Variant specific epitopes of Giardia lamblia.

Giardia lamblia undergoes surface antigenic variation. The capability of different isolates to express certain epitopes on the surfaces of trophozoites from different isolates and clones was determined using 4 surface-reacting monoclonal antibodies (mAbs) to variants derived from WB or WB-like Giardia (mAbs 6E7, 5C1, and 3F6) and GS/M (mAb G10/4). Of 28 isolates, 11 possessed trophozoites reactive with mAbs 6E7, 5C1 and 3F6, 6 with mAb 3F6, 2 with Mab G10/4, 1 with mAb 6E7, and 8 showed no reactivity as determined by direct or indirect immunofluorescence. Newly established clones from different isolates generated small numbers of reactive trophozoites similar to their parents. Only one epitope was found on any single trophozoite. Southern blots hybridized to a probe encoding for the epitope recognized by mAb 6E7 revealed that the inability to express the antigen in most isolates was due to lack of the gene. Analysis of the surface antigens of mAb 6E7 reactive clones from 3 isolates revealed that mAb 6E7 reacted with surface antigens of different molecular masses.

In vitro contractile activity of the mesotubarium superius from the rabbit oviduct in various endocrine states.

We investigated the influence of estrogen on the contractile activity of smooth muscle in the nonpregnant female reproductive tract, using as a model isolated strips of mesotubarium superius (MTS) removed from the rabbit oviduct. We studied four physiologic states in which muscular activity was influenced to varying degrees by endogenous estrogen. Normal estrous (estrogen dominated) rabbits were used as controls. We observed decreased activity in MTS from rabbits chronically deprived of estrogen (60) days postovariectomy). However, a dramatic increase in activity occurred in response to acute withdrawal of ovarian function (12 hours postovariectomy). An increase in activity also occurred at the time of ovulation. We concluded that acute withdrawal of endogenous estrogen is a potent stimulus to reproductive smooth muscle in the female rabbit and that it might contribute to increased muscular activity at ovulation.

Localization of midgut-specific protein antigens from Aedes aegypti (Diptera: Culicidae) using monoclonal antibodies.

Most insect transmitted pathogens interact with the midgut of their vectors for infection and, in some cases, development. Therefore, molecules associated with the midgut epithelium in direct contact with pathogens may be potential targets of infection-blocking measures. Here, we identify midgut-specific protein antigens from Aedes aegypti (L.) using monoclonal antibodies. Sixty-four monoclonal antibodies were generated with reactivity to the mosquito midgut, five of which are reported in this article. Three monoclonal antibodies identified protein antigens localized at the midgut epithelial surface (the brush border) and the peritrophic membrane. In addition, two monoclonal antibodies recognized mosquito nucleus-specific proteins by immunofluorescence microscopy. Because potential target antigens for blocking transmission of pathogens most likely are located at the interface of mosquito-pathogen interactions, these monoclonal antibodies could provide valuable tools for studying midgut-specific proteins and interactions of the mosquito midgut with pathogens.

Detection of microsporidia spore-specific antigens by monoclonal antibodies.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

Bioelectrical and contractile behavior of rabbit mesotubarium superius: estrogen withdrawal.

The mesotubarium superius of the female rabbit is a thin, plane sheet of smooth muscle responding to the in vivo endocrine states of ovulation, estrous, castration, pregnancy, and replacement estrogen therapy of the castrate in a manner similar to that of the uterus. Both isometric tension and microelectrode recorded transmembrane potentials were measured in vitro to make these comparisons. In addition, confirmation of the "estrogen-withdrawal" theory of activation was obtained when it was noted that about 12 h after an abrupt withdrawal of estrogen there is an increase in contractile activity coinciding with an increase in subthreshold activity and a slight (about 5 mV) decline in the average resting potential compared to the control estrous level. It is concluded that the mesotubarium superius is an excellent tissue for reproductive smooth muscle studies, where a geometrically simple model is useful.

