Immunoglobulin G from single plasma donor in immune globulin intravenous causes false positive pyrogen test.

A sudden, unprecedented failure of USP rabbit pyrogen tests for multiple 10% IGIV-C lots prompted a thorough investigation of the root cause for this phenomenon. All microbe-related testing, including Limulus amebocyte lysate test for endotoxin, proved negative, and no deficiencies were discovered in manufacturing. Plasma pool composition analysis revealed that a single plasma donor ("Donor X″) was common to all pyrogenic IGIV-C lots and that as little as one unit of "Donor X″ plasma (in a pool of ∼4500 units) was sufficient to cause IGIV-C lot failure in the USP rabbit pyrogen test. Whole plasma and Protein A-purified IgG from "Donor X″ caused a temperature increase in rabbits; however, all IgG samples tested pyrogen-negative in two in vitro cell-based pyrogen tests. Flow cytometry showed that "Donor X″ IgG bound strongly to rabbit white blood cells (WBC) but minimally to human WBC. Exclusion of "Donor X″ plasma from manufacturing marked the end of IGIV-C lots registering positive in the USP rabbit pyrogen test. This failure of multiple 10% IGIV-C lots to pass the USP rabbit pyrogen test was demonstrated to be due to the highly unusual anti-rabbit-leukocyte specificity of IgG from a single donor.

Analysis of IL-6 and IL-1ß release in cryopreserved pooled human whole blood stimulated with endotoxin.

To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1ß to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at -196℃, the detection limits of the IL-6/IL-1ß responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1ß release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1ß responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1ß tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

Behavioural fever in zebrafish larvae.

Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (TR at 28 °C) and thermal gradient conditions (TCH: 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: TR and TCh. Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection.

Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT).

Pyrogen testing represents a crucial safety measure for parental drugs and medical devices, especially in direct contact with blood or liquor. The European Pharmacopoeia regulates these quality control measures for parenterals. Since 2010, the monocyte activation test (MAT) has been an accepted pyrogen test that can be performed with different human monocytic cell sources: whole blood, isolated monocytic cells or monocytic cell lines with IL1ß, IL6, or TNFα as readout cytokines. In the present study, we examined the three different cell sources and cytokine readout parameters with the scope of accelerating the assay time. We could show that despite all cell types being able to detect pyrogens, primary cells were more sensitive than the monocytic cell line. Quantitative real-time PCR revealed IL6 mRNA transcripts having the largest change in Ct-values upon LPS-stimulation compared to IL1ß and TNFα, but quantification was unreliable. IL6 protein secretion from whole blood or peripheral blood mononuclear cells (PBMC) was also best suited for an accelerated assay with a larger linear range and higher signal-to-noise ratios upon LPS-stimulation. The unique combination with propan-2-ol or a temperature increase could additionally increase the cytokine production for earlier detection in PBMC. The increased incubation temperature could finally increase not only responses to lipopolysaccharides (LPS) but also other pyrogens by up to 13-fold. Therefore, pyrogen detection can be accelerated considerably by using isolated primary blood cells with an increased incubation temperature and IL6 as readout. These results could expedite assay time and thus help to promote further acceptance of the MAT. Copyright © 2016 John Wiley & Sons, Ltd.

Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study.

AIM: We aimed at evaluating pyrogen contamination of dental implants made of titanium and zirconia by using gene expression analysis in a whole-blood in vitro assay. MATERIAL AND METHODS: Titanium and zirconia implants (five each) were incubated in human whole blood. Samples were assayed for gene expression levels of toll-like receptor 4 (TLR4), TLR9, interleukin (IL)-1ß, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kB), tumour necrosis factor (TNF)-α, and Fas-associated protein with death domain (FADD) as indicators of surface contamination resulting in lipopolysaccharides (LPS)-stimulated TLR- or TNF-mediated immune responses. Gene expression was assayed using real-time quantitative polymerase chain reaction (RT-qPCR). Non-stimulated blood from the same donor served as a negative control, and blood stimulated with LPS served as a positive control. After dry-heat treatment with dry heat, all implants were re-analysed as described above. RESULTS: Both implant systems contained surface contaminants evoking a pro-inflammatory response similar to that induced by LPS. After dry-heat treatment, gene expression was significantly decreased to levels similar to those of negative control samples. CONCLUSIONS: The results demonstrated LPS-like surface-bound contaminants in both tested implant systems. Depyrogenation with dry heat seems to be an effective means of reducing such contamination in dental implants.

