The Acari Hypothesis, IV: revisiting the role of hygiene in allergy.

Allergy and its manifestations were first appreciated in the 1870 s. Today, the mechanism by which specific substances elicit allergic reactions remains poorly understood. This is problematic from a healthcare perspective because the prevalence of allergic disease and its societal costs are substantial. Regarding mechanistic understanding of allergy, a new proposal, The Acari Hypothesis, has been forwarded. The Hypothesis, borne from consideration of alpha-gal syndrome, postulates that acarians, i.e., mites and ticks, are operative agents of allergy. By way of their pathogenic payloads and salivary pattern recognition receptor(s), acarians potentiate in human hosts the generation of IgE against acarian dietary elements. Those elements account for most, if not all, known human allergens. Inasmuch as acarian-human interactions occur on human epithelial surfaces, it is to be expected factors that influence the presence and/or operation of acarians on those surfaces influence the expression of allergic diseases. In this report, it is proposed that two adaptations of catarrhine primates, i.e., Old World monkeys, apes and humans, evolved to deter acarian species: firstly, the expansion of eccrine glands across the entirety of body surface area, and, secondly, the secretion of sweat by those glands. Contemporary hygienic practices that reduce and/or disrupt the operation of eccrine glands are likely responsible for the increase in allergic disease seen today.

The Acari Hypothesis, III: Atopic Dermatitis.

Atopic dermatitis is a chronic relapsing dermatopathology involving IgE against allergenic materials present on mammalian epithelial surfaces. Allergens are as diverse as pet danders, and polypeptides expressed by microbes of the mammalian microbiome, e.g., Malassezia spp. The Acari Hypothesis posits that the mammalian innate immune system utilizes pathogen-bound acarian immune effectors to protect against the vectorial threat posed by mites and ticks. Per The Hypothesis, IgE-mediated allergic disease is a specious consequence of the pairing of acarian gastrointestinal materials, e.g., allergenic foodstuffs, with acarian innate immune effectors that have interspecies operability. In keeping with The Hypothesis, the IgE profile of atopic patients should include both anti-acarian antibodies and specious antibodies responsible for specific allergy. Further, the profile should inform on the diet and/or environment of the acarian vector. In this regard, the prevalence of Demodex and Dermatophagoides on the skin of persons suffering from atopic dermatitis is increased. Importantly, the diets of these mites correspond well with the allergens of affected patients. In this report, roles for these specific acarians in the pathogenesis of atopic dermatitis are proposed and elaborated.

Competing Bioaerosols May Influence the Seasonality of Influenza-Like Illnesses, including COVID-19. The Chicago Experience.

Data from Chicago confirm the end of flu season coincides with the beginning of pollen season. More importantly, the end of flu season also coincides with onset of seasonal aerosolization of mold spores. Overall, the data suggest bioaerosols, especially mold spores, compete with viruses for a shared receptor, with the periodicity of influenza-like illnesses, including COVID-19, a consequence of seasonal factors that influence aerosolization of competing species.

The Acari Hypothesis, II: Interspecies Operability of Pattern Recognition Receptors.

Hypersensitivity to galactose-α-1,3-galactose (α-gal) is an informative example of a pathologic IgE-mediated process. By way of their saliva, ticks are able to sensitize humans to tick dietary elements that express α-gal. Mites, which along with ticks constitute the phyletic subclass Acari, feed on proteinaceous foodstuffs that represent most, if not all, human allergens. Given: (1) the gross nature of the pathophysiological reactions of allergy, especially anaphylaxis, (2) the allergenicity of acarian foodstuffs, and (3) the relatedness of ticks and mites, it has been hypothesized that human-acarian interactions are cardinal to the pathogenesis of allergy. In this report, a means by which such interactions contribute to that pathogenesis is proposed.

Mites, ticks, anaphylaxis and allergy: The Acari hypothesis.

