Formation of Charcot-Leyden crystals by human basophils.

Charcot-Leyden crystals (CLC) are currently believed to be unique to the eosinophil and a hallmark of active eosinophilic inflammation or proliferation. The distinctiveness of the CLC to the eosinophil was questioned in 1965 by Archer and Blackwood (9), but their demonstration of CLC formation in basophils was ignored and later dismissed (1) as being the result of eosinophil contamination of basophil-enriched cell suspensions. We reexamined this question and showed that basophils obtained from the peripheral blood of normal individuals form CLC and that basophils contain a protein that is immunochemically indistinguishable from eosinophil CLC protein. These conclusions are based upon the findings that (a) crystal formation in basophils was demonstrated by specific histochemical staining of crystal-containing cells in highly enriched basophil suspensions prepared by fluorescence-activated cell sorter (FACS) purification of surface IgE-positive cells, (b) that enrichment for surface IgE-positive cells (primarily basophils) by the FACS also enriched for cells staining positively by immunofluorescence for eosinophil CLC protein, and (c) that CLC protein was measured by radioimmunoassay in cell extracts prepared from purified basophil suspensions containing 97-99% basophils and absolutely no contaminating eosinophils. These basophil extracts contained a protein immunochemically indistinguishable from eosinophil CLC protein. Based upon these findings, the CLC or the protein comprising the crystal (lysophospholipase) can no longer be considered as distinctive to the eosinophil. We must now consider the possibility that the presence of CLC in tissues, sputum, or stool may also represent basophil involvement in disease processes.

Molecular cloning and characterization of recombinant parasite antigens for immunodiagnosis of onchocerciasis.

Immunological cross-reactivity among nematodes has hampered the development of specific serodiagnostic assays for onchocerciasis. In the present study, an Onchocerca volvulus adult worm complementary DNA expression library was differentially screened with human sera from patients infected with O. volvulus and with an omnibus anti-nematode serum pool comprised of sera from patients infected with Brugia malayi, Loa loa, Wuchereria bancrofti, Mansonella perstans, Strongyloides stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, and Dracunculus medinensis. Seven Onchocerca-specific clones were identified and screened with individual onchocerciasis patient sera. Additional studies were performed to characterize the most immunoreactive clones, OC 3.6 and OC 9.3. OC 3.6 produced a 152-kD beta-galactosidase fusion protein that was recognized in dot-immunoblots by 54 of 55 sera from onchocerciasis patients (98%). The OC 3.6 DNA insert is 996 bp long with an open reading frame of 627 bp and a 369-bp untranslated 3' end. OC 3.6 is closely related to a previously reported clone (OV 33-3), but it differs from that clone at both the 5' and 3' ends. OC 9.3 contained a novel 565-bp insert and produced a 138-kD fusion protein that was recognized by 46 of 55 sera from onchocerciasis patients (83%). Additional studies are in progress to develop and evaluate immunodiagnostic tests for onchocerciasis based on measurement of antibodies to these promising recombinant antigens.

Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.

Total and filaria-specific immunoglobulin E (IgE) levels were studied in cord blood from infants born in Madras, India, where filariasis and intestinal helminth infections are highly endemic. Increased total IgE levels were observed in 82% of 57 cord sera tested (geometric mean 12.6 ng/ml; range 1-1,900 ng/ml). 33 of these sera also contained IgE antibodies specific for filarial antigens as determined by solid-phase radioimmunoassay. Comparison of ratios of filaria-specific IgE to total IgE in paired maternal and cord sera suggested that cord blood IgE was derived from the fetus in most cases and not from transplacental antibody transfer. Our results suggest that prenatal allergic sensitization to helminth parasites occurs in the tropics. Such sensitization may contribute to the heterogeneity in host immune response and disease expression noted in filariasis and other helminth infections.

Neutrophil functional responses depend on immune complex valency.