Physical characteristics of the cervix.

The cervix must remain closed during pregnancy to maintain the pregnancy yet open during parturition. It must perform at the right time and in the right sequence. The process that coordinates these activities is labor and it is an equal mixture of uterine contractions and cervical effacement/dilatation. Neither of these factors can be ignored if one is to understand parturition. Dilatation of the cervix depends upon the biochemical and physical processes that produce effacement. A better understanding of this active, ongoing process will enable one to manipulate them with new drugs that could produce effacement without uterine contractions. Conversely, the capacity to "stiffen" the cervix to ward off premature birth is another potential therapeutic approach that may be utilized. Research into the genesis and physical expression of the incompetent cervix is needed, as are studies of the cervix after forceable dilatation following a D and C. All of these possibilities and future advances are dependent upon our ability to quantitate the physical state of cervical tissue, both in situ and in vitro.

Characteristics of oral prostaglandin E2-induced labor.

Oral prostaglandin E2 appears to play a dual role in human parturition. It induces normal uterine contractions and softens the cervix, thereby decreasing the resistance of the cervix to dilatation. Labor and delivery with oral PGE2 is achieved with less total uterine work when compared with spontaneous nonstimulated labor. This contention is supported by the fact that the rate of cervical dilatation in the active phase of labor is faster (2.73 cm/hr) than that reported by Hendricks et al. for ideal labor (2.12 cm/hour), yet uterine contractility is not increased. Analysis of the composite data of Friedman and Sachtleman in 1974 also shows a more rapid active phase dilatation in the PGE2-stimulated labors (3.3 cm/hour) as compared with spontaneous labor (2.98 cm/hour). Oral PGE2 offers a safe and efficacious alternative to oxytocin for the induction of labor in women. It appears to have a major advantage over oxytocin. The softening effect of PGE2 on the cervix would make this drug an ideal agent for use in patients with low Bishop scores who have medical indications for induction of labor. Regardless of route of administration, prostaglandin E2 is a potent uterine stimulant and must be used with the same precautions and safeguards as intravenous oxytocin.

The stretch modulus of human cervical tissue in spontaneous, oxytocin-induced, and prostaglandin E2-induced labor.

A total of 62 strips of cervical tissue from 28 patients at term were tested for stiffness (stretch modulus) by elongation and measuring the tension produced by a given stretch. The stretch modulus was taken as the slope of the linear regression curve derived from the linear portion of the stress-strain relationship. The data were obtained from three patient categories: (1) 17 strips from seven patients undergoing spontaneous labor, (2) 18 strips form 10 patients with labor induced by PGE2, and (3) 27 strips from 11 patients with labor induced by oxytocin. The stretch moduli of cervical tissue obtained from spontaneous and oxytocin-induced labor patients were similar. The stretch moduli of cervical tissue obtained from PGE2-induced labor patients were significantly lower than those from either the spontaneous or the oxytocin-induced labor groups. These results show that PGE2, when used for induction of labor at term, has the ability to lower the stiffness of cervical tissue. This property of prostaglandin may be useful therapeutically for the indicated induction of labor in patients with an unfavorable cervix.

Variations in the mechanical behavior of the rabbit cervix with endocrine state and anatomic site.

The mechanical test of stress-strain and stress-relaxation were done on excised cervical strips from New Zealand rabbits in estrus, less than 10 hours after luteinizing hormone (LH), 10 to 18 hours after LH, 18 to 50 hours after LH, 30 days pregnant, and castrate. The stretch moduli obtained from these tests indicated that a significant softening of the cervix occurred at 10 to 18 hours after LH and during pregnancy. Normally, the internal os is stiffer than the external os, but this difference disappears at 30 days of pregnancy, and both values are lower and identical. The changes in the stretch modulus are not thought to be due to smooth muscle, particularly in the external os, because of the low contractility of this tissue. The conclusion reached is that the rabbit cervix is a good model to use in the investigation of the mechanical behavior of the cervix and may be comparable to the human cervix in physiologic responses.

