Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide, but not recombinant OspA. AntiOspA borreliacidal antibody was detected in supernatants of cultures of lymph node cells obtained on day 7 after vaccination, peaked on day 17, and rapidly declined. The borreliacidal activity was attributable to immunoglobulin G1 (IgG1), IgG2a, and IgG2b antibodies. When lymph node cells were treated with interleukin-4 (IL-4), production of borreliacidal antibody was inhibited but was unaffected by treatment with anti-IL-4 antibodies. These results suggest that other cytokines, but not IL-4, are mainly responsible for production of the secondary borreliacidal antibody response.Infection with Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, induces a protracted, yet vigorous, humoral immune response (6,8). Most of the anti-B. burgdorferi antibodies involved in this response are important for confirming the clinical diagnosis of Lyme borreliosis (6,8,9), while they play a minor role in protecting the host against infection. Furthermore, several of these anti-B. burgdorferi antibodies, especially those directed against outer surface protein A (OspA), OspB, OspC, and the 39-kDa periplasmic protein, have a unique dual function. These antibodies are highly specific for the serodiagnostic identification of infection with B. burgdorferi, and they can kill B. burgdorferi in the presence of complement (2,20,(24)(25)(26)(27). Induction of borreliacidal antibodies is helpful in evaluating the potential of B. burgdorferi vaccines (5,11,22,29).Recently, clinical trials of two Lyme borreliosis vaccines containing OspA demonstrated that they could protect humans from becoming infected with B. burgdorferi (28, 32). A major concern, however, is the duration of protection afforded by the anti-OspA borreliacidal antibody response. Previously we showed (22) that vaccination with recombinant OspA (rOspA) induced only a short-lived protective borreliacidal antibody response, even after a booster vaccination. Similarly, OspA borreliacidal antibody waned rapidly in hamsters by week 10 of vaccination (22). Thus rOspA or other B. burgdorferi antigens that induce borreliacidal antibodies must be capable of maintaining sustained high levels of borreliacidal antibodies. This would reduce the number of vaccinations required for induction of borreliacidal antibody and lessen the potential for developing adverse side effects that may resemble arthritis (7). Recently, we showed that severe destructive arthritis could be elicited in vaccinated animals challenged with B. burgdorferi only during periods w...

The mechanism(s) by which Lyme arthritis is induced has not been elucidated. In this study, we showed that macrophages have a direct, effector role in the pathogenesis of Lyme arthritis. Severe destructive arthritis was induced in recipients of macrophages obtained from Borrelia burgdorferi-vaccinated and nonvaccinated hamsters exposed to Formalin-inactivated B. burgdorferi in vitro and then challenged with the Lyme spirochete. Swelling of the hind paws was detected within 8 h of infection, increased rapidly, and peaked at 21 h. This initial swelling decreased, and by day 4 only slight swelling was detected. Severe swelling of the hind paws was detected 8 days after infection and increased rapidly, with peak swelling occurring on day 11. Histopathologic examination affirmed that macrophages exposed to Formalin-inactivated spirochetes induced a severe destructive Lyme arthritis. The onset and severity of the severe destructive arthritis were dependent on the number of macrophages transferred. By contrast, macrophages not exposed to Formalin-inactivated B. burgdorferi failed to induce severe destructive arthritis in recipients after challenge with B. burgdorferi. Similarly, severe destructive arthritis was not detected in recipients of macrophages injected with spirochetal growth medium. Our results also showed that transferred macrophages could not protect hamsters from infection with B. burgdorferi, as spirochetes were readily recovered from their tissues when cultured. These findings demonstrate that macrophages exposed to B. burgdorferi are directly involved in the induction of Lyme arthritis.

cInadequate cervical cytological analysis can be facilitated by glacial acetic acid (GAA) treatment of primary liquid-based collections to remove mucus, erythrocytes, inflammatory cells, and cellular debris. In the context of a commercial human papillomavirus (HPV) hybridization assay performed on 465 tandem specimens with and without GAA treatment, we show that GAA treatment significantly reduces genomic DNA content (P < 0.0001) and creates an increased potential for indeterminate and falsenegative results. In the context of cytological workflow, laboratories should consider providing a specimen aliquot for HPV DNA detection prior to GAA treatment. Molecular diagnostic assays specific for human papillomavirus (HPV) play a significant role in cervical cancer triage (15). Such assays, shown to have Ն98.9% negative predictive value for the underlying presence of cervical intraepithelial neoplasia 2ϩ (5, 13), can be performed on endocervical scrapings preserved in liquid transport medium. This collection, also forwarded for cytological analysis, has the potential to possess insufficient squamous cellularity, as well as abundant erythrocytes, mucus, acute inflammatory cells, and cytological debris (11). In particular, the latter factors may compromise Papanicolaou (Pap) smear analyses that are reliant on filter-based processing protocols (e.g., ThinPrep; Hologic, Marlborough, MA). As a result, glacial acetic acid (GAA)-based protocols have been developed to augment diagnostic yield, with some receiving U.S. Food and Drug Administration approval (4).While this paradigm has procured a high percentage of newly satisfactory Pap smear analyses and has increased detection of significant gynecological lesions (1, 2, 6, 11), recent data have revealed that GAA treatment increases false-positive diagnoses of atypical endocervical cells (3). A few studies, typically focused on hybrid capture technology (H c 2; Qiagen/Digene, Gaithersburg, MD), have been conflicting on the potential effect of GAA on HPV DNA detection (1, 6). In addition to oligonucleotide probes specific for 14 high-risk HPV types (provided in three reagent mixtures), the Cervista HPV HR (Hologic/Third Wave Technologies, Madison, WI) possesses an oligonucleotide probe to assess relative DNA content. The presented study utilizes this internal control as one means of assessing interference potential of GAA treatment on assay performance.In an institutional review board-approved protocol, 465 ThinPrep specimens were collected by two laboratories. These specimens were initially defined as unsatisfactory on the basis of the 2001 Bethesda system guidelines (12) for assessing quality of Pap smears. Laboratory A subjected unsatisfactory ThinPrep specimens to a manufacturer-advocated protocol in which unsatisfactory specimens were initially treated with a titration of GAA and CytoLyt solution (Hologic) (2, 4). In contrast, laboratory B validated a protocol derived from the reports of Iverson and Armour (8) and Agoff et al. (1) in which concentrated GAA was added to spec...

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Arthritis is a frequent complication of infection in humans withBorrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetentBorrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ-) deficient mice with viableB. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology.

The serious morbidity associated with Lyme borreliosis has focused considerable effort on the development of a comprehensive vaccine for protection against infection with Borrelia burgdorferi. Induction of borreliacidal antibody by vaccination or infection has been shown to correlate with protection of humans and animals against infection with the Lyme spirochete. In this report, we showed that high levels of borreliacidal antibody (titer of 1,280) were produced in vitro when T and B cells from hamsters 14 days after vaccination were incubated with macrophages and B. burgdorferi. By contrast, T and B cells from hamsters 7 or 21 days after vaccination failed to initiate production of borreliacidal activity. Furthermore, the T cells from hamsters 7 or 21 days after vaccination inhibited the in vitro production of borreliacidal antibody when cocultured with T and B cells obtained from hamsters 14 days after vaccination. When cell-free supernatants from the suspensions of T and B cells from hamsters 14 days after vaccination were absorbed with recombinant OspA, they lost nearly all borreliacidal activity. The removal of anti-OspA antibody resulted in a decrease in borreliacidal titer from 1,280 to less than 4. These results demonstrate that T cells from vaccinated animals can prevent a sustained production of protective borreliacidal antibody.