Anaphylaxis is a poorly understood immune process in which a Th2-/IgE-mediated adaptive response commandeers cellular machinery, typically reserved for defense against multicellular ectoparasites, to activate against otherwise benign molecules. Its clinical manifestations consist of rapid pathophysiological reflexes that target epithelial surfaces. The galactose-α-1,3-galactose hypersensitivity response is a compelling model of anaphylaxis for which causation has been demonstrated. At the core of the model, a tick bite sensitizes a recipient to a tick foodstuff. As proposed herein, the model likely informs on the origin of all allergic inflammation; namely, allergy is not intended to protect against seemingly harmless and irrelevant materials, but is, instead, intended to rid epithelial surfaces of pathogen-bearing Acari, i.e., mites and ticks. The demonstrated adjuvant activity of acarian gastrointestinal secretions, when paired with the polyphagous diet of mites, renders acarians eminently suited to accounting, mechanistically, for many, if not all, human allergies.

Extracellular transport of cell-size particles and tumor cells by dendritic cells in culture.

Many particulate materials of sizes approximating that of a cell disseminate after being introduced into the body. While some move about within phagocytic inflammatory cells, others appear to move about outside of, but in contact with, such cells. In this report, we provide unequivocal photomicroscopic evidence that cultured, mature, human dendritic cells can transport in extracellular fashion over significant distances both polymeric beads and tumor cells. At least in the case of polymeric beads, both fibrinogen and the ß2-integrin subunit, CD18, appear to play important roles in the transport process. These discoveries may yield insight into a host of disease-related phenomena, including and especially tumor cell invasion and metastasis.

Adsorbed fibrinogen regulates the behavior of human dendritic cells in a CD18-dependent manner.

The involvement of fibrinogen in inflammation has been considered by many, but the roles of the protein in that process have yet to be fully elucidated. The protein readily coats surfaces and is deposited at sites of inflammation. Furthermore, adsorbed fibrinogen influences many cells of the immune system, likely a result of increased receptor recognition upon ligand immobilization. To better understand adsorbed fibrinogen's role in inflammation, we studied the effects of the protein, adsorbed to the surface of microscopic beads, on human dendritic cells. Adsorbed fibrinogen increased dendritic cell expression of IL-6, IL-8, MIP-1beta and MCP-1. In contrast, solution phase fibrinogen had no effect. Importantly, dendritic cells formed complexes with, and subsequently accumulated around, beads in fibrinogen-dependent fashion. Antibodies directed against CD18 significantly decreased cytokine/chemokine expression and bead-cell complexation. Epsilon-aminocaproic acid limited bead-cell complexation, suggesting fibrinogen degradation products modulate dendritic cell activity. In support of this proposal, fibrinogen fragment D also increased MCP-1 expression by human dendritic cells. Taken together our data indicate adsorbed fibrinogen and its degradation products directly influence human dendritic cell operation. We propose a model whereby adsorbed fibrinogen plays a distinct causatory role in inflammation through its beta(2) integrin-mediated interaction with dendritic cells.

A turbidimetric method for measuring the activity of trypsin and its inhibition.

Microscopic poly(styrene-divinylbenzene) beads coated with a monomolecular film of fibrinogen agglutinate when stirred in the presence of thrombin, a consequence of interbead fibrin formation. Trypsin, by digesting bead-bound fibrin, dissociates bead aggregates at a rate proportional to the amount of enzyme activity. The agglutination of beads and the dissociation of bead aggregates can be monitored turbidimetrically using a platelet aggregometer or other photometric device equipped with a stirred cell. We have exploited the behavior of aggregates of fibrin-coated beads to develop a rapid, sensitive, and accurate method for measuring the activity of trypsin and its inhibition, in aqueous media, including serum. The new method yields serum antitrypsin activity levels that correlate well with immunological levels of alpha1-antitrypsin and, thus, may prove useful for assessing antitrypsin activity in clinical specimens.

Mitogenicity of a spread film of monophosphoryl lipid A.