[Biological activity of native and modified lipopolysaccharides Rahnella aquatilis].

The results of the comparative toxicity studies of native lipopolysaccharide (LPS) of Rahnella aquatilis 96U037 and that modified by tin complexes indicates that, due to the modification of LPS by tin complex with benzoylhydrazone of 4-dimethylaminobenzaldehyde, a decrease of its toxicity was observed that led to disappearance of the pyrogenic effect. All obtained derivatives lost completely the antigenic activity both in homologous and heterologous systems which may indicate to the interaction of modifying complexes with certain groups being the components of antigenic determinant. The paper is presented in Ukrainian.

[The role of incomplete clearance of apoptotic cells in the etiology and pathogenesis of SLE]. / Die Bedeutung einer unvollständigen Beseitigung apoptotischer Zellen für Atiologie und Pathogenese des SLE.

Systemic lupus erythematosus (SLE) is a complex prototypic autoimmune disease that is based on genetic factors (complement deficiencies) and is influenced by gender (female), environment (infections and UV irradiation), as well as random events (somatic mutations). The course of the disease is influenced by genes (e.g. FcgammaRIIA) and behaviour (sun-exposure). Inefficient clearance of dying cells and subsequent accumulation of apoptotic cell remnants is an intrinsic defect causing the permanent presence of cellular debris responsible for the initiation of autoimmunity. We favour the hypothesis that post-apoptotic debris accumulates in germinal centres, activates complement, and serves as a survival signal for B-cells that had stochastically become autoreactive in the process of somatic hypermutation (etiology). In the presence of autoantibodies against apoptotic cells or adaptor molecules the accumulation of post-apoptotic remnants (SNEC) causes immune complex formation and their pathological elimination, maintaining auto-inflammation. The SLE-type autoimmunity addresses nucleic acid-containing complex antigens (viromimetica). Autoantibody-protein-nucleic-acid complexes are likely to be mistaken for opsonised viruses. As a consequence, the immune system responds with the production of type-I interferons, a hallmark of SLE (pathogenesis). We conclude that the pathogenicity of autoantibodies is strongly increased if autoantigens are accessible and immune complexes are formed, which may be considered a binary pyrogen formed from less pro-inflammatory components. The accessibility of cognate autoantigens is likely to be related to impaired or delayed clearance of apoptotic cells.

Biological characterization of the zinc site coordinating histidine residues of staphylococcal enterotoxin C2.

The bacterial toxin staphylococcal enterotoxin C2 (SEC2) can cause staphylococcal toxic shock syndrome and food poisoning. Although the previously determined crystal structure of SEC2 revealed that some histidine residues (His47, His118 and His122) contribute to the binding of zinc ions, little is known about their biological roles in SEC2. This prompted us to investigate the role of the zinc site coordinating histidine residues in the biological activities of SEC2. The mutants with substitutions at positions 118 and 122 all retained T-cell stimulatory activity, whereas the histidine mutants at position 47 were defective in the ability to stimulate T-cell proliferation. Further toxicity assays in vivo indicated that mutants SEC2-H118A and SEC2-H122A were defective in emetic and febrile activities. However, mutant SEC2-H47A could cause significant emetic and febrile responses in comparison with the other two histidine mutants. These findings suggested that the zinc-coordinating histidine residues play significant roles in superantigen and toxic activities of SEC2 and further implied that superantigen and febrile activities could be separable in staphylococcal enterotoxins. The results also show that it should be possible to design new SEC2 immunotherapeutic agents that have superantigen activity and low toxicity.