Reduction of the stretch modulus of human cervical tissue by prostaglandin E2.

Samples of cervical tissue were obtained from women immediately following term delivery from spontaneous labor or oral PGE2-induced labor and from a small nunber of nonpregnant women in the reproductive years undergoing hysterectomy for benign gynecologic disease. The measured strips were placed in a well-oxygenated bath at 37 degrees C. containing Ringer's solution and stretched at a constant rate while continuously recording length and tension. The data were converted to stress-strain diagrams. Each curve contained a long linear portion which allowed one to compute a stretch modulus for each sample. A total of 71 strips from 23 patients was used. The average stretch modulus from the oral PGE2-induced patients was significantly lower than the spontaneous labor group (p less than 0.005), which, in turn, was significantly lower than the stretch modulus of the nonpregnant cervical tissue. It was also noted that the yield point was lower in the PGE2 series when compared to the spontaneous labor series (p less than 0.05). The effect of the PGE2 at a bath concentration of 10(-5) to 10(-6)Gm. per cubic centimeter was to materially reduce the stretch modulus within 5 to 15 minutes of the drug addition in both the PGE2-induced and spontaneous labor series. The results of these experiments indicate that PGE2 has the effect of reducing cervial stiffness.

Giardia lamblia infections in adult mice.

An adult mouse-Giardia lamblia model was developed and used to study host-parasite interactions, including antigenic variation. The H7/1 clone of isolate GS infected mice consistently and produced infections in 14 mouse strains tested. Infection patterns were mouse strain and Giardia isolate dependent. Antigenic variation occurred in immunocompetent mice but not in mice with severe combined immunodeficiency.

Giardia lamblia: identification and characterization of a variant-specific surface protein gene family.

Giardia lamblia trophozoites undergo antigenic variation by modulating the expression of variant-specific surface proteins (VSP), which are encoded by a number of small multigene families. We characterized the genomic copy of the VSP gene expressed by the cloned trophozoite line H7, derived from the isolate GS/M, in addition to a related, but nonexpressed, family member. The coding regions of the two genes encode closely related polypeptides (86% identity). However, differences in the coding region of these genes reside solely in an 873 bp segment. Only four differences were found between the 5' flanking sequences (465 bp). The proximal 205 base pairs downstream from the coding regions were identical, but thereafter the sequences diverged (37% identity over the next 391 bp). Mapping studies indicated that no other VSP gene was located within 4 kb pairs of the expressed H7 VSP gene, and transcripts from the nonexpressed gene were detected in neither GS/H7 nor heterogeneous trophozoites populations derived from this cloned line. Any mechanisms responsible for the differential expression of VSP genes must reconcile the near identity of DNA sequences that flank the coding regions of expressed and nonexpressed VSP genes.

Isolate and epitope variability in susceptibility of Giardia lamblia to intestinal proteases.