Ligand-induced cross-linking of Fc gamma receptors (Fc gamma R) on neutrophils plays a significant role in their stimulation, shown here by contrasting the responses induced by low valency immune complexes (LICs) and high valency immune complexes (HICs) and by cross-linking LICs in situ (L/Ab) after their addition to the cells. Multiparameter flow cytometry was used to measure immune complex (IC)-elicited changes in cytoplasmic Ca2+ concentration and initiation of the oxidative burst simultaneously in the same cell and to correlate these with Fc gamma R occupancy. We have previously shown that subpopulations of neutrophils respond maximally to subsaturating concentrations of HIC; saturating dosages stimulate the entire population. This discrepancy was not due to differences in receptor occupancy. The magnitude of the transient Ca2+ increase was independent of the dose of HIC but depended on the dose when an LIC was used. As shown here, L/Ab cross-linking elicited Ca2+ responses similar to those observed in HIC-stimulated cells. In contrast, LIC elicited only minimal intracellular delta pH and no oxidative burst or membrane potential changes at all unless Fc gamma R was cross-linked, accomplished by HIC or by L/Ab. However, azurophilic degranulation, as determined by elastase release, was not observed in cells stimulated by the in situ cross-linking method, whereas the HIC preparation triggered azurophilic degranulation. Thus, some Fc gamma R-mediated neutrophil effector functions such as azurophilic degranulation and oxidative burst initiation have an absolute requirement for Fc gamma R cross-linking, whereas signaling functions such as changes in membrane potential, intracellular pH, and intracellular Ca2+ concentration can occur, albeit more slowly and to a lesser extent, if single Fc gamma R are occupied.

Isolation of human basophils by flow microfluorometry.

Human mononuclear cells labeled with fluorescein-conjugated anti-IgE antibody were subjected to flow microfluorometric analysis. Basophils were enriched to 97-99% purity with a 2-step cell sorting procedure. Trypan blue exclusion of sorted cells exceeded 90% and the net yield of the procedure was 15%.

Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils.

Immune complex stimulation of human neutrophils elicits, among other events, the formation of phagocytic vacuoles into which the products of the stimulus activated oxidative burst and degranulation are released. In order to monitor burst activity in the phagocytic vacuole, we have developed a fluorochrome-coupled derivative of this neutrophil agonist. The fluorochrome 2',7'-dichlorodihydrofluorescein (DCFH) (the nonfluorescent, reduced form of 2',7'-dichlorofluorescein (DCF] has been covalently linked to bovine serum albumin (BSA), which can be used to form an immune complex with anti-BSA immunoglobulin. The resultant complex is an effective agonist for stimulating all immune complex-mediated neutrophil responses, as compared to nonderivatized controls. Upon exposure to hydrogen peroxide the stimulus-linked probe is converted to its oxidized, fully fluorescent form, the fluorescence of which is linearly related to the extent of probe oxidation. Using flow cytometry, we have demonstrated that the probe-stimulus complex is capable of monitoring the kinetics of the production of activated oxygen species by the membrane bound NADPH-oxidase enzyme, presumably within the phagocytic vacuoles of immune complex-activated neutrophils. We have found that the immune complex-mediated activation of the oxidative burst within the phagocytic compartment is preceded by a lag of approximately 30 s followed by a large sustained release of superoxide dependent hydrogen peroxide. Neutrophils from patients with chronic granulomatous disease, however, demonstrated no sustained increase in probe fluorescence, a finding consistent with the lack of oxidative burst activity in these cells. The DCFH-immune complex conjugate therefore provides an effective probe for monitoring the kinetics of the localized release of oxidative products within the forming phagocytic vacuoles of activated neutrophils, and may be used to further examine both the activation and activity of human neutrophils in response to 'physiologic' host defense agonists such as immune complexes.

Ehrlichiosis presenting as a life-threatening illness with features of the toxic shock syndrome.

