe of not-self-antigens, a potential host organism spread its resources widely. In the 1960s, the two signal hypothesis showed how this might work. However, in the 1970s an affinity/avidity model further proposed that anticipating a pathogen strategy of exploiting "holes" in the repertoire created by negative selection, hosts should also positively select near-self-reacting cells. A microbe mutating an antigen from a form foreign to its host to a form resembling that host should prevail over host defences with respect to that antigen. By mutating a step towards host self, along the path from non-self to self, it should come to dominate the microbe population. By progressive stepwise mutations, such microbes would become better adapted, to the detriment of their hosts. But they would lose this advantage if, as they mutated closer to host self, they encountered progressively stiffer host defences. Thus, as described in the affinity/avidity model, positive selection of lymphocytes for specificities that were very close to, but not quite, anti-self (ie, "anti-near-self") should be an important host adaptation. While positive selection affects both B and T cells, mechanisms are uncertain. Converging evidence from studies of lymphocyte activation, either polyclonally (with lectins as "antigen-analogs") or monoclonally (by specific antigen), supports the original generic affinity/avidity model for countering mutations towards host self.

On certain two-signal perspectives of lymphocyte activation and inactivation, thymic G-quadruplexes, and the role of aggregation in self/not-self discrimination.

Distinctive "two signal" paths in immunology, taken by researchers with different academic backgrounds, seem to have both contained facets of the truth. Having been influenced by education at a medical school where Almroth Wright's early contributions were not forgotten, the author's "path less followed" led to views that began to gain recognition late in the twentieth century when the intimate relationship between innate and acquired immunity became more apparent.

Lymphocyte repertoire selection and intracellular self/non-self-discrimination: historical overview.

Immunological self/non-self-discrimination is conventionally seen as an extracellular event, involving interactions been receptors on T cells pre-educated to discriminate and peptides bound to major histocompatibility complex proteins (pMHCs). Mechanisms by which non-self peptides might first be sorted intracellularly to distinguish them from the vast excess of self-peptides have long been called for. Recent demonstrations of endogenous peptide-specific clustering of pMHCs on membrane rafts are indicative of intracellular enrichment before surface display. The clustering could follow the specific aggregation of a foreign protein that exceeded its solubility limit in the crowded intracellular environment. Predominantly entropy-driven, this homoaggregation would colocalize identical peptides, thus facilitating their collective presentation. Concentrations of self-proteins are fine-tuned over evolutionary time to avoid this. Disparate observations, such as pyrexia and female susceptibility to autoimmune disease, can be explained in terms of the need to cosegregate cognate pMHC complexes internally before extracellular display.

'A vehicle of symbols and nothing more'. George Romanes, theory of mind, information, and Samuel Butler.

Today's 'theory of mind' (ToM) concept is rooted in the distinction of nineteenth-century philosopher William Clifford between 'objects' that can be directly perceived and 'ejects', such as the mind of another person, which are inferred from one's subjective knowledge of one's own mind. George Romanes, a founder with Charles Darwin of the discipline of comparative psychology, considered the minds of animals as ejects, an idea that could be generalized to 'society as eject' and, ultimately, 'the world as an eject' - mind in the universe. Yet, Romanes and Clifford only vaguely connected mind with the abstraction we call 'information', which needs 'a vehicle of symbols' - a material transporting medium. However, Samuel Butler was able to address, in informational terms depleted of theological trappings, both organic evolution and mind in the universe. This view harmonizes with insights arising from modern DNA research, the relative immortality of 'selfish' genes, and some startling recent developments in brain research.

Ohno's hypothesis and Muller's paradox: sex chromosome dosage compensation may serve collective gene functions.

Muller found halving gene dosage, as in males with one X chromosome, did not affect specific gene function. Why then was dosage "compensated?" This paradox was solved by invoking collective gene functions such as self/not self discrimination afforded by protein aggregation pressure. This predicts female susceptibility to autoimmune disease.

Speciation, natural selection, and networks: three historians versus theoretical population geneticists.

