Antibodies targeting human endothelin-1 receptors reveal different conformational states in cancer cells.

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ET(A) and ET(B)) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ET(B) receptors (hET(B)) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ET(B) receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ET(A) receptor (hET(A)). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hET(B) receptor only some of new antibodies directed against ET(A) receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hET(A) is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

Pharmacological in vitro evaluation of new substance P-cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells.

Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.

Importance of hydropathic complementarity for the binding of the neuropeptide substance P to a monoclonal antibody: equilibrium and kinetic studies.

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303-308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters (ka and kd) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 x 10[5] M[-1] s[-1]), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 x 10[-4] to 1.15 x 10[-1] s[-1]). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.

A monoclonal antibody directed against the neurokinin-1 receptor contains a peptide sequence with similar hydropathy and functional properties to substance P, the natural ligand for the receptor.

Monoclonal antibody (mAb) PS12, obtained using the complementary peptide methodology, mimics the neuropeptide substance P (SP) in recognizing the SP-binding domain of the neurokinin-1 receptor (NK1R) and eliciting production of polyclonal antibodies cross-reacting with SP with a high affinity (Déry et al., 1997. J. Neuroimmunol. 76, 1-9). The aim of the present study was to investigate which structural features of mAb PS12 might account for this molecular mimicry. Cloning and sequencing of variable regions of both light (VL) and heavy (VH) chains of this 'SP-like' antibody did not indicate any primary sequence homology between SP and any antibody region. Instead, they revealed a striking similarity between the hydropathic profile of SP and that of an 11-amino-acid region in the light chain encompassing the second complementarity determining region (CDR2). When applied to CHO cells expressing the human NK1R, a synthetic extended 17-amino-acid peptide (denoted CDR2L) corresponding to this VL region inhibited the high-affinity binding of radiolabeled SP and antagonized the SP-induced inositol phosphate production. Moreover, a re-examination of the sequences of several antibodies that previously served in the design of CDR-derived bioactive peptides indicated that these antibodies also carried the hydropathic image of the respective ligands that they mimic. In agreement with previous observations on artificial synthetic peptides, our data thus suggest that the molecular mimicry between natural proteins (i.e. antibody and hormone, for example) could be understood on a structural level directly related, at least in part, to hydropathic homology. These results could then guide the search for bioactive paratope-derived peptides of potential pharmacological interest. We also observed inverse hydropathy between multiple CDRs of mAb PS12 (including CDR3H and CDR3L) and the peptide epitope, confirming the importance of hydropathic complementarity in antigen-antibody interactions.

Identification in the NK1 tachykinin receptor of a domain involved in recognition of neurokinin A and septide but not of substance P.

The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS-7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193-197) region), selected because of its hydropathic complementarity with the common C-terminal extremity of tachykinins, abolish both the high-affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6-Pro9] SP 6-11). These results suggest that the (193-197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high-affinity binding domain for both NKA and septide, distinct from the SP binding site.

Immunoreactive substance P and calcitonin-gene-related peptide (CGRP) in rat milk and in human milk and infant formulas.

This study examined the presence of substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities in various milks and infant formulas. Rat milk was obtained from lactating dams between parturition and weaning (0, 2, 5, 10, 15, and 20 d postpartum). Samples of human milk were obtained from seven multiparous, nonsmoking white women, and newborn infant formulas were purchased from local stores. Substance P and CGRP were measured by competitive enzyme immunoassay using acetylcholinesterase-peptide conjugates as tracers. In rats, substance P and CGRP were below detectable concentrations in amniotic fluid from the last day of gestation. In contrast, in milk the concentrations of substance P and CGRP-like immunoreactivities were high on the first day of lactation (3.1 +/- 0.2 and 23.1 +/- 1.5 micrograms/L, respectively), then dropped after day 2 (1.6 +/- 0.7 and 7.5 +/- 0.4 microgram/L, respectively) and remained fairly constant until weaning. Significant concentrations of substance P and CGRP were found in human milk (129.2 +/- 27 ng/L and 4.5 +/- 0.7 microgram/L, respectively, at 15 wk), but substance P or CGRP could not be detected in any of the formulas tested. These data show that milk contains high concentrations of immunoreactive substance P and CGRP. In rats the absence of peptides in amniotic fluid suggests that there is a flood of peptides into the gastrointestinal tract of neonates when suckling is initiated. Significant concentrations of substance P and CGRP in human milk but not in infant formulas may therefore have physiologic implications for neonatal nutrition.

