I-STOD: a new standardization method for analysing indirect-ELISA results of a schistosomiasis field study.

Variability among samples analysed using the same ELISA protocol generates ambiguity in deciding which assay best quantifies the protein concentration. In this study, we propose a standardization method, called I-STOD (Improved STandardization method for Optical Density), for the transformation of OD values on different plates into relative concentrations of the antibody levels being assessed. We derived an equation relating OD values of different test samples to antibody levels according to the multi-stage reaction dynamics of the indirect-ELISA. Using serum samples from a Schistosomiasis japonica endemic area, we evaluated the fitness of the I-STOD model to experimental data of a standard reference serum in comparison with 5 other models. Calibration curves fitted by the I-STOD method judged to be superior, based on adjusted R2 (adjusted R2>0.99 on 22 out of 26 plates) values. The CV (coefficient of variation) value of the results between multi-well plates and the number of plates with OD values beyond the control range in Shewhart charts also demonstrate that the I-STOD method is a powerful tool which can greatly improve the comparability of results on different multi-well ELISA plates. We conclude that a standardization method is certainly necessary for antibody levels detected in order to properly illustrate clinical differences.

Anti-striatal antibodies in Tourette syndrome cause neuronal dysfunction.

Serologic studies of children with Tourette syndrome (TS) have detected anti-neuronal antibodies but their role in TS has not been explored. Stereotypies and episodic utterances, analogous to involuntary movements seen in TS, were induced in rats by intrastriatal microinfusion of TS sera or gamma immunoglobulins (IgG) under noninflammatory conditions, as found in TS. Immunohistochemical analysis confirmed the presence of IgG selectively bound to striatal neurons. These data support the hypothesis that binding of an anti-neuronal antibody from some children with TS induced striatal dysfunction and suggest a possible cause for the basal ganglia alterations observed in children with TS.

Schistosoma mansoni gene GP22 encodes the tegumental antigen sm25: (1) antibodies to a predicted B-cell epitope of Sm25 cross-react with other candidate vaccine worm antigens; (2) characterization of a recombinant product containing tandem-repeats of this peptide as a vaccine.

Monospecific antibodies against two putative epitopes of schistosome protein encoded by gene GP22 (182 codons, no introns) were used to probe worm extracts fractionated by lentil-lectin affinity chromatography or by electrophoresis. Anti-peptide-alpha (codons 70-84) exclusively identifies the N-glycanase-sensitive, 25 kDa tegumental glycoprotein Sm25 in the lectin-bound fraction of detergent-solubilized adult worm extract S3. In contrast, antipeptide-delta (codons 151-162) does not react with Sm25 but cross-reacts with other schistosome proteins, including candidate vaccine antigens paramyosin (Sm97) and glutathione-S-transferases (Sm26, Sm28, Sj26). Recombinant protein r4 x 47, constructed to express multiple copies of codon sequence 117-163 (containing delta), reacts with anti-delta and is uniquely recognized by protective Fischer twice-infected (F-2x) rat antiserum. Immunization with r4 x 47 induces antibodies with cross reactivities similar to anti-delta, but which also recognize Sm25. Despite these cross-reactivities with protective antigens, rodents vaccinated with r4 x 47 were not protected against cercarial infection. On the basis of these data, two hypotheses are proposed: (1) antigenic epitopes other than delta are present within the r4 x 47 sequence which induce antibodies reactive with Sm25 and/or (2) peptide-delta assumes alternative antigenic conformations, dependent upon the context of neighbouring sequences, some of which mimic epitopes of proteins encoded by other schistosome genes. These mimotopes are not targets of protective antibodies.

Immunomodulation and allergy.

