Multiple Cross-reactivity Research Articles

The Guillain–Barrè peptide signatures: from Zika virus to Campylobacter, and beyond

The Guillain-Barre peptide signatures: from Zika virus to Campylobacter, and beyond Guglielmo Lucchese,1 Darja Kanduc2 1Brain Language Laboratory, Freie Universität Berlin, Berlin, Germany; 2Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Abstract: Scientific attention has focused recently on the link between Guillain–Barrè syndrome (GBS) and Zika virus (ZIKV). Two related questions emerged: 1) what triggered the violent 2014 outbreak of a virus, which, first identified in 1947, had caused only a limited number of documented cases of human infection until 2007 and 2) which molecular mechanism(s) relate ZIKV active infection to GBS, an autoimmune inflammatory polyradiculoneuropathy. Capitalizing on the increased interest on ZIKV and hypothesizing the involvement of autoimmune mechanisms, we searched for minimal epitopic determinants shared between ZIKV and other GBS-related pathogens – namely, Epstein–Barr virus, human cytomegalovirus, influenza virus, Campylobacter jejuni, and Mycoplasma pneumoniae, among others – and human proteins that, when altered, have been associated with myelin disorders and axonopathies. We report a considerable peptide matching that links GBS-related pathogens to human proteins related to myelin disorders and axonopathies. Crucially, the shared pentapeptides repeatedly occur throughout numerous epitopes validated as immunopositive by a conspicuous scientific literature. The data support a scenario where multiple different infections over time and resulting multiple cross-reactions may contribute to the pathogenesis of GBS. In practice, previous infection(s) might create immunologic memory able to trigger uncontrolled hyperimmunogenicity during a successive pathogen exposure. ZIKV pandemic appears to be an exemplar model for a proof-of-concept of such multiple cross-reactivity mechanism. Keywords: peptide sharing, GBS-related human proteins, GBS-related pathogens, multiple cross reactivity, hyperimmunogenicity

Minimal immune determinants connect Zika virus, human Cytomegalovirus, and Toxoplasma gondii to microcephaly-related human proteins.

Aiming at the molecular definition of the relationship(s) between prenatal infections and fetal microcephaly and using pentapeptides as minimal immune determinants, we analyzed the peptide matching between proteins from infectious agents involved in microcephalic syndromes (namely Zika virus, human Cytomegalovirus, and Toxoplasma gondii) and human proteins that, when altered, have been specifically associated with microcephaly. We report that an unexpected high number of epitopic pentapeptides (ie, 34) are common to the three fetopathogenic agents and repeatedly occur throughout an important number of microcephaly-related human proteins. The data introduce the issue of multiple cross-reactivity into the etiology on ZIKV-associated pathologies. Indeed, the commonality of immune determinants might lead to a sequence of boostered immune responses if the host undergoes different fetopathogenic infections, thus temporally scanning a potential clinical progression toward brain malformations. At this juncture, the past history of maternal infections/vaccinations might dictate the fetal pathologic outcome.

Multiple cross-reactivity to several types of neuromuscular blocking agents in a patient with rocuronium anaphylaxis

