br Materials and Methods br Results br Discussion This is

Materials and Methods
Results
Discussion This is a study of IgA seroreactivity to Bb KU60019 Supplier in a large group of early LD patients; we are unaware of any previous studies that have focused on anti-Bb IgA in early LD. As EM develops between 3 and 30days after a tick bite (average of 7days), the level of anti-Borrelia antibody within this patient population can vary greatly (Berger, 1993). Depending upon when patients seek medical attention, they may present with IgM, IgG, or a mixture of both to Bb. In the present study, we report significant levels of antigen-specific serum IgA in approximately one-third of total patients diagnosed with early LD. When IgA seropositivity was evaluated in the context of existing IgM/IgG seropositivity, ~50% of patients who were seropositive for anti-FlaB-mV or anti-C6 IgM or IgG were also seropositive for IgA. The lower rate of IgA binding in the total early LD population may simply be due to the fact that some of the patients had not seroconverted at the time of initial blood draw. We expect that this IgA is monomeric, as the majority of circulating serum IgA in humans is monomeric, and of the IgA1 subtype; however, we did not confirm this in our studies. We assessed IgA binding to two specific immunogenic peptides derived from Bb antigens, C6 and FlaB-mV. We chose to use these peptides rather than whole protein antigens to minimize the possibility of IgA binding to cross reactive epitopes. Concomitantly, very little cross-reactivity was observed in healthy and disease control patients in terms of anti-C6 and -FlaB-mV IgA levels. Sensitivity of IgA binding to FlaB-mV was slightly better than binding to C6, which is unsurprising considering that this peptide contains two epitopes from two different antigens, VlsE and flagellum, where C6 contains only one epitope from a single antigen, VlsE (Gomes-Solecki et al., 2007). The function of IgA in LD is unclear; however, the role of serum IgA in systemic immune responses in general is poorly understood in comparison to the roles of IgM and IgG. It is primarily regarded as a neutralizing antibody when produced in polymeric form at mucosal surfaces, where it is secreted across the mucosal barrier. The role of IgA in LD has been poorly studied, presumably because Bb is not associated with mucosal pathogenesis. We are aware of only one study, from 1979, that measured anti-Bb IgA levels in patients with early disease (Steere et al., 1979); however, anti-Bb IgA was incidental to the focus of the study. It indicated that anti-Bb IgA was present and appeared to increase along with the level of anti-Bb IgG and that both IgA and IgG moved conversely to anti-Bb IgM. IgA production in LD has principally been studied in terms of the production of intrathecal IgA specific to Bb antigens during Lyme neuroborreliosis, a late stage form of the disease (Kaiser, 1998; Steere et al., 1990; Jesse et al., 2011; Schwenkenbecher et al., 2017; Kowarik et al., 2012; Roberg et al., 1995; Steere et al., 1990; Jesse et al., 2011; Schwenkenbecher et al., 2017; Kowarik et al., 2012). High antigen-specific intrathecal IgA levels are also typical of tuberculosis meningitis, which is often included in the differential diagnosis with Lyme neuroborreliosis, as both diseases are marked by abnormal cerebral spinal fluid (CSF) changes in immunoglobulins that include elevated intrathecal IgM, elevated B-specific antibody indexes for IgG or IgM, lymphopleocytosis, severe blood-CSF barrier dysfunction, and intrathecal IgA (Djukic et al., 2012). Conversely, intrathecal synthesis of IgG is observed in multiple sclerosis (MS), an autoimmune disease (Reiber, 1998). This suggests a potential biological role for intrathecal IgA in infectious disease, and provides limited insight into a possible neurologic function of IgA. The rate of IgA seropositivity was significantly higher in patients from the Upper Midwest compared to the Northeast (p<0.005). However, the rate of seropositivity for IgM/IgG was also significantly higher in patients from the Upper Midwest (p<0.0001), suggesting that the lower IgA seropositivity could be linked to a lower rate of seroconversion in early LD patients from the Northeast compared to the Upper Midwest. Concordantly, when IgA seropositivity was evaluated as a function of IgM/IgG seropositivity, the positive levels of IgA to either FlaB-mV or C6 was much closer in patients from the two regions. The difference in seroconversion between the two regions may lie in differences in the duration of illness prior to diagnosis. Though data was only obtained for some patients from the Upper Midwest, the available data suggests that duration of illness prior to diagnosis was far shorter in patients from the Northeast (4.6days±2.4) compared to the Upper Midwest (16.8days±12.0). It should be noted that Northeastern patient data reported duration of EM prior to diagnosis, while Midwestern patient data reported duration of symptoms prior to diagnosis, which could exist prior to development of EM; however, the data still imply a trend toward a longer time of infection prior to seeking medical care. On the other hand, the late LD patients from the Midwest had, as expected, a very high rate of IgG seropositivity (94.7% (18/19) with 5.3% equivocal (1/19) and no negative), but a lower rate of seropositive IgA individuals (31.6%, 6/19), compared to patients with early LD. Therefore, duration of illness does not seem to be a sole driving factor in IgA seropositivity in LD, suggesting that additional factors contribute to the induction of anti-Bb IgA, though at the current time these factors remain undefined. No specific associations between IgA positivity and reported clinical manifestations in the Lyme arthritis population were observed, and we unfortunately did not have access to a bank of serum from patients with Lyme neuroborreliosis. A larger prospective study, based upon our findings, is being designed to further assess the association of anti-Borrelia IgA with specific clinical disease manifestations.