A major function of the immune system is to protect the body from infection and the diseases caused by infectious agents. The immune system also provides protection against cancer cells, for once they arise, cancers can essentially behave as "foreign" cells capable of causing pathology. In contrast, allergy is a manifestation of the immune response to certain environmental cells or molecules that are usually neither a threat for infection nor cancer. Allergic reactions are generally an annoynance, even life-threatening. I will focus on type I allergy, characterized in part by induction of IgE antibody responses to allergens. It should be noted that not all IgE responses cause allergic symptoms. There is even evidence that IgE responses to tropical helminthic parasites offer a degree of immunity to reinfection. I have three objectives: (1) review T cell differentiation leading to the Th1/Th2 paradigm; (2) evaluate the increased prevalence of atopy, including asthma, as a consequence of a Th2-dominated immune system; (3) relate the high prevalence of asthma in inner city United States black children to the relatively recent migration of their ancestors from tropical regions of Africa, where genetically biased Th2-dependent IgE responses may be important in protection against high burdens of parasitic worms.

Role of the cervical lymphatics in the Th2-type hierarchy of CNS immune regulation.

CNS immune regulation is intimately dependent on the dynamics of cerebral extracellular fluid circulation. Animal models indicate that following the introduction of antigen into the CNS, normal circulation of interstitial and cerebrospinal fluids provides the opportunity for (a) delivery of CNS-derived antigen to lymphoid organs, as well as, (b) retention of immunologically significant amounts of antigen within the CNS. Thus, even in the absence of disease, CNS-derived antigen can induce antigen-specific activation of naive lymphocytes in lymphoid organs and specific reactivation of lymphoblasts that have migrated into the CNS. The initial peripheral immune response to CNS-derived antigen is induced in cervical lymph nodes and is characterized by a strong antibody response, no delayed-type hypersensitivity, and only priming for cytotoxic T-cell responses. This Th-2 type hierarchy of immune regulation is reinforced within the antigen-stimulated CNS where specific B lymphoblasts are permitted to develop their effector function but cell-mediated immunity is inhibited. Developing a paradigm for CNS immune regulation is important in understanding how CNS disorders in humans are induced, perpetuated, and may be manipulated.

Interleukin-12 as an adjuvant for an antischistosome vaccine consisting of adult worm antigens: protection of rats from cercarial challenge.

Our group previously demonstrated that a detergent extract (fraction S3) prepared from immature (4-week) Schistosoma mansoni parasites can induce partial, serum-transferable immunity to challenge infection in rats when administered as an alum precipitate. In the present study, we examined whether S3 prepared from adult (7-week) worms could similarly induce protection and whether immunity could be positively influenced by treatment with interleukin-12 (IL-12). IL-12 coadministered to Fischer rats and C57BL/6 mice at the time of S3 vaccination altered the prechallenge kinetics of S3-specific antibody titers in both species, ultimately leading to a stable enhancement of titers (relative to those in animals vaccinated without IL-12) in mice but not rats. Immunoblot analysis of prechallenge immune sera demonstrated that IL-12 treatment was associated with changes in the S3 antigen recognition profile in each species. Isotyping of specific antibodies in S3- plus IL-12-vaccinated mice prior to challenge infection revealed a moderate elevation in immunoglobulin G1 (IgG1) responses, strongly enhanced IgG2a and IgG2b responses, as well as diminished total serum IgE responses compared to those in mice given S3 only. In vaccinated rats, IL-12 profoundly suppressed specific IgG1 and enhanced IgG2b responses but did not affect IgG2a responses. S3- plus IL-12-vaccinated rats also produced less total IgE upon challenge infection. Enumeration of worm burdens revealed that vaccination with S3 plus IL-12 conferred 50% protection from cercarial challenge to rats, whereas rats given S3 only were not protected; mice were not protected by S3 vaccination regardless of IL-12 coadministration. The protection observed in S3- plus IL-12-vaccinated rats could not be transferred with serum, suggesting participation of an activated cellular component in the expression of immunity.

Normal cerebrospinal fluid suppresses the in vitro development of cytotoxic T cells: role of the brain microenvironment in CNS immune regulation.