Cryopreservation of differentiated HL-60 cells for pyrogen testing.

All-trans retinoic-acid (ATRA) differentiated HL-60 cells can be used to detect pyrogens such as bacteria, bacterial components, yeasts and fungi. Differentiated HL-60 cells obtain neutrophil like characteristics and if stimulated the differentiated HL-60 cells produce reactive oxygen species in a dose dependent manner. Culturing and differentiation of cell lines are time consuming activities and require suitable facilities; cryopreservation of pre-differentiated cells could provide the basis for an easily distributable pyrogen testing kit. Cryopreservation of granulocytes has proven to be very complicated and neutrophils are especially difficult to cryopreserve, most likely due to their large degree of granulation. Here we present evidence that HL-60 cells can be differentiated with ATRA and subsequently cryopreserved. Upon thawing the cells retain their ROS producing capabilities and reactivity towards pyrogens. Further, the cells retain their ability to react dose dependently towards lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan. At pathophysiologically relevant concentrations of LPS, LTA and zymosan the cells retain full reactivity for at least two months when stored in liquid nitrogen. In conclusion, ATRA differentiated HL-60 cells are cryopreservable and retain reactivity upon thawing. It is therefore possible to produce an in-vitro in-house pyrogen test kit for medicines and related products.

Identification of pentadecapeptide mimicking muramyl peptide.

We used monoclonal antibody, generated against N-acetylglucosaminyl-beta1-4-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP), and phage display libraries of random peptides to select for oligopeptides, that mimic GMDP in their biological activity. Selected phage clones displayed a peptide RVPPRYHAKISPMVN (called RN-peptide) on their surface. This peptide was synthesized. RN-peptide was shown to augment the antibody response to ovalbumin in mice while the peptide was non-immunogenic and non-pyrogenic. We also characterized adjuvant activity of 14-, 10- and 7-mer analogs of RN-peptide truncated at the C-terminus and found them to be active. Because both carbohydrate and peptide fragments are critical for the biological activity of muramyl peptides, the results indicate that RN-peptide mimicks the spatial structure of intact GMDP.

TLRs: Professor Mechnikov, sit on your hat.

Toll-like receptors (TLRs) are sensors of foreign microbial products, which initiate host defense responses in all multicellular organisms examined to date. They are the target for most adjuvants, are essential for the establishment of memory in T and B cells and provoke inflammation. They program dendritic cells in their interaction with Th1 cells and their signalling pathways enable a tailoring of host defense responses to the provoking microbe previously unsuspected in the innate arm of immunity. Their discovery and characterisation fills a void in immunology and is the culmination of an effort that began with one of immunology's founding fathers, Elie Mechnikov. Targeting TLRs therapeutically now has the potential to impact on how we treat infectious and inflammatory diseases.

Differential sensitivities of pyrogenic chemokine fevers to CC chemokine receptor 5 antibodies.

Macrophage inflammatory protein (MIP)-1beta and RANTES (regulated on activation, normal T-cells expressed and secreted) are members of the CC-family of chemokines. Although these two peptides are structurally and functionally related to one another, each exhibits distinct features, which allows it to independently regulate specific aspects of the host inflammatory response. They evoked intense and functionally different febrile responses when applied directly on pyrogen-sensitive cells located in the in the preoptic area of the anterior hypothalamus (POA). The present experiments were carried out to test the central role of CCR5, a functional receptor for MIP-1beta and RANTES, in the febrile responses induced by these chemokines when injected directly into the POA. The microinjection of an equimolecular dose (50 pg) of either MIP-1beta or RANTES into the POA induced a rapid onset; monophasic fever in rats that persisted for a long period. The microinjection of 2.0 microg specific neutralizing antibodies against CCR5 (anti-CCR5) into the POA fails to affect the effects on body temperature induced by MIP-1beta. However, pretreatment with the same dose of anti-CCR5 suppressed the febrile response induced by RANTES given at the same site. The microinjection of control IgG or anti-CCR5 does not affect basal temperature, when administered alone at the same hypothalamic site. The present experiments show that hypothalamic CCR5 are functionally involved in the febrile response induced by RANTES, but not by MIP-1beta. They also suggest the existence of functionally different components in the presumptive primary locus of the thermoregulatory controller, in which both chemotactic cytokines, together other mediators, could play a relevant role in the complex process of fever pathogenesis.