OBJECTIVE: To describe clinical and laboratory features of patients with severe ehrlichiosis, some of whom presented with toxic shock syndrome (TSS)-like illnesses, and to report, to our knowledge, the first documented fatal case of ehrlichiosis in a child. DESIGN: Case series. SETTING: Tertiary-care medical center. PATIENTS: All patients with documented ehrlichiosis during a 3-year period, August 1, 1989, to July 31, 1992. RESULTS: Eight patients (age range: 2 to 46 years) met clinical and serologic diagnostic criteria for ehrlichiosis. The mean interval from first contact with a physician to initiation of appropriate antibiotic therapy was 4.6 days (range: 1 to 11 days). All eight patients with ehrlichiosis had fever, chills, thrombocytopenia, and abnormal liver function test results. Most patients also had rash (seven), conjunctival hemorrhage or erythema (six), and leukopenia (six). Four cases met diagnostic criteria for TSS with fever, hypotension, rash, and multiorgan dysfunction. Two patients required mechanical ventilation, and one of these, a 6 1/2-year-old boy, died of complications of the infection. A ninth patient with probable ehrlichiosis also met diagnostic criteria for TSS. CONCLUSIONS: Human ehrlichiosis can present as a severe, life-threatening illness that may resemble TSS. The diagnosis of ehrlichiosis was not considered by the physicians who first cared for these patients. Greater awareness of the potential severity of ehrlichiosis is needed to ensure that proper treatment is initiated early in the course of the disease.

Gender-specific gene expression in Brugia malayi.

Brugia malayi is a mosquito-borne filarial nematode that causes lymphatic filariasis and elephantiasis in humans. The purpose of this study was to identify and characterize genes that are expressed differentially in male and female B. malayi in hopes of gaining new insight into the reproductive biology of the parasite. Two approaches were used. A 5' differential display PCR (splice leader differential display PCR, SL DD-PCR) was performed by PCR with splice leader and random primers on cDNA templates, and electronic subtraction was performed on expressed sequence tag (EST) cluster databases developed by the Filarial Genome Project (FGP). Gender-specific expression of candidate clones was confirmed by RT-PCR for six of 22 (27%) clones identified by DD and in seven of 15 (47%) clones identified by electronic subtraction. One clone was identified by both methods. Several female-specific clones had homology to known nematode genes that encode a fatty acid binding protein, a high mobility group protein, an eggshell protein, a glutamate-gated ion channel, and a collagen. However, most of the clones have no significant homology to known genes or proteins in computer databases. This project has confirmed the value of SL DD-PCR and electronic subtraction for analysis of gene expression in filariae. These two complimentary techniques may be generally applicable to the study of gender-specific (and by analogy stage specific) gene expression in other nematodes.

Use of Iodogen and sulfosuccinimidobiotin to identify and isolate cuticular proteins of the filarial parasite Brugia malayi.

The cuticle of filarial nematodes is a dynamic structure which may be an important target for protective host immune responses. Prior studies have employed radioiodination of intact parasites to demonstrate that the collagenous cuticle of filariids contains relatively few exposed proteins, some of which are stage and/or species-specific. In the present study, we have used sulfo-NHS-biotin to label and affinity purify cuticular components of living adult Brugia malayi. Results obtained by this method were compared with the widely used Iodogen method of surface radioiodination by SDS-PAGE analysis of detergent-solubilized worms and by ultrastructural analysis. Both labeling methods produced very similar electrophoretic patterns with major doublets at 70 and 100 kDa, a major band at 25 kDa, and minor bands between 60-200 kDa. Ultrastructural analysis showed that both methods labeled components throughout all levels of the parasite cuticle; underlying somatic tissues were not labeled. The biotinylated components were isolated from the total parasite extract by affinity chromatography on an avidin matrix. Further characterization of these surface-associated proteins may lead to improved methods for the control of filariasis.

Molecular characterization of a Brugia malayi intermediate filament protein which is an excretory-secretory product of adult worms.