The regulatory role of cerebrospinal fluid (CSF) in brain physiology is well established, while our understanding of its role in brain immunity is undefined. We demonstrate that normal rat CSF suppresses the in vitro development of mastocytoma-specific CTL activity in restimulated splenocytes from Balb/c mice, a strain unable to reject this tumor from the brain. Suppression is dependent on TGF-beta, revealed by reversal of suppression with specific neutralizing antibody. In contrast, mice which can reject this tumor from the brain, such as Balb/c mice with immunological memory to the tumor or CD-1 mice with major histo-incompatibility with the tumor, have populations of precursor CTL which are resistant to CSF-induced suppression, in the in vitro restimulation protocol. We propose that the susceptibility to CSF-induced suppression of peripherally generated immune cells that traffic to the brain plays an important role in determining whether growing tumor cells survive in the brain.

Antigen-dependent intrathecal antibody synthesis in the normal rat brain: tissue entry and local retention of antigen-specific B cells.

The intrathecal Ab response to Ag introduced into the normal brain has not been fully explored. Involvement of Ag-specific, peripheral B cells in an intrathecal response was studied using a normal rat model of Ag infusion through an indwelling cannula into defined brain sites, while maintaining a functionally intact blood-brain barrier. Specific Ab was detected in serum and cerebrospinal fluid. The intrathecal response is first detectable at day 14. Isoelectric focusing of cerebrospinal fluid reveals banding patterns consistent with local Ab production. To increase Ag-specific, circulating peripheral lymphocytes available for trafficking to Ag-stimulated brain and for enhancing intrathecal Ab synthesis, rats were preimmunized peripherally. Subsequently, Ag or saline (control) was infused through the cannula. Under this protocol, intrathecal synthesis is detectable earlier (day 5 postinfusion). Immunohistochemical studies at the infusion site assessed Ag-specific B cells, T cells, and activated APCs. Rats receiving peripheral preimmunization followed by Ag into caudate nucleus have far greater numbers of these cells, including plasma cells, within the infusion site compared with saline controls. Results confirm previous indirect evidence of intrathecal Ab synthesis in normal rat brain and provide the first direct evidence for B cell trafficking across normal brain barriers plus retention at the Ag deposition site. Our studies indicate that the normal brain microenvironment supports development of Ag-directed humoral immunity. We propose that immune privilege in normal brain is characterized by down-regulation of cell-mediated but not Ab immune responses within the central nervous system.

Growth of P511 mastocytoma cells in BALB/c mouse brain elicits CTL response without tumor elimination: a new tumor model for regional central nervous system immunity.

We have developed a murine model to explore the tumor-specific CTL response in the immune-privileged central nervous system using P511 mastocytoma cells. Three strains with varying degrees of histocompatibility to P511 cells (CD-1, allogeneic; BALB/c, minor histoincompatible; DBA/2, syngeneic) received tumor cells (10(4)) into the putamen 7 days after cannula implantation, when the blood-brain barrier was functionally intact. Without exception, tumor formed reproducibly by day 7 in all strains. Tumor rejection occurred in CD-1 but not in BALB/c and DBA/2 mice. Using a flank injection site, both CD-1 and BALB/c, but not DBA/2 mice, ultimately rejected flank tumors. Analysis of tumor-specific CTL in BALB/c spleens revealed that P511 administration into brain or flank elicited similar responses: no fully activated CTL were detectable but a significantly expanded population of nonkilling precursors of CTL (pCTL) were present. A P511 cell-specific pCTL population was also identified at the brain tumor site 14 days post-tumor introduction, indicating that pCTL, generated in the periphery, traffic to the tumor site in brain. These data indicate that failure to reject tumor in brain is neither due to lack of afferent stimulation nor to inability of peripheral effectors (P511 cell-specific pCTL) to reach the tumor site. We hypothesize that these effector cells are prevented from developing into fully activated CTL by conditions within the central nervous system microenvironment that down-regulate CTL development.