Anaphylaxis that occurs during anesthesia is a major cause of morbidity and mortality. Muscle relaxant is the most frequent cause of perioperative anaphylaxis [1]. Although it is believed that there is a risk of anaphylaxis to the drug in some patients, a similar incidence of anaphylaxis has been observed with use of other members of the same drug class. We report on a case of anaphylactic reaction to rocuronium in a male patient confirmed as having multiple cross-reactivity to various neuromuscular blocking agents (NMBAs) on an allergic skin test at three months after discharge. A 31-year-old, 174 cm, 85 kg man with congenital cystic ad enomatoid malformation was scheduled to undergo lobectomy of the right lower lung. He did not have a history of allergy or surgery. Results of routine preoperative tests showed no abnormalities. In the operating room, routine monitoring devices, including a blood pressure (BP) cuff, pulse oximetry, and elec trocardiography (ECG) were placed on the patient. He showed stable vital signs, including oxygen saturation (SpO2) 100%, BP 115/78 mmHg, and heart rate (HR) 90 beats/min. Epidural catheter insertion was performed for control of intraoperative and postoperative pain. Propofol target-controlled infusion was performed for induc tion of general anesthesia. After loss of eyelash reflex, rocuronium 60 mg was administered, and endotracheal intubation was performed after two minutes of facemask ventilation. After endotra cheal intubation, his vital signs showed moderate hypotension; BP 85/40 mmHg, HR 75 beats/min. However, his breath sounds were normal. A 20-gauge arterial cannula was inserted into the left radial artery for continuous monitoring of arterial BP and a central venous catheter was placed in the right subclavian vein. He was treated with intravenous injection of ephedrine 10 mg and rapid infusion of Ringer’s lactate solution. BP did not improve; therefore, we stopped propofol infusion. We injected midazolam 3 mg and rocuronium 20 mg. A few minutes after injection, his vital signs indicated severe hypotension (BP 50/30 mmHg). We observed a skin rash, urticaria, and edema on his face, neck, arms, and legs. On arterial blood gas analysis, the measured values were pH 7.230, PCO2 50.4 mmHg, PO2 124 mmHg and SpO2 98%. The patient was treated with intravenous injection of epinephrine 0.1 mg and continuous infusion of dopamine, dobutamine, and phenylephrine. After a few minutes of infusion, his vital signs became stable; BP 90/45 mmHg, HR 105 beats/min, and SpO2 100%. Hydrocortisone 100 mg and chloropheniramine 4 mg were administered intravenously. Seventy

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvumstrains

BackgroundUreaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars.ResultsWe determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75−0.78 Mbp genomes and UUR serovars were 0.84−0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level.ConclusionsComparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

The molecular anatomy of human Hsp60 and its similarity with that of bacterial orthologs and acetylcholine receptor reveal a potential pathogenetic role of anti-chaperonin immunity in myasthenia gravis.

Heat-shock protein 60 (Hsp60) is ubiquitous and highly conserved being present in eukaryotes and prokaryotes, including pathogens. This chaperonin, although typically a mitochondrial protein, can also be found in other intracellular sites, extracellularly, and in circulation. Thus, it can signal the immune system and participate in the development of inflammation and immune reactions. Both phenomena can be elicited by human and foreign Hsp60 (e.g., bacterial GroEL), when released into the blood by infectious agents. Consequently, all these Hsp60 proteins become part of a complex autoimmune response characterized by multiple cross reactions because of their structural similarities. In this study, we demonstrate that Hsp60 proteins from humans and two common pathogens, Chlamydia trachomatis and Chlamydia pneumoniae, share various sequence segments of potentially highly immunogenic epitopes with acetylcholine receptor α1 subunit (AChRα1). The structural data indicate that AChRα1 antibodies, implicated in the pathogenesis of myasthenia gravis, could very well be elicited and/or maintained by self- and/or bacterial Hsp60.

Chromatin modified with Nitric Oxide and its Role in Systemic Lupus Erythematosus

Freshly isolated chromatin from goat liver, exposed to nitric oxide (generated by the reduction of sodium nitrite with sodium dithionite) caused structural alterations and loss of tertiary structure of chromatin along with strand breaks and base modifications in the DNA of chromatin. The NO-chromatin was highly immunogenic in experimental animals as compared to its native form. Induced antibodies were highly specific for their respective immunogen. However, delineating the antigenic specificity of anti -NO-chromatin antibodies by competition ELISA, multiple cross-reactivity was observed. Anti-NO-chromatin antibodies recognized human blood proteins, modified tyrosine polymers and histones. Moreover, NO modified conformers were better recognized than their native forms. The visual detection of immune complex formation with native and NOchromatin reiterated preferential binding with modified chromatin. Chromatin modified by nitric oxide presents unique epitopes which may be one of the factors in antigen driven autoimmune response in SLE.

Immunogenicity of Nitric Oxide Modified Chromatin and Its Implications in Systemic Lupus Erythematosus

Freshly isolated chromatin from goat liver, exposed to nitric oxide (generated by the reduction of sodium nitrite with sodium dithionite) caused structural alterations and loss of tertiary structure of chromatin along with strand breaks and base modifications in the DNA of chromatin. The NO-chromatin was highly immunogenic in experimental animals as compared to its native form. Induced antibodies were highly specific for their respective immunogen. However, delineating the antigenic specificity of anti -NOchromatin antibodies by competition ELISA, multiple cross-reactivity was observed. Anti-NO-chromatin antibodies recognized human blood proteins, modified tyrosine polymers and histones. Moreover, NO modified conformers were better recognized than their native forms. The visual detection of immune complex formation with native and NO-chromatin reiterated preferential binding with modified chromatin. Chromatin modified by nitric oxide presents unique epitopes which may be one of the factors in antigen driven autoimmune response in SLE.