Possible relationship between streptococcal pyrogenic exotoxin A and Kawasaki syndrome in patients older than six months of age.

BACKGROUND: We previously investigated antibody titers against four kinds of superantigens [streptococcal pyrogenic exotoxin A (SPEA), streptococcal pyrogenic exotoxin C, toxic shock syndrome toxin-1 and staphylococcal enterotoxin B] in patients with Kawasaki syndrome (KS) younger than 6 months of age and reported a relationship between toxic shock syndrome toxin-1 and KS patients. In this study we have investigated antibody titers in KS patients older than 6 months of age. METHODS: Serum of 81 patients with KS older than 6 months of age, before intravenous gamma-globulin therapy, and 88 normal age-matched children were used in this study. The IgG antibody titers against four kinds of superantigens were measured with an enzyme-linked immunosorbent assay. RESULTS: The KS patients showed significantly elevated mean SPEA titer (P = 0.006) and significantly higher incidence of high SPEA (P = 0.0024) compared with the controls. The SPEA titer in KS patients showed a significant positive correlation with the number of days from onset of illness (P = 0.0002). CONCLUSIONS: The elevated antibody titer against superantigens of KS patients older than 6 months of age was different from that of KS patients younger than 6 months of age. Our results suggest that KS patients' exposure to SPEA occurred a few weeks before the onset of KS. SPEA may be one of the possible etiologic agents of KS among patients older than 6 months of age in Kagoshima, Japan.

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Characterization and immunogenicity of pyrogenic mitogens SePE-H and SePE-I of Streptococcus equi.

Two pyrogenic mitogens, SePE-H and SePE-I, were characterized in Streptococcus equi, the cause of equine strangles. SePE-H and SePE-I have molecular masses of 27.5 and 29.5 kDa, respectively, and each is almost identical to its counterpart in Streptococcus pyogenes M1. Both genes are adjacent to a gene encoding a phage muramidase of 49.7 kDa and are located immediately downstream from a phage genomic sequence almost identical to a similar phage sequence in S. pyogenes M1. Strong mitogenic responses were elicited by both proteins from horse peripheral blood mononuclear cells. However, although both were pyrogenic for rabbits, only SePE-I was pyrogenic in ponies. Convalescent sera contained antibody to each mitogen and horses recovered from strangles or immunized with SePE-I were resistant to the pyrogenic effect of SePE-I. The immunogenicity of SePE-I suggests that it should be included in new generation strangles vaccines. In isolates of S. equi sepe-I and sepe-H were consistently present but they were absent from the closely related Streptococcus zooepidemicus, suggesting that phage mediated transfer was an important event in the formation of the clonal, more virulent, S. equi from its putative S. zooepidemicus ancestor.

Use of intravenous immunoglobulin in the treatment of staphylococcal and streptococcal toxic shock syndromes and related illnesses.

Pyrogenic toxin superantigens comprise a large family of exotoxins made by Staphylococcus aureus and group A streptococci. These toxins include toxic shock syndrome toxin-1, the staphylococcal enterotoxins, and the streptococcal pyrogenic exotoxins (synonyms: scarlet fever toxins and erythrogenic toxins), all of which have the ability to cause toxic shock syndromes and related illnesses. These toxins have a similar three-dimensional structure that allows them to interact with relatively invariant regions of major histocompatibility complex class II molecules on the surface of antigen-presenting cells and with certain variable regions of the T-cell receptor-beta chain. The consequence of these interactions (and other immunobiological properties of the toxins) is the exaggerated release of bioactive cytokines. The latter molecules are responsible for the clinical signs of illness associated with these toxins.

