Borrelia burgdorferi tissue morphologies and imaging methodologies.

This manuscript offers an image presentation of diverse forms of Borrelia burgdorferi spirochetes which are not spiral or corkscrew shaped. Explanations are offered to justify the legitimacy of tissue forms of Borrelia which may confuse the inexperienced microscopic examiner and which may lead to the misdiagnosis of non-spiral forms as artifacts. Images from the author's personal collection of Borrelia burgdorferi images and a few select images of Borrelia burgdorferi from the peer-reviewed published literature are presented. A commentary justifying each of the image profiles and a survey of the imaging modalities utilized provides the reader with a frame of reference. Regularly spiraled Borrelia are rarely seen in solid tissues. A variety of straightened, undulating, and clipped-off profiles are demonstrated, and the structural basis for each image is explained. Tissue examination is a diagnostic tool and a quality control for judging the eradication or the persistence of borreliosis following attempts to eradicate the infection with antibiotic therapy. The presence or absence of chronic Lyme borreliosis may be objectively adjudicated by tissue examinations which demonstrate or which fail to show pathogenic microbes in patients who have received a full course of antibiotics.

Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection.

Chlamydia trachomatis is the leading cause worldwide of preventable infectious blindness (trachoma) and sexually transmitted disease, including nongonoccocal urethritis and pelvic inflammatory disease. To date, no effective vaccine against C. trachomatis infection has been identified. A monoclonal anti-idiotypic antibody (anti-Id) to the chlamydial exoglycolipid antigen (GLXA) was tested in a murine model of ocular chlamydial infection for its ability to induce systemic immunity, which reduces microbiologic and clinical disease. The anti-Id to GLXA, delivered either systemically in soluble form or orally after encapsulation in poly(lactide) microspheres, induced significant protective immunity against ocular challenge of mice with a human biovar of C. trachomatis. Protection was associated with induction of anti-GLXA antibody and anti-chlamydial neutralizing antibody.

Quantitative investigations of idiotypic antibodies. 3. Persistence and variations of idiotypic specificities during the course of immunization.

Changes and persistence of idiotypic specificities of specifically purified rabbit anti-p-azobenzoate antibodies were studied by quantitative methods. In each rabbit idiotypes identified 2 months after the start of immunization were still present in comparable concentrations 2 months later. After month 4, they were replaced by new and unrelated specificities; the changes were abrupt in two rabbits and gradual in the third, and were associated with an increase in the average affinity for specific hapten. In two surviving rabbits the new sets of specificities persisted in part for at least 1 yr. Quantitative changes occurred during this period, and the antibody preparation used as immunogen reacted most effectively with the homologous anti-D serum. The antibody population present at month 17 (D(17)) in one rabbit was deficient in idiotypic specificities present in D(8) and lacked specificities present in D(2), indicating the presence in D(17) of a third group of specificities. The percentage of the antibody population from each rabbit reactive with homologous anti-idiotypic serum was greater at month 8 than at month 2, suggesting a decrease in heterogeneity. Since the donor rabbits were challenged repeatedly with antigen, it appears that, after month 8, a portion of the antigen was utilized to stimulate existing cell lines and a portion to initiate new clones. Precipitation of anti-p-azobenzoate antibodies removed idiotypic specificities, indicating that they were not present on the anti-bovine gamma-globulin antibodies in the same sera.

Quantitative investigations of idiotypic antibodies. II. Nonprecipitating antibodies.

Idiotypic antibodies were investigated quantitatively by a method of indirect precipitation, which utilizes labeled F(ab')(2) fragments of specifically purified antibenzoate antibody from the donor, anti-antibody, and an antiglobulin reagent. The contribution of allotypic and hidden determinants to these reactions was excluded. Greater fractions of an idiotypic antibody population are precipitated by this method, as compared to direct precipitation, and in two instances large proportions of idiotypic antibodies were detected in populations which failed to form precipitates by double diffusion in agar gel. The greater sensitivity of the indirect method was attributed to its capacity to detect molecules bearing a small number of antigenic determinants. Extensive studies of cross-reactions, carried out by an inhibition technique, failed to reveal any strong reactions of anti-idiotypic antibodies with heterologous antibenzoate antibody preparations, heterologous sera, or IgG, although a few weak cross-reactions were noted. One definite cross-reaction was observed by a direct binding measurement with heterologous antiserum. Antisera prepared in more than one recipient against a single donor preparation reacted with identical or overlapping subpopulations of the donor molecules. Instances in which two recipient antisera reacted with different proportions of the molecules of a single donor provided evidence for the existence of more than one idiotypic antibody population in the antibenzoate antibody of an individual rabbit.