When spread at the air-water interface, monophosphoryl lipid A (MPLA) forms stable insoluble monolayers that collapse at approximately 55 dyn/cm. At collapse, the exclusion area of each molecule is approximately 119 Angstrom(2), consistent with the cross-sectional area of the lipid's 6 acyl chains. The nominal thickness of such films is approximately 22 Angstrom, determined, presumably, by the length of the acyl chains. For biological modeling of MPLA films, a system was developed in which monolayers of the lipid are supported by monodisperse hydrophobic beads of microscopic dimensions. Beads coated with MPLA monolayers within which the nominal area of each molecule is approximately equivalent to the "take-off" area of the lipid at the air-water interface, 280 Angstrom(2), are mitogenic for spleen cells. Given the natural occurrence of lipid A in the bacterial cell wall as well as the inherent stability of lipid A films, it seems reasonable to assume that at least some of the biological activities attributed to the lipid derive from its presentation/operation at an interface, i.e., on a surface. We propose beads coated with adsorbed films of lipid A will prove useful tools for modeling the activities of the lipid both in vitro and in vivo, and for elucidating the surface dependency and structural requirements of those activities.

Fibrinogen-coated droplets of olive oil for delivery of docetaxel to a fibrin(ogen)-rich ascites form of a murine mammary tumor.

Micronized droplets of olive oil loaded with docetaxel and coated with functional fibrinogen were administered intraperitoneally to mice bearing the fibrin(ogen)-rich ascites form of the TA3/St mammary tumor. When compared with docetaxel administered intraperitoneally as its commercial formulation (i.e., Taxotere), docetaxel-loaded oil droplets coated with murine fibrinogen prolonged the median survival time of tumor-bearing mice from 14.5 to 29.5 days. Drug-free oil droplets provided no therapeutic benefit. Significantly more docetaxel was associated with tumor cells 24 and 48 hours after administration of the drug in fibrinogen-coated oil droplets than after its administration as Taxotere. Consistent with a role for thrombin in the retention of fibrinogen-coated oil droplets within the tumor microenvironment, hirudin significantly reduced the association of tumor cells with docetaxel delivered in fibrinogen-coated oil droplets and, at the same time, reduced the therapeutic efficacy of the droplets to that of Taxotere. Importantly, fibrinogen-coated oil droplets formed rosettes with tumor cells in vivo, a process prevented by hirudin. Although mice treated with oil droplets developed antifibrinogen antibodies, those antibodies seemed to be inconsequential. Taken together, our results and observations indicate fibrinogen-coated oil droplets markedly improve the therapeutic efficacy of docetaxel for the treatment of a mammary tumor grown in ascites form, a consequence of thrombin-mediated retention of the drug-loaded droplets within the tumor microenvironment.

Preparation, characterization, and preliminary application of fibrinogen-coated olive oil droplets for the targeted delivery of docetaxel to solid malignancies.

Micronized droplets of olive oil loaded with docetaxel (1.0 mg.ml(-1)) and coated with fibrinogen were prepared and then characterized for physicochemical and cytotoxic properties in vitro and anticancer activity in vivo. The droplets remain readily dispersible and relatively stable in size for at least 24 h when stored at 4 degrees C. During storage, the fibrinogen remains bound to the droplets and thrombin coagulable. Nucleoside incorporation assays, growth inhibition assays, and clonogenic assays involving several different tumor cell lines all indicate that the cytotoxicity in vitro of docetaxel applied in olive oil droplets is at least as great as that of docetaxel applied in DMSO. When compared with Taxotere, an equivalent dose of docetaxel administered in fibrinogen-coated oil droplets improved the median survival time of B16F10 melanoma-bearing mice from 21 days to 69 days. Furthermore, whereas none of the Taxotere-treated mice survived longer than 34 days, 33% (three of nine) of the mice treated with docetaxel-loaded, fibrinogen-coated oil droplets were apparently free of disease after 139 days. Preliminary studies indicate fibrinogen adsorbed to docetaxel-loaded oil droplets facilitates the retention of the droplets within the fibrin-rich tumor microenvironment. We propose this new formulation may prove generally useful for the treatment of taxane-sensitive, fibrin-rich tumors.

Fibrinogen-coated chylomicrons in gastrointestinal lymph: a new rationale regarding the arterial deposition of postprandial lipids.