Sources of sensitization, cross‐reactions, and occupational sensitization to topical anaesthetics among general dermatology patients

Contact sensitization to local anaesthetics is often from topical medicaments. Occupational sensitization to topical anaesthetics may occur in certain occupations. The aim of the study was to analyse the occurrence of contact sensitization to topical anaesthetics in general dermatology patients. Patch testing with topical anaesthetics was carried out in 620 patients. Possible sources of sensitization and the clinical histories of the patients are analysed. Positive patch test reactions to one or more topical anaesthetics were seen in 25/620 patients. Dibucaine reactions were most common (20/25), and lidocaine sensitization was seen in two patients. Six patients had reactions to ester-type and/or amide-type anaesthetics concurrently. Local preparations for perianal conditions were the most common sensitizers. One patient had developed occupational sensitization to procaine with multiple cross-reactions and with concurrent penicillin sensitization from procaine penicillin. Dibucaine-containing perianal medicaments are the major source of contact sensitization to topical anaesthetics. Although sensitization to multiple anaesthetics can be seen, cross-reactions are possible. Contact sensitization to lidocaine is not common, and possible cross-reactions should be determined when reactions to lidocaine are seen. Occupational procaine sensitization from veterinary medicaments is a risk among animal workers.

Cross‐reactivity of Anisakis simplex: possible role of Ani s 2 and Ani s 3

Anisakis simplex is a fish parasite that can cause allergy in humans. The multiple cross-reactivities of this nematode make diagnostic tests for allergy unreliable, because of frequent false-positive results. To date, only four allergens of A. simplex have been characterized, but their role in cross-reactivity is largely unknown. We performed an amino acid sequence homology analysis, according to the Food and Agriculture Organization/World Health Organization (FAO/WHO) criteria, to identify allergens likely to be cross-reactive with Ani s 2 (paramyosin) and Ani s 3 (tropomyosin) of A. simplex. Next, within segments shared by Ani s 2 or Ani s 3 and homologous proteins, we searched for the occurrence of binding motifs of human leukocyte antigen (HLA) alleles (DRB1*1502, DQB1*0601, or DRB1*0404) known to be genetic risk factors for the development of allergy to A. simplex. We identified a number of proteins belonging to various organisms, which are significantly similar to (and therefore are likely to cross-react with) Ani s 2 and Ani s 3. The presence of binding motifs of HLA DRB1*0404 in Ani s 2, Ani s 3, and homolog proteins also supports our hypothesis. Our results provide a possible molecular explanation of some cross-reactivities reported for A. simplex, and suggest additional ones.

In silico identification of potential new latex allergens

Allergy to latex of Hevea brasiliensis is a frequent problem. In spite of the significant progress of research, the identity and cross-reactivity of some latex allergens are unknown. To identify, among the fully characterized latex proteins, those with a higher probability to be allergenic. We used in silico techniques (amino acid sequence comparison and molecular modelling) to identify potential new allergens among the known proteins of H. brasiliensis. Cu/Zn superoxide dismutase, heat shock protein and calmodulin of H. brasiliensis show highly significant (E < 10(-9)) amino acid sequence homologies with known allergens. On the basis of our data, Cu/Zn superoxide dismutase, heat shock protein and calmodulin are the most probable allergens among fully characterized proteins of H. brasiliensis, and could potentially explain, at least in part, the multiple cross-reactivities of latex with vegetable foods and other plant-derived products. Consequently, we think that the above proteins should be particularly considered in the future laboratory and clinical research.

T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells.

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.

Antigen binding characteristics of antibodies induced against nitric oxide modified plasmid DNA

Nitric oxide (NO) generated by the reduction of sodium nitrite with sodium dithionite caused damage to plasmid Bluescript DNA leading to strand breaks and base modifications. The NO-plasmid DNA was highly immunogenic in rabbits. The antibody activity was inhibited to the extent of 86% with the immunogen as inhibitor, indicating the induction of immunogen specific antibodies. However, delineating the antigenic specificity of anti-NO-plasmid DNA antibodies by competition ELISA, multiple cross-reactivity was observed. The antibodies recognised B-, A- and allied conformations. The visual detection of immune complex formation with native and NO-plasmid DNA reiterated preferential binding with modified plasmid DNA. DNA modified by nitric oxide presents unique epitopes which may be one of the factors in antigen-driven autoimmune response in systemic lupus erythematosus.