Prevaccination with diluted Freund adjuvant prevents the development of chronic pain and transient release of cerebrospinal fluid substance P in adjuvant-induced arthritis in rats.

Parallel time courses of preclinical and behavioural pain-related parameters and levels of substance P-like immunoreactivity in plasma (plasma-SPLI) and cerebrospinal fluid (CSF-SPLI) were studied in 2 groups of rats injected with an arthritogenic solution (concentrated Freund adjuvant) over a 9-week post-infection (PI) period; 1 group was pretreated with saline (control) and 1 pretreated with diluted Freund adjuvant (immunized). In control rats all symptoms of adjuvant-induced arthritis (AIA) developed while in immunized rats AIA symptoms were significantly reduced or did not appear. A significant increase in plasma-SPLI was obvious as early as the 2nd week PI and remained at this level in both groups of animals until the end of the 9-week PI observation period, but with a significantly higher increase in control versus immunized group at all stages. In contrast, CSF-SPLI transiently peaked only in the control group at 3 weeks PI whereas CSF-SPLI values did not differ from one week to another in both groups of rats. These results suggest that successive injections of diluted Freund adjuvant impairs the development of chronic inflammation and pain in AIA in rats, as well as the transient increase in SP release in CSF at 3 weeks PI, but not the long-lasting increased SP release in plasma. Since there is a clear dissociation between our biochemical and preclinical and behavioral data, this study does not provide evidence for the role of substance P as a possible biologic marker of chronic pain either in plasma or in CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P.

Monoclonal antibodies to the binding site of the NK1 receptor for the neuropeptide substance P were produced in mice using the complementary or antisense peptide methodology. Among several anti-peptide monoclonal antibodies, we selected the mAb12 antibody which specifically crossreacted, through its paratope, with a binding site present on membranes from rat parotid gland cells, with an affinity close to 2 x 10(-7) M and with membranes from CHO cells expressing human brain NK1 receptors. Immunocytochemical investigations using mAb12 revealed immunostaining whose distribution in the dorsal horns of rat spinal cord fits well with the known location of NK1 receptors. In both biochemical and immunocytochemical experiments, the competition occurring between the antibody and substance P, or a substance P-protein conjugate, indicates that mAb12 recognizes a membrane epitope located at or near the substance P binding domain on the NK1 receptor. Immunization of mice with mAb12 led to the production of specific anti-substance P antibodies, again suggesting that mAb12 shares common structural features with the neuropeptide. This monoclonal antibody can now be used in further biochemical or cytochemical characterizations of NK1 receptors. Owing to its fine specificity, mAb12 could also serve as a molecular model for designing peptides, possibly displaying pharmacological properties in the various processes in which substance P is involved, e.g. immunomodulation, inflammation or chronic pain.

Rapid axonal transport of three molecular forms of acetylcholinesterase in the frog sciatic nerve.