In 1913, the geneticist William Bateson called for a halt in studies of genetic phenomena until evolutionary fundamentals had been sufficiently addressed at the molecular level. Nevertheless, in the 1960s, the theoretical population geneticists celebrated a "modern synthesis" of the teachings of Mendel and Darwin, with an exclusive role for natural selection in speciation. This was supported, albeit with minor reservations, by historians Mark Adams and William Provine, who taught it to generations of students. In subsequent decades, doubts were raised by molecular biologists and, despite the deep influence of various mentors, Adams and Provine noted serious anomalies and began to question traditional "just-so-stories." They were joined in challenging the genetic orthodoxy by a scientist-historian, Donald Forsdyke, who suggested that a "collective variation" postulated by Darwin's young research associate, George Romanes, and a mysterious "residue" postulated by Bateson, might relate to differences in short runs of DNA bases (oligonucleotides). The dispute between a small network of historians and a large network of geneticists can be understood in the context of national politics. Contrasts are drawn between democracies, where capturing the narrative makes reversal difficult, and dictatorships, where overthrow of a supportive dictator can result in rapid reversal.

Centenary of Haldane's 'rule': why male sterility may be normal, not 'idiopathic'.

While the term 'idiopathic' may be applied correctly to many diseases of unknown origin, its broad application to undiagnosed cases of human male infertility is unwarranted. Infertility can be a nonpathological expression of the action of Nature's normal qualitycontrol mechanisms. We now celebrate the centenary of Haldane's famous 'rule' paper that has clarified much uncertainty. Furthermore, there are similarities between two 'seed organs' that audition and then export, either T cells (thymus), or germ cells (gonad). Nature sets high bars both for generating T-cell repertoires and for gamete entry into the next generation. Extrapolations from thymus studies suggest that germ cells are severely scrutinized for incompatibilities at both protein and nucleic acid levels, and many perish. Scrutiny continues through fertilization to embryos, which may abort, sometimes with couples unaware. The auditioning continuum is something that the inchoate forms we once were had to progress through. Even post-partum and into adulthood, it continues. Defining a point when Nature relaxes and 'life' can be considered as having begun, is not easy. Those who medicalize the normal with inappropriate terminology may reinforce certain attitudes on the morality, ethics, and legality of induced abortion.

Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy.

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

When few survive to tell the tale: thymus and gonad as auditioning organs: historical overview.

Unlike other organs, the thymus and gonads generate nonuniform cell populations, many members of which perish, and a few survive. While it is recognized that thymic cells are "audited" to optimize an organism's immune repertoire, whether gametogenesis could be orchestrated similarly to favor high-quality gametes is uncertain. Ideally, such quality would be affirmed at early stages before the commitment of extensive parental resources. A case is here made that, along the lines of a previously proposed lymphocyte quality control mechanism, gamete quality can be registered indirectly through detection of incompatibilities between proteins encoded by the grandparental DNA sequences within the parent from which haploid gametes are meiotically derived. This "stress test" is achieved in the same way that thymic screening for potential immunological incompatibilities is achieved-by "promiscuous" expression, under the influence of the AIRE protein, of the products of genes that are not normally specific for that organ. Consistent with this, the Aire gene is expressed in both thymus and gonads, and AIRE deficiency impedes function in both organs. While not excluding the subsequent emergence of hybrid incompatibilities due to the intermixing of genomic sequences from parents (rather than grandparents), many observations, such as the number of proteins that are aberrantly expressed during gametogenesis, can be explained on this basis. Indeed, promiscuous expression could have first evolved in gamete-forming cells where incompatible proteins would be manifest as aberrant protein aggregates that cause apoptosis. This mechanism would later have been co-opted by thymic epithelial cells which display peptides from aggregates to remove potentially autoreactive T cells.

X chromosome reactivation perturbs intracellular self/not-self discrimination.

New reports indicate a chromosomal rather than hormonal basis for the susceptibility of females to autoimmune disease. It is held that if females reactivate an inactivated X chromosome, there will be overexpression of certain X-located genes affecting immune function. Hence, normal mechanisms of self/not-self discrimination might be impaired resulting in immune reaction to self antigens. However, the data are also consistent with the long-held view that the demands of intracellular self/not-self discrimination have driven the evolution of X-chromosome dosage compensation. It was proposed that, whether cells are in male or female bodies, concentrations of proteins are fine-tuned up to their aggregation thresholds. A disruption of this equilibrium, by agents originating either externally (for example, virus) or internally (for example, reactivated X chromosome), generates homoaggregates that trigger responses against the respective not-self or self antigens. Thus, female susceptibility to autoimmune disease may not be because certain immune system genes happen to be X-located, but because self/not-self discrimination was the raison d'être for X-chromosome dosage compensation in the first place.

Samuel Butler and human long term memory: is the cupboard bare?