Quantitative investigations of idiotypic antibodies. I. Analysis of precipitating antibody populations.

Specifically purified anti-p-azobenzoate antibodies of the IgG class from individual rabbits were used to elicit anti-idiotypic antibodies in recipient rabbits. Allotypes of each donor and recipient were matched. When polymerized antibodies were used for immunization, more than 80% of the recipients responded with the formation of antibodies that precipitated the monomeric donor antibody. Percentages of precipitable molecules in the donor antibody population (D) varied from 4 to 56. As little as 4% was readily detectable by the Ouchterlony method or precipitin test. Specificity of the reaction was tested by double diffusion in agar gel against a panel of purified antibenzoate antibodies from 14 heterologous rabbits and, quantitatively, in three systems by measurement of the extent of coprecipitation of heterologous, radiolabeled antibenzoate antibodies. No cross-reactions were observed. Reactions were shown to be attributable to antibenzoate antibodies in the donor serum, and contributions of allotypic reactions were excluded. In three systems investigated quantitatively, and in one studied qualitatively, two recipients of the same donor antibody produced anti-antibody that reacted with essentially the same subfraction of the donor antibody population. The findings that only a portion of the D population is immunogenic, and that the same subfraction is frequently immunogenic in different recipients, suggest that the immunogenic population comprises a limited number of homogeneous groups of antibody molecules. This is supported by the small number of bands usually observed by the Ouchterlony technique. Quantitative methods of analysis should provide an approach to the study of cell populations producing antibodies of a particular idiotype.

Specific heterologous enhancement of immune responses. VI. Partial purification of a nonspecific enhancing factor from supernates of allogeneically stimulated human lymphocyte cell lines.

The mixing of two histoincompatible human lymphocyte cell lines generated the release of a soluble factor which was capable of nonspecifically enhancing the in vitro immune response of normal mouse spleen cells against sheep erythrocytes. When active supernates were subjected to exclusion chromatography on Sephadex G-150 and BioGel P-150, the active principle eluted with molecules of approximately 35,000 mol wt. Column aliquots from similarly treated supernates from independent cultures of each lymphoid cell line were inactive. The human enhancing factor was concentrated and purified by ammonium sulfate fractionation, followed by Sephadex gel filtration and polyacrylamide gel electrophoresis.

Oral immunization against chlamydial eye infection.

The effects of enteric administration of different preparations of Chlamydia trachomatis prior to ocular challenge with live chlamydia were compared to the immunity that develops after recovery from ocular infection. Oral immunization with either live homologous serovar B or with formalin-killed heterologus serovar L2 did not influence the response to subsequent ocular challenge. Although oral immunization with live serovar led to protection against heterologous ocular challenge with serovar B, oral immunization with noninfectious UV-irradiated serovar L2 led to more severe and prolonged disease. An immunizing regimen designed for maximal mucosal and systemic immunity also resulted in protection against homologous ocular challenge. Although protection was correlated with the presence of serum IgA antibodies, no clear mechanism for the protective ocular immunity to chlamydial infection has emerged. These studies show that it is possible to stimulate mucosal immunity to induce protection against subsequent ocular challenge with C. trachomatis that is equal to that which follows prior ocular infection.

Concurrent neocortical borreliosis and Alzheimer's disease.

Borrelia spirochetes were directly visualized in autopsy brain tissue from a patient with Alzheimer's disease and were cultured from cerebral cortex in artificial media. The authors propose that, as occurs in tertiary neurosyphilis and general paresis of the insane, Borrelia species may invade the brain, remain in a latent state for many years, and cause dementia in the absence of other focal neurologic deficits. An undetermined fraction of patients with Alzheimer's disease may be shown to have late tertiary neuroborreliosis.

Immunity to chlamydial infections of the eye. IV. Immunity in owl monkeys to reinfection with trachoma.

In a study of trachoma in owl monkeys, it was found that owl monkeys are equally susceptible to low and high doses of trachoma and that resistance to reinfection persisted for six months in the majority of animals. Previous infections with a single trachoma type did not elicit greater resistance than previous infections with two types. Both serum antibodies and eye secretion titers correlated well with resistance to reinfection, but it is not as yet clear if either or both play a substantial role in immunity to trachoma.

Gestational Lyme borreliosis. Implications for the fetus.