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.

Antigen Binding Characteristics of Experimentally-Induced Antibodies Against Hydroxyl Radical Modified Native DNA

Hydroxyl radical, generated by UV irradiation of hydrogen peroxide caused damage to native calf thymus DNA leading to strand breaks, base modification and decrease in melting temperature. The ROS-DNA was highly immunogenic in goat. The antibody activity was inhibited to the extent of 89% with the immunogen as inhibitor. Antigenic specificity of anti-ROS-DNA antibodies by competition ELISA showed multiple cross-reactivity, recognizing B-, A-and allied conformations. The immune complex formation with native and ROS-DNA was substantiated by band shift assay. A striking observation, is the enhanced recognition of ROS-thymine and ROS-poly(dT) by the induced antibodies. These investigations suggest that ROS might be the plausible candidate for the presentation of unique epitopes on ROS-DNA, in particular of thymine, inducing antibodies cross-reacting with native DNA and play an important role in the pathogenesis of SLE.

Serologic survey for hantavirus infection in domestic animals and coyotes from New Mexico and northeastern Arizona

Objective To determine whether animals had serologic evidence of infection with Sin Nombre virus (SNV). Design Prospective serosurvey. Sample Population Serum samples were obtained from 145 cats, 85 dogs, 120 horses, and 24 cattle between April 1993 and August 1994 and 54 coyotes between December 1994 and February 1995. Procedure Serum samples were analyzed by western immunoblot assays for reaction with SNV nucleocapsid antigen. Samples with reactivity to SNV nucleocapsid proteins were used to probe multiple-antigen blots containing recombinant fusion proteins derived from prototypic hantaviruses. Lung tissue or blood clots were used in nested reverse-transcriptase polymerase chain reaction assays for a 320-nucleotide portion of the SNV G1 gene. Results Sera from 4 of 145 (2.8%) cats and 4 of 85 (3.5%) dogs had trace reactivity to full-length SNV-encoded nucleocapsid proteins. All samples from horses, cattle, and coyotes were nonreactive. Sera from cats and dogs that had trace IgG-antibody reactivity to nucleocapsid proteins were then tested for IgG-antibody reactivity to nucleocapsid proteins of prototypic hantaviruses. One cat had multiple cross-reactivities with these hantaviruses, consistent with exposure to a hantavirus; however, epitope mapping studies did not support this conclusion. Reverse-transcriptase polymerase chain reaction studies of blood clots or lung tissue from 2 animals that had weak reactivity to SNV failed to amplify any hantavirus sequence. Clinical Implications Domestic animals, particularly dogs and cats, as well as coyotes do not appear to have a major role in the maintenance and transmission of SNV. (J Am Vet Med Assoc 1998;212:970–973)

Cuticle proteins of the boll weevil, Anthonomus grandis, abdomen: Structural similarities and glycosylation

Cuticle proteins are thought to be important in defining the structural and functional differences occurring in insect cuticle. In order to explain and better understand the structural similarities among the cuticle proteins of the cotton boll weevil, Anthonomus grandis Boheman, described in a previous study (Stiles and Leopold, 1990, Insect Biochem. 20, 113–125) three series of monoclonal antibody producing hybridoma cell lines were produced. Larval, pupal or adult cuticle proteins were used as antigens. While some of the monoclonal antibodies were specific for one or two cuticle proteins from a single developmental stage, the majority showed multiple cuticle protein binding patterns on Western blots. To determine whether this cross-reaction was due to common oligosaccharide chains bound to the proteins, lectins were used to probe Western blots. Many of the cuticle proteins were found to be glycosylated. The majority of the Con A reactive carbohydrate could be removed from the protein by N-glycosidase F digestion (specific for N-asparagine linked carbohydrate). N-glycosidase F digestion did not reduce the multiple cross-reactions of the monoclonal antibodies, nor did periodate oxidation of the CP. The carbohydrate remaining after enzyme digestion is presumably O-linked to serine/threonine.