Memory studies in biological systems distinguish three informational processes that are generally sequential--production/acquisition, storage, and retrieval/use. Identification of DNA as a storage form for hereditary information accelerated progress in that field. Assuming the path of successful elucidation in one memory field (heredity) to be heuristic for elucidation in another (brain), then progress in neuroscience should accelerate when a storage form is identified. In the 19th century Ewald Hering and Samuel Butler held that heredity and brain memory both involved the storage of information and that the two forms of storage were the same. Hering specified storage as 'molecular vibrations' but, while making a fuller case, Butler was less committal. In the 20th century, the ablation studies of Karl Lashley failed to identify unique sites for storage of brain information, and Donald Hebb's 'synaptic plasticity' hypothesis of distributed storage over a neuronal network won favor. In the 21st century this has come under attack, and the idea that brain and hereditary information are stored as DNA is advocated. Thus, albeit without attribution, Butler's idea is reinstated. Yet, while the case is still open, the synaptic plasticity and DNA hypotheses have problems. Two broad alternatives remain on the table. Long term memory is located: (1) in the brain, either in some other macromolecular form (e.g. protein, lipid) or in some sub-molecular form (e.g. quantum computing and 'brain as holograph' hypotheses) or (2) outside the brain. The suggestion of the medieval physician Avicenna that the brain 'cupboard' is bare--i.e. the brain is a perceptual, not storage, organ--is consistent with a mysterious 'universe as holograph' model. Understanding how Butler came to contribute could be heuristic for future progress in a field fraught with 'fractionation and disunity'.

Microsatellites that violate Chargaff's second parity rule have base order-dependent asymmetries in the folding energies of complementary DNA strands and may not drive speciation.

Models for meiotic recombination based on Crick's "unpairing postulate" require symmetrical extrusion of stem-loop structures from homologous DNA duplexes. The potential for such extrusion is abundant in many species and, for a given single-strand segment, can be quantitated as the "folding of natural sequence" (FONS) energy value. This, in turn, can be decomposed into base order-dependent and base composition-dependent components. The FONS values of top and bottom strands in most Caenorhabditis elegans segments are close, as are the corresponding base order-dependent and base composition-dependent components; any discrepancies are in the base composition-dependent component. This suggests that the strands would extrude with similar kinetics. However, interspersed among these segments and at the ends of chromosomes (telomeres) are segments containing short tandem repeats (microsatellites) which, by virtue of their high variability, have been postulated to inhibit the pairing of homologous chromosomes and hence drive speciation. In these segments, there are usually wide discrepancies between the FONS values of top and bottom strands, mainly attributable to differences in base order-dependent components. Analyses of artificial microsatellites of different unit sizes and base compositions show that this asymmetrical distribution of folding potential is greatest for microsatellites when the units are short and violate Chargaff's second parity rule. It is proposed that when there is folding asymmetry, recombination proceeds by special, strand-biased, somatic mechanisms analogous to those operating with Chi sequences in Escherichia coli. If meiotic recombination in the germ-line requires extrusion symmetry, then a general inhibitory influence of microsatellite-containing segments could mask the antirecombinational influence of their variability. Thus, microsatellites may not have driven speciation.

Almroth Wright, opsonins, innate immunity and the lectin pathway of complement activation: a historical perspective.

Two clinical tests - the erythrocyte sedimentation rate and the opsonic index - have long been known to non-specifically detect pathology based on their responsiveness to changes in serum proteins. In infections serum levels of specific antibodies increase. However, for healthy subjects Wright held that antibodies contributed minimally to opsonic activity (the complement-enhanced phagocytosis of microorganisms). The activity was present in newborn serum, was increased in the acute phase of an immune response prior to antibody increase, and was less specific. Furthermore, defective opsonization was associated with undue susceptibility to certain infections, for which a genetic basis was later found. With the demonstrations of complement-mediated lysis both of normal cells by foreign (plant) lectins, and of foreign cells (microorganisms) by animal lectins, it now appears that endogenous lectins correspond to the heat-stable component of Wright's serum opsonic activity. His work leads to the lectin pathway of complement activation with specificities limited to the recognition of relatively immutable surface sugars - predictable pathogen characters that contrast with the less predictable targets of the adaptive immune system.

"Altered-self" or "near-self" in the positive selection of lymphocyte repertoires?

Positive selection of lymphocyte repertoires is now recognized as applying to both B and T cells. However, much of the early literature on positive selection focussed on cell-mediated immunity (T cells), which biased consideration of its general biological role. The term "altered-self," which initially captured the idea of self (i.e. MHC) altered by the addition of what was later found to be a peptide fragment, has not proven robust and may now be clouding our understanding. It is recommended that the term "near-self" be reintroduced since it captures the essence of the probable underlying adaptive process-sub-threshold self-reactivity to countermand rapid pathogen mutation.