Great diversity of clinical expression of signs and symptoms of gestational Lyme borreliosis parallels the diversity of prenatal syphilis. It is documented that transplacental transmission of the spirochete from mother to fetus is possible. Further research is necessary to investigate possible teratogenic effects that might occur if the spirochete reaches the fetus during the period of organogenesis. Autopsy and clinical studies have associated gestational Lyme borreliosis with various medical problems including fetal death, hydrocephalus, cardiovascular anomalies, neonatal respiratory distress, hyperbilirubinemia, intrauterine growth retardation, cortical blindness, sudden infant death syndrome, and maternal toxemia of pregnancy. Whether any or all of these associations are coincidentally or causally related remains to be clarified by further investigation. It is my expectation that the spectrum of gestational Lyme borreliosis will expand into many of the clinical domains of prenatal syphilis.

Clinical implications of delayed growth of the Lyme borreliosis spirochete, Borrelia burgdorferi.

Lyme borreliosis, a spirochetal infection caused by Borrelia burgdorferi, may become clinically active after a period of latency in the host. Active cases of Lyme disease may show clinical relapse following antibiotic therapy. The latency and relapse phenomena suggest that the Lyme disease spirochete is capable of survival in the host for prolonged periods of time. We studied 63 patients with erythema migrans, the pathognomonic cutaneous lesion of Lyme borreliosis, and examined in vitro cultures of biopsies from the active edge of the erythematous patch. Sixteen biopsies yielded spirochetes after prolonged incubations of up to 10.5 months, suggesting that Borrelia burgdorferi may be very slow to divide in certain situations. Some patients with Lyme borreliosis may require more than the currently recommended two to three week course of antibiotic therapy to eradicate strains of the spirochete which grow slowly.

The roles of alcohol and alcohol expectancy in the dampening of responses to hyperventilation among high anxiety sensitive young adults.

Previous research suggests that high anxiety sensitivity (AS) young adults are particularly sensitive to alcohol's dampening effects on their responses to arousal-induction challenge [Alcohol.: Clin. Exp. Res. 24 (2000) 1656.]. This sensitivity to alcohol reward may place high AS individuals at increased risk for alcohol abuse. Over-and-above alcohol's pharmacological effects, tension-reduction expectancies might contribute to alcohol's reactivity-dampening effects in high-AS individuals. The present study examined the role of alcohol and alcohol expectancy factors by activating expectancies experimentally. Forty-eight high-AS young adults were randomly assigned to one of three beverage conditions: alcohol (pharmacology plus expectancy), placebo (expectancy only), and control (no pharmacology and no expectancy). Following beverage consumption and absorption, participants underwent a 3-min voluntary hyperventilation challenge. Replicating and extending previous findings, participants in the alcohol condition showed dampened affective and somatic responses to the challenge, and marginally dampened cognitive responses to the challenge, compared to both placebo and control participants. However, placebo participants did not display dampened responses to the challenge relative to control beverage condition participants. Additional analyses suggested that activation of tension-reduction expectancies might have contributed to an "inverse placebo" effect among high-AS participants administered placebo. Implications of the results for future research and for the prevention and treatment of alcohol problems among high-AS individuals are discussed.

Antigens of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular parasite that elaborates antigens on its surface. These antigens are divided into genus-, species-, subspecies-, and serovar-specific determinants. The genus, or group antigen(s), are lipopolysaccharides similar to those found in gram-negative bacteria and a glycolipid that is secreted by infected cell cultures. Species-specific antigens differentiate Chlamydia trachomatis from Chlamydia psittaci and are expressed on the outer membrane. These proteins range in molecular weight from 155,000 to approximately 40,000. Monoclonal antibodies to outer-membrane proteins have demonstrated the presence of subspecies-reactive antigenic determinants. Type-specific antigens are associated with the major outer-membrane protein and are secreted from infected cells as well. The molecular weights of these proteins range from 30,000 to 40,000. These antigens may participate in the binding of the organism to target cells and in an enzymatic process of some type that initiates endocytosis. The significance of the soluble antigens detected in the microenvironment in vitro may suggest immune-complex formation, a process that could contribute to the immunopathology of the disease.

Lyme disease. A neuro-ophthalmologic view.

Lyme borreliosis is a spirochetal infection with a potential to produce a clinical disease in the human host with protean manifestations as diverse as the spectrum of disease caused by Treponema pallidum. Neuro-ophthalmologic manifestations of Lyme borreliosis are emphasized in this short review. A brief historical chronicle of Lyme disease is offered. Potential pitfalls in the diagnosis of Lyme disease with an emphasis on false negative serology and currently available diagnostic modalities are presented. Therapeutic options for Lyme borreliosis are briefly reviewed.