Evaluation of recombinant protein r140, a polypeptide segment of tegumental glycoprotein Sm25, as a defined antigen vaccine against Schistosoma mansoni.

To investigate the role of tegumental glycoprotein Sm25 in protective immunity against schistosomiasis, codons 43-182 of its gene (GP22) were amplified by PCR and cloned in the pET 15b bacterial expression system. Recombinant protein r140 was inducibly expressed in the presence of rifampicin and purified by Ni-affinity chromatography. In different vaccination trials, Balb/c mice and Fischer rats repeatedly immunized with r140 in combination with one of several adjuvants (alum, cholera toxin or complexed into proteosomes) produced high titre anti-r140 responses. These antibodies detected an N-glycanase sensitive. 25 kDa antigen in a detergent solubilized worm fraction using Western immunoblotting. The choice of adjuvant affected the isotype distribution of the specific anti-r140 antibodies. Despite the presence of high antibody titres and isotypes which have been shown to correlate with protective immunity, protection against subsequent cercarial challenge was not observed. In addition, no appreciable effects on worm sex ratios or liver egg yields were detected in mice. Studies involving biotin labelling of membrane proteins in live worms showed that the majority of anti-r140 reactive molecules present in adult schistosomes are biotinylated after permeabilization of the parasite surface. Several possibilities to account for the lack of protective immunity are analysed.

Immobilization of Schistosoma mansoni miracidia by activation of the alternate complement pathway at unusually high serum dilution.

Free-swimming Schistosoma mansoni miracidia were immobilized by adding normal mammalian serum to the water. Miracidial immobilizing activity (MIA) was shown to result from activating the alternate pathway of complement (APC). MIA in normal sera was heat-sensitive and antibody independent; it was greatly reduced in factor B-depleted or C6-depleted, but not in C1-depleted, human serum. Addition of purified factor B to B-depleted serum totally restored MIA. Half-maximal MIA in normal human, rabbit, and guinea pig sera was detectable at final dilutions exceeding 1/100, 1/200, and 1/500, respectively, and normal rat serum was particularly potent, with MIA at dilutions exceeding 1/2000. Detection of APC activity at such high dilutions is quite extraordinary and attributed to the hypotonic conditions. We confirmed and extended previous findings that heat-inactivated infection sera also display MIA. Immobilizing activity in irradiated-cercarial vaccine rat serum cofractionated with rat IgG and anti-SWAP antibody activity. Antibody-dependent MIA titres were much lower than for APC-dependent MIA. Based upon light microscope and transmission EM studies, immobilization of miracidia by APC activation was attributed to severe tegumental damage. Miracidia within egg shells were insensitive to MIA.

Elevated cerebrospinal fluid IgA in humans and rats is not associated with secretory component.

Immunoglobulin A (IgA) is transported across mucosal tissue membranes covalently bound to secretory component (SC). To determine if this receptor-mediated process also occurs at central nervous system (CNS) boundaries, cerebrospinal fluid (CSF) and serum from patients with CNS neuroinflammatory disease were analyzed for IgA and SC. Excess CSF IgA was detected in six of 24 patients, but no significant CSF SC was detected. In a parallel study using a rat model with normal brain barriers, inactivated lymphocytic choriomeningitis virus was microinfused into CSF. Elevated CSF IgA was detected in four of six rats, yet the proportion of secretory IgA was again insignificant compared to normal exocrine fluids (bile, semen). There does not appear to be a secretory IgA immune system at CNS boundaries and elevated CSF IgA is attributed to intrathecal synthesis.

Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: a new view.

This new view of the immunoreactivity of the normal brain is based on three key components. First, there is an active and highly-regulated communication between the brain and the central immune organs. Secondly, the connection from the brain to the draining nodes is much larger than previously appreciated. And third, the blood-brain barrier, by virtue of its selective permeability properties, contributes to the regulation of immunoregulatory cells and molecules in the brain cell microenvironment.