Acetylcholinesterase occurs in the frog sciatic nerve under five stable molecular forms with distinct sedimentation coefficients in sucrose gradients: 3 globular forms (3.6S, 6S and 10.5S) and two asymmetric ones (14S and 18S). Whereas in birds and mammals, the asymmetric tailed forms of acetylcholinesterase are present in trace amounts in peripheral nerves and account for only a small part of the enzyme activity submitted to a rapid axonal transport, the two asymmetric 14S and 18S forms represent nearly 50% of total activity in the frog sciatic nerve and account for 60-70% of the acetylcholinesterase activity accumulated at both sides of a nerve transection, the rest being due to an accumulation of globular molecules. We showed that the three forms, 10.5S, 14S and 18S, are all carried with the fast phase of axonal transport at a velocity of 100-120 mm/day in the anterograde direction and 20-30 mm/day in the retrograde direction. The velocity of transport for the light molecular forms 3.6S and 6S could not be calculated. In addition, we observed that large amounts not only of the 10.5S but also of the asymmetric 14S and 18S forms appear to be stationary along the frog sciatic nerve, contrary to the situation described for peripheral nerves in birds or mammals. Our results thus reveal that some axonal transport parameters for the asymmetric forms of acetylcholinesterase greatly differ in the peripheral nerves of amphibians on the one hand and of birds and mammals on the other, suggesting that these heavy molecular forms might have distinct functions in the nerves of lower and higher vertebrates.

Slow axonal transport of the molecular forms of butyrylcholinesterase in a peripheral nerve.

Butyrylcholinesterase was found in chick sciatic nerve in four main molecular forms--G1, G2, G4 and A12--distinguishable by thier sedimentation coefficients in sucrose gradients (4.2S, 6.4S, 11.3S and 19S, respectively). Axonal transport of butyrylcholinesterase was studied by measuring the accumulation of its molecular forms on each side of a transected sciatic nerve. Twenty-four hours after transection, butyrylcholinesterase activity had risen by about 32% at the extremity of the proximal stump, and by 20% at the extremity of the distal stump. Proximal accumulation was due to a two-fold rise in G4 activity and to a six-fold rise in A12 activity, whereas distal accumulation was exclusively due to a 50% increase in G4 activity, accompanied by the complete loss of A12. The activities of G1 and G2 remained stable in both directions. Under our experimental conditions, the accumulation of butyrylcholinesterase activity cannot be attributable to local protein synthesis, cross-contamination with accumulated acetylcholinesterase or the presence of plasma butyrylcholinesterase. Hence we conclude that all A12 butyrylcholinesterase molecules were carried in the anterograde direction, moving at 11.6 +/- 4.2 mm/day, and that probably some of the G4 molecules were slowly transported in both directions. These findings suggest that some of the butyrylcholinesterase is located in the axonal mitochondria and/or axolemma.

Anti-idiotypic antibodies as a tool for cytochemical identification of substance P receptors in the central nervous system.

Anti-idiotypic antibodies may serve as valuable probes for cytological identification of peptide receptors in the CNS. We have previously described the preparation of anti-substance P (SP) anti-idiotypic antibodies (anti-Id Ab) and have shown that they recognize SP receptors. These anti-Id Ab can be used in cytology to label SP receptors in CNS. We chose rat cervical spinal cord as a model because SP is present in large amounts in the dorsal and ventral horns, where it is implicated in pain and in motor function, respectively. After application of an indirect immunoperoxidase technique to tissue sections from perfused animals, immunolabeling was seen in the two superficial layers of the dorsal horn, the area surrounding the central canal, extending along the white matter in lamina VII, and in part of the ventral horn. This localization is in accordance with the classical distribution of SP receptors as seen by autoradiography with labeled SP. In the light of control experiments, as well as of biochemical and pharmacological arguments, we discuss the specificity of the immunolabeling. We conclude that anti-Id Ab recognize NK-P receptors, although crossreaction with NK-A or NK-B receptors cannot be totally ruled out.

Fine distribution of substance P-like immunoreactivity in the dorsal nucleus of the vagus nerve in cats.