Pyrogenicity and cytokine-inducing properties of Streptococcus pyogenes superantigens: comparative study of streptococcal mitogenic exotoxin Z and pyrogenic exotoxin A.

Streptococcal mitogenic exotoxin Z (SMEZ), a superantigen derived from Streptococcus pyogenes, provoked expansion of human lymphocytes expressing the Vbeta 2, 4, 7 and 8 motifs of T-cell receptor. SMEZ was pyrogenic in rabbits and stimulated the expression of the T-cell activation markers CD69 and cutaneous lymphocyte-associated antigen. A variety of cytokines was released by human mononuclear leukocytes stimulated with SMEZ, which was 10-fold more active than streptococcal pyrogenic exotoxin A. Th2-derived cytokines were elicited only by superantigens and not by streptococcal cells.

[Mechanisms for development of tolerance to repeated injections of exogenous pyrogens]. / Mechanizmy wzbudzania tolerancji na powtarzane iniekcje pirogenów egzogennych.

Pyrogenic tolerance has been recognized for many years in a variety of species although the mechanisms that are responsible for its development are not well understood. The development of pyrogenic tolerance is associated with the stepwise diminution of pathophysiological and behavioral responses induced by exogenous pyrogens, such as fever, reduction in food and water intake. Several studies either in vivo or ex vivo have indicated the role of various proinflammatory cytokines in the development of pyrogenic tolerance. Most of these studies have indicated that pyrogenic tolerance is associated with down-regulation of cytokine production as well as their biological activity. The mechanisms responsible for down-regulation of cytokine production during development of pyrogenic tolerance are unclear. Since glucocorticoids are required for induction of tolerance, it has been postulated that well known glucocorticoids-dependent negative feedback on the production and biological activity of cytokines may play an important role in development of pyrogenic tolerance. We can not, however, rule out possibility that other mechanisms may participate directly or indirectly in a suppression of cytokines response due to repeated exogenous pyrogen challenge. Either the enhanced uptake of exogenous pyrogens by the hepatic Kupffer cells or the desensitization to exogenous pyrogens by the loss of binding sites, have been proposed as an additional mechanisms which may participate in exogenous pyrogen hyporesponsiveness.

Development of streptococcal pyrogenic exotoxin C vaccine toxoids that are protective in the rabbit model of toxic shock syndrome.

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.

Pyrogenic signaling via vagal afferents: what stimulates their receptors?

Although there is good evidence that pyrogenic messages may be conveyed from the periphery to the brain via vagal afferents, the exact nature of the factors that activate their sensory terminals is unclear. Since IL-1beta and PGE2 have established roles in fever production and since their receptors have been identified on or near vagal nerves, they are potential candidate mediators. A difficulty, however, is that (1) IL-1beta is not expressed constitutively in mononuclear phagocytes, their presumed cell source upon stimulation by exogenous pyrogens, e.g. endotoxin, and (2) similarly, the isoform of the enzyme that selectively mediates the production and release of PGE2 by endotoxin-stimulated macrophages, COX-2, is also not constitutively expressed in these cells. Since the transcription and translation of these factors significantly lags the onset of fever induced by endotoxin administered intravenously, in particular, it is possible that a secondary, quickly-acting mediator evoked in almost immediate reaction to the presence of endotoxin excites, directly or indirectly, the sensory neurons. We have evidence that the complement component C5 contributes importantly to the initiation of the febrile response to endotoxin. This article briefly reviews the prevailing concepts of pyrogen sensing and signaling, examines their shortcomings particularly in terms of the temporal discrepancy between the very rapid onset of the febrile response to intravenously administered endotoxin and the significant delay in the elaboration of the putative mediators of fever, and presents newer data that may help to integrate the various preposed mechanisms.