Partial characterization of an immunoenhancing factor from allogeneic human lymphocyte cell lines.

The mixing of two histoincompatible human lymphocyte cell lines generated the release of a soluble factor which was capable on non-specifically enhancing the in vitro immune response of normal mouse spleen cells against sheep erythrocytes. The mediator was secreted into the supernatant of the allogeneic cell cultures within 24 h of cultuvation. The human enhancing factor (HEF) must be added to assay cultures on day 2, of a 5-day culture period, for its activity to be manifest. HEF was resistant to DNase, RNase and heating at 56 degrees for 30 min, but was inactivated by exposure to protease or elevated temperature (80 degrees for 30 min). The molecular weight of HEF, purified by ammonium sulphate fractionation, followed by Sephadex gel filtration, DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis, was approximately 38,000 Daltons.

Isolation of a type-specific antigen from Chlamydia trachomatis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

This report describes the isolation of a type-specific antigen of a serotype A strain of Chlamydia trachomatis. The antigen could be identified in sodium dodecyl sulfate-polyacrylamide electrophoretic (SDS-PAGE) analysis of immunoprecipitates of homologously reacted lysates from Bolton-Hunter 125I-labeled elementary bodies, solubilized by sonication and treatment with Nonidet P40. The electrophoretic pattern of this precipitate revealed a peak of unique mobility that was not reproduced by heterologous or control precipitates. Immunoadsorbtion of test antigen with purified IgG fractions from homologous antisera completely removed this peak, whereas similar adsorbtion wth heterologous IgG had minimal effect. Comparison of this antigen in SDS-PAGE with protein standards revealed an approximate m.w. of 27,000.

Some characteristics of a secreted chlamydial antigen recognized by IgG from C. trachomatis patient sera.

Chlamydia trachomatis serovars release a glycolipid antigen (GLXA) into the culture supernatant during the infective cycle. This antigen is recognized by IgG isolated from humans with a natural chlamydial infection; GLXA may be a major antigenic determinant of chlamydia. It can be immunopurified by molecular shift or affinity chromatography. Silver staining of SDS-PAGE gels demonstrates a pattern of bands that is essentially the same for preparations isolated by either method. GLXA can also be extracted from mature elementary bodies (EB). These preparations show the same pattern of silver staining bands, and the major bands are immunoreactive as shown by Western blot analysis. Isoelectric focusing studies demonstrate that purified GLXA has an acidic pI.

Neutralization of tetanus toxin by human and rabbit immunoglobulin classes and subunits.

This investigation found that the human antibody class of importance in neutralizing tetanus toxin in mice was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of the human IgG class. Although human IgM and IgA classes appeared to neutralize tetanus toxin at very low levels, evidence was obtained that this neutralization was probably due to IgG contamination. Human Fabmu isolated from the IgM class did not neutralize tetanus toxin. Human antibodies of the IgG, IgM and IgA classes reacted with tetanus toxoid in the indirect haemagglutination (HA) test with IgG giving the highest HA titre. Rabbit antibodies of the IgG class also neutralized tetanus toxin, with neutralization being retained by the F(ab')2 and Fab' subunits of the rabbit IgG class. Absorption of several rabbit antisera to tetanus toxoid with goat-antirabbit Fc which is specific for absorption of IgG from antiserum, rendered them incapable of neutralizing tetanus toxin.

Immune response of owl monkeys to topical vaccination with irradiated Chlamydia trachomatis.

The conjunctivae of owl monkeys were topically vaccinated with purified Chlamydia trachomatis organisms that had been inactivated by 60Co irradiation and lyophilized onto an inert carrier. Vaccinated monkeys developed antibody in serum and tears, while control animals given a placebo had no detectable titers. When challenged 35 days after the start of administration of the vaccine, all monkeys showed evidence of infection. The vaccinated group had a longer course of disease and more ocular discharge than did controls. Antibody levels in both serum and tears were nearly 10-fold higher after infection in vaccinated animals than in controls.

Use of an autologous antigen in the serologic testing of patients with erythema migrans of Lyme disease.

We attempted to detect an early rise in antibody titers to Borrelia burgdorferi in the serum of patients with erythema migrans of Lyme disease by utilizing B. burgdorferi isolates obtained from patients' own skin lesions instead of the B31 reference strain. B. burgdorferi was isolated from nine of 23 skin biopsy specimens submitted for culture. Elevated antibody titers were not detected in any of the 23 acute serum samples by immunofluorescence assay. The antigens derived from patient isolates were no more effective than the reference strain in detecting antibodies in patients with early Lyme disease.