The ultrastructure of substance P (SP)-immunoreactive elements in the cat dorsal motor nucleus of the vagus nerve was examined using pre- and post-embedding immunocytochemical procedures. Substance P-like immunoreactivity was observed in axon terminals and axon fibres which were mostly unmyelinated. Quantitative data showed that at least 16% of axon terminals contained SP. Their mean diameter was larger than that of their non-immunoreactive counterparts. Most (83%) SP-containing terminals were seen to contact dendrites but some were observed adjoining soma or entirely embedded in the cytoplasm of vagal neurons (4.5%). Only 0.5% were observed to contact soma of internuerons. A few immunoreactive axon terminals (4%) were observed in contact with non-immunoreactive axon terminals. Round agranular vesicles and numerous dense core vesicles were visible in most SP-containing axon terminals (84.6%). The immunogold procedure showed the preferential subcellular location of SP to be dense core vesicles. In 32.4% of cases, SP-containing terminals were involved in synaptic contacts that were generally of the asymmetrical Gray type 1 and mainly apposed dendrites. The theoretical total of synaptic contacts was 74.5% and this suggests the existence of weak non-synaptic SP innervation involving approximately 25% of SP-containing axon terminals. No axo-axonic synapses were observed in the dorsal vagal nucleus. These results support the hypothesis that SP found in the dorsal vagal nucleus originates partly from vagal afferents and is involved in direct modulation of visceral functions mediated by vagal preganglionic neurons.

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody.

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10(-6)M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non-junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.

Increased reactivity to chemical but not to heat noxious stimuli in mice producing anti-idiotypic antibodies for substance P.

Mice immunized against anti-substance P (anti-SP) monoclonal antibodies produced anti-SP anti-idiotypic antibodies (SPAb2). In a previous report. SPAb2 antibodies were found to have in vitro biological activity i.e. to behave either as agonists or as antagonists for substance P (SP) depending on the biological test. In this study, the involvement of SPAb2 in in vivo biological activity has been tested. Because of the possible implication of SP in the generation and transmission of nociceptive information, we have tested the responsiveness of SPAb2 responding mice in behavioral nociceptive tests. SPAb2 mice showed very small behavioral variations in the hot plate test as compared with a control group of mice immunized against an unrelated monoclonal antibody. In the formalin test, however, SPAb2 mice displayed a significant increase in paw licking time, which was significantly correlated with SPAb2 serum concentration. These results are discussed in terms of the use of SPAb2 as pharmacological tools for studying the biological properties of SP receptors and more generally of auto anti-idiotypic antibodies in modulating behavioral responses.

Anti-substance P anti-idiotypic antibodies modulate the secretory process in the rat parotid gland in vitro.

Anti-idiotypic antibodies (anti-Id) obtained in rabbits in response to immunization with polyclonal anti-substance P antibodies (anti-SP) were shown to bind specifically and with high affinity to membranes from rat parotid gland cells. Whereas substance P (SP) was unable to displace anti-Id from membrane binding sites, anti-Id partly inhibited the binding of radiolabelled substance P. Like substance P, anti-Id were able to trigger protein secretion by parotid cells i.e. to behave as physiological agonists of the neuropeptide. Under our experimental conditions, the biological effects of both ligands appear to be additive. Unlike substance P, however, anti-Id did not potentiate the secretory response induced by a beta-adrenoceptor agonist. Taken together, the present results might indicate that anti-Id interact with epitope(s) at or/and near the peptide-combining site on the substance P receptor. These data demonstrate further the possibility of raising pharmacologically active anti-receptor antibodies through the immunological anti-idiotypic approach.

Muscarinic M2-receptors in rat thoracic dorsal root ganglia.

The occurrence and distribution of the muscarinic M2-receptor subtype (M2R) was investigated in rat thoracic dorsal root ganglia (DRG). Messenger RNA for M2R was demonstrated by RT-PCR in total RNA from DRG. Immunoreactivity to M2R-protein was localized to 26% of sensory neurons, the majority of them (85%) belonging to the size class of 25-40 microm in diameter. Double-labeling (immuno)histochemistry revealed that all M2R-immunoreactive neurons bind the lectin, I-B4, whereas they are generally devoid of substance P-immunoreactivity. These data show the presence of M2R on a subpopulation of presumably nociceptive primary afferent neurons, thereby extending previous pharmacological and electrophysiological studies that indicated a role of M2R and/or M4R in inhibition of calcium channel currents in rat sensory neurons (Wanke, E., Bianchi, L., Mantegazza, M., Guatteo, E., Macinelli, E. and Ferroni, A., Muscarinic regulation of Ca2+ currents in rat sensory neurons: channel and receptor types, dose-response relationships and cross-talk pathways. Eur. J. Neurosci., 6 (1994) 381-391).