Filarial parasites release macromolecules into their environment both in vitro and in vivo. These excretory-secretory products (E-S) have been studied with respect to function, vaccination potential, pathogenicity, and ability to serve as antigen targets for diagnostic tests. We have recently described monoclonal antibody OV-1 which binds to an intermediate filament in E-S and circulating antigens of Onchocerca volvulus. OV-1 also binds to cross-reactive antigens of Brugia malayi. Therefore, OV-1 was used to immunoscreen a B. malayi adult worm cDNA library in an attempt to clone a homologue (BMIF). BMIF is a 1664-bp full-length transcript which codes for 505 amino acids. BMIF has 95% sequence homology at the amino-acid level to OV1CF, an O. volvulus intermediate filament that was also selected with OV-1, and 75% homology to Ascaris intermediate filament A. Southern blot analysis suggests that BMIF is confined to a single location in the genomic DNA of B. malayi. Antibodies raised to BMIF identified native antigens in immunoblots of B. malayi adult worms, infective larvae and adult E-S. In addition, the antibody also bound to a 60-kDa antigen in immunoblots of poly(ethylene glycol)-precipitated immune complexes in sera from B. malayi infected patients. Localization studies showed that the antigen encoded by BMIF is present in the hypodermis, developing embryos and muscle of adult B. malayi. These studies show that BMIF is an E-S product of B. malayi adult worms which is detectable in sera from patients with brugian filariasis.

Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis.

Immunological crossreactivity among nematodes has hampered development of specific serodiagnostic assays for lymphatic filariasis. In the present study, we report the molecular cloning and characterization of two filaria-specific recombinant clones (BmM5 and BmM14) with immunodiagnostic potential. BmM5 is a 505-bp cDNA which codes for a protein of 130 residues that ends with an endoplasmic reticulum targeting sequence. BmM14 is closely related to a recently reported clone (SXP-1), and it has 62% homology (deduced amino acid sequence) with a previously described Onchocerca volvulus clone, lambda RAL-2. Glutathione S-transferase fusion proteins of BmM5 and BmM14 were tested in various ELISA formats. The best results were obtained by measuring IgG4 antibodies to the fusion proteins. ELISA studies showed that approximately 90% of 111 sera from Indian and Egyptian patients with brugian and bancroftian filariasis were reactive with both antigens. Nonendemic sera as well as sera from patients with schistosomiasis or intestinal helminths were uniformly nonreactive. Assays based on BmM5 and BmM14 may be useful for large scale screening as an alternative to microfilaria or filarial antigen detection as a means of obtaining a rough index of filariasis endemicity in previously unstudied areas.

Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds.

Vaccination of jirds with irradiated infective larvae of Brugia malayi has been reported to provide partial immunity to larval challenge. In the present study, we found that sera from vaccinated animals recognized larval antigens with apparent molecular weights of 97, 55-60, and 10 kDa that were not recognized by sera from infected animals. A B. malayi cDNA expression library in lambda gt11 was screened to identify clones that were preferentially recognized by sera from immunized animals. One of these clones (BM-5) was chosen for further study. BM-5 contains a 2.1 kb DNA insert and produces a fusion protein with a molecular weight of 185 kDa. Antibody, affinity-purified with the BM-5 fusion protein, binds to a 97 kDa native B. malayi antigen. Immunological studies and partial DNA sequence data confirm that BM-5 encodes paramyosin. Recombinant B. malayi paramyosin is strongly recognized by antibodies in sera from jirds that have been immunized either by injection with irradiated larvae or by chemotherapy-abbreviated infection. Most sera from infected jirds do not contain antibody to paramyosin. Additional studies are needed to determine whether paramyosin is actually protective in this filariasis model.

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.

Cloning of an early immunodominant filarial antigen: a member of the Brugia malayi myosin heavy chain gene family.

Partial protective immunity to filariasis can be achieved in animals by vaccination with irradiated infective larvae. A Brugia malayi cDNA expression library was screened with serum pools from vaccinated and infected jirds to select clones that expressed potentially protective recombinant antigens that were preferentially recognized by sera from vaccinated animals. Bmmyo-2, the largest of a group of related clones, was studied in detail. Jirds produced strong antibody responses to the protein product of Bmmyo-2, Bmmhc-B, as early as 1 month after vaccination with irradiated larvae. Antibody responses to Bmmhc-B in infected jirds were weaker than those of vaccinated jirds, and they developed somewhat later. Antibodies produced to Bmmhc-B were reactive with a 200 kDa native B. malayi antigen by immunoblot and with muscle bands in the body wall of microfilarial by indirect immunofluorescence. Sequence analysis of the 1454 bp cDNA insert of Bmmyo-2 showed that it codes for a portion of the rod region of a B. malayi myosin heavy chain isoform. The deduced amino acid sequence of Bmmyo-2 is 74.6% identical with that of the corresponding region of Caenorhabditis elegans myosin heavy chain B, but only 64.6% identical with a recently described B. malayi myosin heavy chain, Bmmhc-A.