Afferent and efferent arms of the humoral immune response to CSF-administered albumins in a rat model with normal blood-brain barrier permeability.

Cerebrospinal fluid (CSF) and serum antibody responses to albumin administered into CSF or muscle have been compared with respect to titer, isotype profile and complement-fixing activity in a rat model with normal brain barrier function. CSF/serum titer ratios and the ratio of IgG subclasses, IgG1/IgG2, were both elevated following CSF immunization. In contrast, there was no difference in complement-fixing activity between antibodies elicited by the two routes of immunization. It is suggested that intrathecal antibody synthesis accounts for the elevated CSF antibody titers in CSF-immunized rats, providing the first example of central nervous system antibody synthesis in an animal with normal brain barrier permeability.

Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance.

Cerebral extracellular fluids drain from brain to blood across the arachnoid villi and to lymph along certain cranial nerves (primarily olfactory) and spinal nerve root ganglia. Quantification of the connection to lymph in rabbit, cat and sheep, using radiolabelled albumin as a marker of flow, indicates that a minimum of 14 to 47% of protein injected into different regions of brain or cerebrospinal fluid passes through lymph. The magnitude of the outflow to lymph is at variance with the general assumption that the absence of conventional lymphatics from the brain interrupts the afferent arm of the immune response to brain antigens. The immune response to antigens (albumin or myelin basic protein) introduced into the central nervous system (CNS) has been analysed using a rat model with normal brain barrier permeability. The micro-injection of antigen into brain or cerebrospinal fluid elicits a humoral immune response, with antibody production in cervical lymph nodes and spleen, and also affects cell-mediated immunity. Furthermore, antigen may be more immunogenic when administered into the CNS than into conventional extracerebral sites. Clearly, the afferent arm of the immune response to antigens, within the CNS, is intact. Modern studies suggest that the efferent arm is also intact with passage of activated lymphocytes into the brain. Results support a new view of CNS immunology which incorporates continuous and highly regulated communication between the brain and the immune system in both health and disease.

Ovalbumin is more immunogenic when introduced into brain or cerebrospinal fluid than into extracerebral sites.

The magnitudes of serum antibody responses to ovalbumin have been compared following immunization via cerebral or extracerebral sites in Sprague-Dawley rats. In central nervous system (CNS)-immunized rats, conditions were designed to ensure normal brain barrier permeability. Extracerebral immunization was via the footpad or along pathways of antigen outflow from the CNS. The relative immunogenicity of different injection sites is: CSF greater than brain tissue greater than extracerebral sites. Enhancement of the antibody response to CNS-administered antigen appears to depend on events initiated within the CNS, since ovalbumin injected into blood (which reaches the spleen) or nasal submucosa (which drains to cervical nodes) fails to elicit a similar response.

Modern approaches to development of vaccines against schistosomes.

Vaccination, as currently practiced, is a prophylactic procedure. A vaccine is given before exposure to the infectious agent, in order to induce a state of specific immunological memory within the recipient.

Myelin basic protein infused into cerebrospinal fluid suppresses experimental autoimmune encephalomyelitis.

We have evaluated the antibody and the effector T-cell responses to a single cerebrospinal fluid (CSF) infusion of myelin basic protein (MBP) in Lewis rats by measuring serum anti-MBP antibodies and clinical signs of experimental autoimmune encephalomyelitis (EAE), respectively. Some rats developed anti-MBP antibodies, but none manifested EAE in response to the primary infusion. Antibody responses to an EAE challenge 3 weeks after CSF infusion were normal, but clinical symptoms of EAE were markedly suppressed. Brain trauma at the time of MBP pretreatment enhanced this suppression. The CSF route of MBP administration is more effective in inducing suppression of EAE than peripheral routes.