Enkephalinase (EC 3.4.24.11) is highly localized to a striatonigral pathway in rat brain.

Autoradiographic visualization of enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) in sagittal sections of rat brain using a 125I-labelled monoclonal antibody showed the presence of a dense immunoreactivity in a tract joining the striatum to the substantia nigra. Unilateral kainate injections into the striatum elicited a strong ipsilateral decrease in enkephalinase activity and immunoreactivity in both the injected area and substantia nigra, particularly its pars compacta. This demonstrates the presence of enkephalinase all along fibers of a striatonigral pathway.

Localization of the neurokinin 1 (NK-1) receptor in the human antrum and duodenum.

A newly available antibody to the neurokinin-1 receptor (NK-1r) has made it possible to determine the distribution of the NK-1r receptor in human tissue. In the present study the distribution of the NK-1r and substance P have been determined in the human antrum and duodenum by immunocytochemistry. The NK-1r was present on myenteric and submucosal neurons and nerve fibers of the gastro-enteric nervous system. In addition, the receptor was present on spindle-shaped cells in the circular muscle layer, endothelial cells and a population of mucosal cells. In the submucosal plexus NK-1r immunoreactive neurons were surrounded by substance P containing fibers. These results indicate an extensive cellular expression of the NK-1r in the human antrum and duodenum.

A behaviorally active dose of lipopolysaccharide increases sensory neuropeptides levels in mouse spinal cord.

To assess whether peripheral immune stimuli activate sensory afferents at behaviorally active doses, we measured the effects of lipopolysaccharide (LPS) on the levels of sensory neuropeptides in the spinal cord. LPS (10 micrograms/mouse i.p.) increased the levels of substance P, neurokinin A, and calcitonin gene-related peptide in the spinal cord, the maximum being observed 1 hr post-injection. Pretreatment with indomethacin at a dose (5 mg/kg i.p.) which completely blocked the decrease in food-motivated behavior induced by LPS abrogated this effect.

Systemic capsaicin pretreatment fails to block the decrease in food-motivated behavior induced by lipopolysaccharide and interleukin-1beta.

The physiological and behavioral disturbances observed during an infection can be reproduced by systemic administration of proinflammatory cytokines (e.g., interleukin (IL)-1, IL-6, tumor necrosis factor-alpha) or lipopolysaccharide (LPS), a potent inducer of these cytokines. It is now well established that these molecules induce their effects by acting centrally, however, the mechanisms by which they reach central structures are not clear. We have earlier proposed that the humoral immune message is converted to a central neural activation by the action of cytokines on peripheral terminations of afferent neurons. Subdiaphragmatic vagotomy abolishes several effects of peripherally injected IL-1beta and LPS (e.g., decreased food-motivated behavior and social exploration, central expression of cytokines). To further define the nature of the peripheral fibers implicated in this phenomenon, we used a potent sensory neurotoxin, capsaicin, to selectively destroy C-fiber afferents. Adult rats were injected I.P. with a total dose of 25 mg/kg capsaicin in a series of 10 injections over a 48-h period. Adult mice were injected I.P. with a total dose of 75 mg/kg in a series of seven injections over a 7-day period. Although capsaicin treatment altered visceral chemosensory function, corneal and pain sensitivity, vagal-mediated anorexic effects of cholecystokinin, and depleted levels of substance P in the thoracic spinal cord, it was completely ineffective in blocking the decrease in food-motivated behavior induced by IL-1beta (4 microg/rat I.P. in rats) and LPS (250 microg/kg I.P. in rats and 400 microg/kg I.P. in mice). Thus, other afferents besides capsaicin-sensitive C-fibers appear to be involved in the transduction of cytokine effects during inflammatory and infectious events.