Effect of ivermectin prophylaxis on antibody responses to Onchocerca volvulus recombinant antigens in experimentally infected chimpanzees.

Antibody responses to recombinant Onchocerca volvulus antigens were studied in experimentally infected chimpanzees. Sera from 3 groups of 6 animals were tested by ELISA with recombinant antigens OC 3.6 and OC 9.3. Groups I and II were treated with 200 micrograms/kg of ivermectin on the day of infection or on day 28, respectively. Group III were untreated controls. Antibodies to OC 3.6 developed during the prepatent period in all 3 groups. In contrast, antibodies to OC 9.3 were usually first detected around the time of onset of patency. Several animals had early antibody responses to OC 9.3, but these animals subsequently failed to develop microfilarial patency. Only 1 of 6 animals in group I produced a strong antibody response to OC 9.3 while all 12 animals in groups II and III developed antibodies to this antigen. Although there was some inconsistency in antibody responses observed in each treatment group, the results suggest that OC 9.3 may be more useful than OC 3.6 for monitoring the efficacy of prophylactic drugs or vaccines for onchocerciasis while OC 3.6 may be useful for detecting exposure to the parasite and early infection, regardless of the later outcome of the infection.

Paper chromatography hybridization: a rapid method for detection of Onchocerca volvulus DNA amplified by PCR.

Prior studies have shown that Onchocerca volvulus DNA can be detected in skin snips and in black flies after polymerase chain reaction (PCR) with primers specific for repeated "O-150" DNA sequences. We have adapted a paper chromatography hybridization assay (PCHA) to detect amplified O-150 DNA and compared this method to two established methods, namely agarose gel electrophoresis (AGE) and hybridization enzyme-linked immunosorbent assay (ELISA). The minimum amounts of purified O-150 DNA detected by PCHA, AGE, and ELISA were 5, 10, and 2 ng, respectively. The three methods had similar estimated sensitivities for detecting O. volvulus DNA amplified from skin snips from African subjects with onchocerciasis (88%, 84%, and 91%, respectively). No false positive results were observed with skin snips from uninfected control subjects. The paper chromatography hybridization assay detects PCR products in 30 minutes without electricity or special equipment. This technology brings DNA detection a step closer to widespread use in field settings.

Evaluation of a monoclonal-antibody based antigen assay for diagnosis of Wuchereria bancrofti infection in Egypt.

Conventional methods for diagnosis of Wuchereria bancrofti infection are insensitive and often impractical because of the need for night blood collections. A sensitive and specific antigen detection assay has been developed for W. bancrofti, which is based on a monoclonal antibody (AD12) that binds to a repeated epitope on a 200 kDa adult worm excretion product present in sera from infected humans. The only formal evaluation of this assay to date was performed with sera from India. In the present study, we have evaluated the performance of the AD12 antigen assay in two laboratories with sera collected in endemic and non-endemic areas in Egypt. Antigen was detected in 57 of 59 (97%) sera from microfilaremic subjects, and in 22 of 139 asymptomatic and amicrofilaremic subjects who reside in a highly endemic area. Antigen titers were significantly correlated with microfilaria counts (r = 0.41, P less than 0.01). Filarial antigen was not detected in most sera from amicrofilaremic subjects with clinical filariasis. Comparative antigen test results obtained from laboratories in Cairo and St. Louis agreed in 170 of 173 sera tested. Filarial antigen was not detected in sera from Egyptians with no history of residence in filaria-endemic areas. Specifically, nonendemic sera from patients with other parasitic infections (schistosomiasis, fascioliasis, ascariasis, etc.) were uniformly negative in the assay. We conclude that the AD12 filarial antigen assay is sensitive and specific for W. bancrofti infection in Egypt.