The surface antigens of Giardia lamblia differ. To determine whether the unique surface antigens found in variants and isolates could differentially protect the parasite from digestion by intestinal protease, G. lamblia clones WB-2X (WB), GS/M-H7 (GS/M), and B6, each of which expresses a unique surface variant antigen, were exposed to alpha-chymotrypsin and trypsin at concentrations up to 20 mg/ml in culture medium. The number of surviving trophozoites and morphologic changes were assessed over time. After 24 h, there was a significant decrease in the number of surviving trophozoites of WB (80.5 and 94.2% for trypsin and alpha-chymotrypsin treatments, respectively, compared with controls) and B6 (78.9 and 95.5% for trypsin and alpha-chymotrypsin treatments, respectively, compared with controls) at 10 mg of enzyme per ml compared with culture medium alone. Cytotoxicity was prevented by the presence of soybean trypsin inhibitor, indicating the effects were due to protease activity. In contrast, there was no significant cytotoxicity after exposure of GS/M to either enzyme at the same enzyme concentration. After exposure to alpha-chymotrypsin, susceptible G. lamblia became rounded and then lysed, but after exposure to trypsin, G. lamblia appeared plastered onto the surface of the well and was intertwined and surrounded by finely granular material. Effects were concentration and time dependent; at least 6 h of treatment was required to observe changes 12 to 18 h later. Trophozoites surviving alpha-chymotrypsin or trypsin exposure became stably resistant to protease treatment. In vitro, the variant surface antigen of GS/M, but not those of WB or B6, resisted digestion by trypsin or alpha-chymotrypsin, suggesting that the variant surface antigens impart susceptibility or resistance to digestion. The initial surface variant antigens of WB and B6 were replaced in resistant cultures. Trophozoites differ in their ability to survive after exposure to intestinal proteases, which may enable certain G. lamblia isolates or isolates possessing certain surface variant antigens to survive in the small intestine.

Biological selection of variant-specific surface proteins in Giardia lamblia.

Immune evasion is frequently cited as the main reason for antigenic variation in pathogenic microorganisms. To better understand the role of switching of variant-specific surface proteins (VSPs) in Giardia lamblia-host interactions, antigenic variation during infections of mice and gerbils was examined, using clones that predominantly expressed unique VSPs. As expected, VSPs were selected against during infections of immunocompetent hosts. In contrast, in immunodeficient hosts, some VSPs were selected for and others were selected against. These diverse patterns of selection demonstrate that there are host-VSP interactions that exert both positive and negative selective pressures on parasites, independent of the adaptive immune response. Furthermore, selection was dependent on both the particular VSP and the host. Thus, the large number of VSP genes in G. lamblia may allow the parasite to infect multiple different hosts, and antigenic variation could be a mechanism to expand the parasite's host range.

Variant-specific surface protein switching in Giardia lamblia.

Surface antigen switching in Giardia lamblia was analyzed using monoclonal antibodies specific for two variant-specific surface proteins (VSPs). Two VSPs were detected on the surface of single trophozoites. Dual expression persisted for 13 h but disappeared at 36 h, as in other parasites that undergo surface antigenic variation.

Antigenic variation in Giardia lamblia.

Clones of the WB isolate of Giardia lamblia were exposed to cytotoxic mAb 6E7 which reacts with a 170-kDa surface Ag. Surviving progeny occurred at a frequency of about 1 in 1000 and were resistant to the effects of mAb 6E7. Analysis of progeny and clones of these progeny by surface radiolabeling, surface immunofluorescence, and Western blotting failed to detect the 170-kDa Ag. Loss of this Ag was associated with the appearance of a series of new surface Ag. A cytotoxic mAb (5C1) was produced to one of the newly appearing antigens (approximately equal to 64 kDa) and Giardia resistant to the cytotoxic effects of 5C1 isolated. Neither the approximately equal to 64 kDa nor the 170 kDa Ag were present and were replaced by a second series of new Ag. These studies clearly establish the loss and subsequent replacement of two antigenically distinct epitopes on Giardia derived from a single organism.

Increased expression of the molecular chaperone BiP/GRP78 during the differentiation of a primitive eukaryote.