The recent discovery that fibrinogen binds to chylomicrons in gastrointestinal lymph has prompted a new rationale regarding the arterial deposition of postprandial lipids, i.e., dietary fat. According to this new rationale, fibrinogen bound to chylomicrons in the gastrointestinal lymph renders those lipid particles and/or their remnants an adhesive potential, even before the particles reach the arterial system. It is proposed that such an adhesive potential, if realized in the vicinity of the arterial wall, can contribute to the nucleation and growth of atherosclerotic plaques, especially during and immediately following a fat-rich meal. Arguments in support of this proposal are made based on the proximity of the lymph outflow tract to the arteries most susceptible to atherosclerosis, and on the tissue distributions and activities of heparin, diamine oxidase, and lipoprotein lipase. This new rationale reconciles existing theories on atherosclerosis, and it also suggests novel means by which to prevent/treat the disease.

Fibrinogen in rat gastrointestinal lymph before, during and after intraduodenal administration of emulsified triglyceride: fibrinogen bound to chylomicrons in gastrointestinal lymph is functional.

Samples of gastrointestinal lymph were collected from fasted, male, Sprague-Dawley rats before, during and after intraduodenal administration of either a phospholipid-stabilized, triglyceride-rich emulsion or the dextrose-saline diluent of the emulsion. In lipid-treated rats, the triglyceride, total protein, and functional fibrinogen contents of lymph increased significantly during the 4 h of continuous lipid infusion, with all analytes returning to near baseline values by 20 h later. Levels of the same analytes changed little, if at all, in control animals. As assessed using immunoblotting, chylomicrons in gastrointestinal lymph are coated with fibrinogen. Fibrinogen-coated chylomicrons readily incorporate into solution phase clots and, in the presence of thrombin, adhere in heparin-preventable fashion to each other and to other fibrinogen-coated surfaces. Taken together, these data indicate that lipid feeding creates in gastrointestinal lymph a condition that is conducive temporally to the physical association of fibrinogen with newly ingested lipids before they reach the circulatory system.

Distribution of silicon/e in tissues of mice of different fibrinogen genotypes following intraperitoneal administration of emulsified poly(dimethylsiloxane) [correction of poly(dimethysiloxane)].

Following injection into the abdominal cavity of a C57BL/6 mouse, droplets of emulsified PDMS visible by light microscopy (diameter > or = 1 microm) disseminate to multiple organs of the animal. Because fibrinogen may facilitate dissemination, we compared histologically the accumulation of PDMS droplets in lymph nodes, lungs, spleen, liver, and left kidney of Fib +/+, Fib +/-, and Fib -/- mice of C57BL/6 background 35 and 75 days after intraperitoneal injection of an emulsion of the polymer. We also used ICP-AES to assess the accumulation of silicon in the lymph nodes, livers, and spleens of the animals. The emulsion droplets ranged in diameter from approximately 0.04 to approximately 80 microm. PDMS droplets visible by light microscopy were in all organs of both Fib +/+ mice and Fib +/- mice. In those animals, droplets were invariably either within or adjacent to inflammatory cells, predominantly macrophages. In contrast, PDMS droplets were visible in none of the organs of Fib -/- mice. Despite the absence of visible droplets in them, the lymph nodes, livers, and spleens of Fib -/- mice, like the corresponding organs of Fib +/+ and Fib +/- mice, contained measurable silicon after 35 and 75 days. The amount of silicon, however, was always greater in the organs of Fib +/+ and Fib +/- mice than in the organs of Fib -/- mice. We attribute the presence of silicon in organs that had no histologic evidence of droplets to diffusion of the very smallest droplets/soluble species of PDMS from the abdominal cavity. Taken together, our data and observations implicate a role for fibrinogen in the dissemination of larger PDMS droplets in vivo. We propose this role involves recognition of droplet-bound fibrinogen by macrophages and, perhaps, other inflammatory cells, and the subsequent fibrinogen-facilitated ingestion and/or extracellular movement of the droplets by those cells.

Mutation of highly conserved arginine residues disrupts the Structure and Function of Annexin V

Background. Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca²+-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Anexin V contains four such domains. Each of these domains has a highly conserved arginine (R). Methods. To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. Results. Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation, R43E and R115E exhibited much grater susceptibility to proteolytic digestion than the wild type. While Ca²+-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 µ; Ca²+), R43E and R115E exhibited a phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. Conclusions. Our data suggest that mutation of these highly conserved arginine residus in each of the four canonical domains of annexin have differential effects on the phospholipid binding tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation , R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of ammexin V by establishing hydrogen bonds and ionic bridges