Detection of circulating parasite antigens in canine dirofilariasis by counterimmunoelectrophoresis.

Circulating Dirofilaria immitis antigen was detected in sera from 24 of 24 infected dogs by counterimmunoelectrophoresis (CIE). Parasite antigen was not detected in sera from uninfected dogs or dogs with Dipetalonema reconditum infection. In experimentally infected dogs, the antigen was first detectable 6.5-8.5 months after infection. Preliminary evidence suggests that the antigen is present in male and female adult worms but not in microfilariae. Sera from dogs with microfilaremic and amicrofilaremic infections contained statistically equivalent amounts of D. immitis antigen. However, a significant correlation was observed between serum parasite antigen content and the number of adult worms present in individual dogs at necropsy. Previous studies from several laboratories have shown that microfilarial counts and serum antibody titers are not related to adult worm counts in canine dirofilariasis or other filarial infections. Thus, CIE detection of D. immitis antigenemia represents a significant improvement over previously available diagnostic techniques because it is more sensitive than microfilarial tests, more specific than antibody tests, and the only test that has been related to infection intensity.

Persistence of parasite antigenemia following diethylcarbamazine therapy of bancroftian filariasis.

This study was designed to reexamine the efficacy of diethylcarbamazine for bancroftian filariasis with special reference to changes in serum parasite antigen levels and antifilarial antibody titers after treatment. Patients with asymptomatic microfilaremia were treated with 6 mg/kg diethylcarbamazine daily for 12 days. Microfilaria counts fell dramatically after treatment, as expected. IgG antibody titers to adult and microfilarial antigens of B. malayi were increased 1 month after treatment in most patients. Titers fell slowly to or below pretreatment levels, but remained positive during subsequent months. Parasite antigen levels, measured by monoclonal antibody-based enzyme immunoassay, decreased to 72%, 58%, 53%, and 48% of pretreatment values 1, 3, 6, and 12 months after diethylcarbamazine treatment, respectively. Parasite antigen levels decreased similarly in subjects with and without residual microfilaremia after treatment. These results suggest that diethylcarbamazine has only partial macrofilaricidal activity against W. bancrofti with this dosage schedule. The sustained, impressive reductions in microfilaria counts after treatment despite significant persistence of parasite antigenemia may be explained by sublethal effects of the drug on adult worms. We believe that parasite antigen detection represents a valuable new approach for monitoring the efficacy of antifilarial drug therapy which we hope will lead to improved use of existing drugs and aid in the evaluation of new drugs for filariasis.

Parasite antigenemia without microfilaremia in bancroftian filariasis.

The term "endemic normal" in the context of filariasis refers to people who are amicrofilaremic and free of clinical signs or symptoms of filariasis despite regular exposure to the parasite. Some sera from endemic normals contain soluble Wuchereria bancrofti antigens that are detectable by enzyme-linked immunosorbent assay. We now report evidence that filarial antigenemia in these people is not an artifact and that it is indicative of active W. bancrofti infection. Filarial antigenemia was first detected within one month of the onset of microfilarial patency in experimentally infected primates. Human sera from antigen-positive endemic normals contained the same filarial antigens (by Western blot) as sera from people with microfilaremia. Sera from antigen-positive endemic normals also contained significantly higher levels of immunoglobulin G4 antibodies to native and recombinant filarial antigens than sera from antigen-negative controls matched for age and sex. The epidemiology of filarial antigenemia in endemic normals was studied with sera from a population-based study of filariasis in an Egyptian village with a microfilaria prevalence of 29%. Seventeen percent of endemic normals had antigenemia, and this group comprised 11% of the total village sample. Filarial antigenemia was significantly more common in endemic normals more than 30 years of age than in younger people. These results suggest that amicrofilaremic and asymptomatic W. bancrofti infections are relatively common in endemic areas. Additional studies are needed to determine the clinical significance, prognosis, and optimal management of such infections.