Anti-DNA antibodies bind directly to renal antigens and induce kidney dysfunction in the isolated perfused rat kidney.

The pathogenesis of SLE is commonly attributed to the deposition of circulating immune complexes consisting of DNA and anti-DNA autoantibodies. However, recent work has shown multiple cross-reactions between anti-DNA antibodies and a variety of cellular and extracellular Ag. To test the possibility that these antibodies interact directly with glomerular Ag and induce kidney dysfunction, we applied mouse and human anti-DNA IgG to the isolated perfused rat kidney. The NZB/NZW mouse monoclonal anti-DNA bound to glomerular Ag with a concomitant induction of proteinuria and a decrease in inulin clearance. The albumin excretion was 2301 +/- 734 micrograms/min at 160 min of perfusion, as compared with 85 +/- 21 micrograms/min in controls (p less than 0.001). The inulin clearance was reduced to 0.17 +/- 0.02 ml/min as compared with 0.28 +/- 0.09 ml/min in controls (p less than 0.05). Polyclonal anti-DNA IgG obtained from patients with lupus nephritis bound to rat glomeruli and induced albumin excretion of 542 +/- 217 micrograms/min at 160 min of perfusion, as compared with 163 +/- 77 micrograms/min in controls (p = NS). The addition of plasma as a source of C to the human IgG increased the proteinuria markedly (albumin excretion of 1115 +/- 195 micrograms/min at 160 min of perfusion, p less than 0.02), probably due to C activation. Preincubation of the reactive mouse and human IgG with DNA completely abolished their binding to renal tissue and its physiologic consequences. These results suggest that direct binding of anti-DNA antibodies to renal Ag may play an important role in the induction of lupus nephritis.

Immunocytochemical detection of corticotropin-releasing factor: Multiple cross-reactions of a widely used carboxy-terminally directed corticotropin-releasing factor antiserum (code rC70) in rat hypothalamus

Forty-one-residue corticotropin-releasing factor is a physiologically significant mediator of the hypothalamic control of corticotropin secretion by the anterior pituitary gland. This releasing hormone is produced by parvicellular neurons in the hypothalamic paraventricular nucleus that project to the external zone of the median eminence. Recent immunocytochemical evidence based on work with a rabbit antiserum against rat corticotropin-releasing factor (code rC70) suggests that about half of the parvicellular corticotropin-releasing factor-containing neurons in the hypothalamic paraventricular nucleus synthesize vasopressin, another potent corticotropin secretagogue, while the rest of the cells do not. If this is indeed the case, the neurohumoral control of corticotropin release may be mediated via distinct hypothalamic effector pathways utilizing releasing hormone cocktails of varying composition. In the present study we have examined the specificity of various antisera against rat corticotropin-releasing factor in immunocytochemical staining. Male Wistar rats pretreated with colchicine were used throughout. The brain was fixed by perfusion with a Zamboni type fixative solution. Vibratome sections of the hypothalamus were immunostained with three different primary antisera (codes rC70, rCRF-3, oCRF-N) using the peroxidase-antiperoxidase or avidin-biotin complex methods. All three antisera stained cell groups previously described to be immunopositive for corticotropin-releasing factor. Most notably, however, rC70 labelled a significant number of additional cells, most readily identified in the arcuate and suprachiasmatic nuclei, as well as in the dorsolateral hypothalamic area caudal to the paraventricular nucleus. Liquid-phase preabsorption of rC70 with N- and C-terminal fragments of corticotropin-releasing factor revealed that the immunostaining in these latter areas could be blocked by the C-terminal hexapeptide of rat corticotropin-releasing factor, whereas in other hypothalamic regions the hexapeptide caused no noticeable change in the staining pattern. Moreover, the staining with rC70 in the arcuate nucleus and the dorsolateral hypothalamic area could be blocked by α-melanocyte stimulating hormone and that in the suprachiasmatic nucleus by peptide histidine/isoleucineamide. This may be readily explained by sequence homologies between the aforementioned peptides and the C-terminal hexapeptide of rat corticotropin-releasing factor. Thus, we communicate yet another case where an apparently highly specific antiserum may contain antibodies that recognize small portions (two amino acid residues) of the original antigen even when these are presented within relatively small and otherwise structurally unrelated neuropeptide transmitters.