Giardia lamblia, a major cause of intestinal disease worldwide, is a parasitic protozoan that represents the earliest branch of the eukaryotic lineage. Trophozoites, which possess two nuclei but lack mitochondria, peroxisomes and a typical Golgi apparatus, colonize the small intestine of the vertebrate host where they may differentiate into infective cysts. Encystation is a regulated process characterized by the biosynthesis, secretion and formation of a protective extracellular cyst wall. In previous studies, we demonstrated the biogenesis of the Golgi apparatus during encystation and identified two leucine-rich proteins (CWPs), which localize within encystation-specific secretory granules before their incorporation into the cyst wall. Here, we used immunological, biochemical and molecular biological approaches to analyze the expression of BiP/GRP78, an endoplasmic reticulum (ER)-resident chaperone, during the Giardia life cycle. A monoclonal antibody specific for Giardia BiP permitted the visualization of the ER of this protozoan and showed that BiP expression increased simultaneously with the increased expression of CWPs during encystation. However, in contrast to the 140-fold increase in levels of CWP transcripts, the steady-state level of BiP mRNA did not increase during encystation. Furthermore, potent inducers of BiP expression in higher eukaryotic cells, including agents that perturb the ER environment, did not affect BiP expression in Giardia. These results, when considered together with the profound changes that occur in the secretory pathway during Giardia encystation, indicate an important role for this molecular chaperone during the differentiation of this primitive eukaryote.

Antigenic variation in Giardia lamblia: cellular and humoral immune response in a mouse model.

Neonatal mice (CR:NIH:S) were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7) and the surface antigens of the intestinal trophozoites, as well as the cellular and humoral immune responses, were analysed during the course of infection. Infections in mice peaked 2-3 weeks after inoculation and were self-cured by day 42 post-infection (p.i.). The proportion of trophozoites expressing the Mr 72,000 surface antigen of the initial inoculum had decreased by day 12 and approached zero by day 22 p.i., similar to infections in humans. The predominant parasite-specific humoral response was an IgM- and IgG-isotype directed to the original Mr 72,000 surface antigen as well as other antigens. T-lymphocytes (predominantly LY4(CD4)+) isolated from Peyer's patches 12 days p.i. and later showed a significant proliferative response to Giardia lamblia antigens. Spleen and lymph node cells showed no lymphoproliferative response. T-cell blot analysis revealed the presence of dominant T-cell epitopes in the areas of Mr 200,000-75,000 and less than 50,000 polypeptides. No response was demonstrated in the Mr 72,000 region (migration site of the major surface antigen), suggesting T-cell dependent mechanisms are most likely not responsible for the surface antigen switch which occurred during the course of infection. This model infection can be used to study the role of immunological mechanisms in Giardia lamblia variant antigen switching and in the control of infections.

Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall.

Giardia lamblia trophozoites, like most intestinal parasitic protozoa, undergo fundamental biological changes to survive outside the intestine of their mammalian host by differentiating into infective cysts. This complex process entails the coordinated production, processing, and transport of cyst wall constituents for assembly into a protective cyst wall. Yet, little is known about this process and the identity of cyst wall constituents. We previously identified a 26-kDa cyst wall protein, CWP1. In the present work, using monoclonal antibodies to cyst wall antigens, we cloned the gene that encodes a novel 39-kDa cyst wall protein, CWP2. Expression of CWP1 and CWP2 was induced during encystation with identical kinetics. Soon after synthesis, these two proteins combine to form a stable complex, which is concentrated within the encystation-specific secretory granules before incorporation into the cyst wall. Both proteins contain five tandem copies of a 24-residue leucine-rich repeat, a motif implicated in protein-protein interactions. Unlike CWP1, CWP2 has an extremely basic 121-residue COOH-terminal extension that might be involved in the sorting of these proteins to the secretory granules.

Size heterogeneity among antigenically related Giardia lamblia variant-specific surface proteins is due to differences in tandem repeat copy number.

Giardia lamblia undergoes antigenic variation by modulating the expression of the different genes that comprise the trophozoite's variant-specific surface protein (VSP) repertoire. We studied an epitope that is conserved among VSPs expressed by cloned trophozoite lines derived from the independent G. lamblia isolates WB, G3M, Be-2, and CAT. The epitope recognized by monoclonal antibody 6E7 lies entirely within the region of tandemly repeated 65-amino-acid units that is characteristic of these size-variant VSPs. Northern (RNA) hybridization, cDNA cloning, and DNA sequence analysis indicate that size heterogeneity among these VSPs is due to differences in the number of repetitive units.