Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment.

We have earlier shown that Borrelia burgdorferi-infected and ceftriaxone-treated mice have viable spirochetes in their body, since immunosuppressive treatment allows B. burgdorferi to be detected by culture. However, the niche of the persisting spirochetes remained unknown. In the present study, we analyzed the tissues of B. burgdorferi-infected and ceftriaxone-treated mice by culture and PCR to reveal the foci of persisting spirochetes. C3H/HeN mice were infected via intradermal needle injection with B. burgdorferi s.s. N40. The mice were treated as follows: (i) short (5 days) and (ii) long (18 days) course of ceftriaxone at 2 weeks of infection and killed after either 10 or 30 weeks, or (iii) the mice received ceftriaxone for 5 days at 18 weeks of infection and were killed 21 weeks after the treatment. All samples of ceftriaxone-treated mice were culture negative, whereas all untreated controls were culture positive. Importantly, B. burgdorferi DNA was detected in the joints of 30-100% of the treated mice. In conclusion, these results combined with earlier results suggest that the joint or a tissue adjacent to the joint is the niche of persisting B. burgdorferi in ceftriaxone-treated mice.

TLR2 utilization of Borrelia does not induce p38- and IFN-beta autocrine loop-dependent expression of CD38, resulting in poor migration and weak IL-12 secretion of dendritic cells.

Lyme borreliosis is a tick-borne bacterial infection that in many cases is limited to the skin. However, in some patients the bacterium evades the immune response and disseminates into various organs. Dendritic cells (DCs) are among the first cells to meet invading pathogens in the skin. We have previously shown that CD38, an ectoenzyme involved in the migration of DCs and generally upregulated by microbial stimuli, is not upregulated in Borrelia garinii-stimulated DCs. In this paper, we characterize the cellular events that lead to the absence of CD38 on the DC surface after B. garinii stimulation and investigate the consequences of absent CD38 expression for the migration of DCs in vitro and in vivo. The data show that 1) effective signaling via p38 MAPK (and STAT1 and NF-kappaB) is needed for CD38 expression and 2) TLR2 stimulation, as opposed to TLR4 stimulation, does not induce IFN-beta autocrine loop-dependent expression of CD38 and secretion of IL-12. Further, we show that 3) B. garinii-stimulated DCs do not migrate effectively toward CCL19 and CCL21 and 4) after B. garinii infection of mice, the number of DCs migrating from the infection site to draining lymph nodes is only half that induced by Escherichia coli infection. Our results provide evidence for the first time that different TLR use results in different CD38 expression, which correlates with the migratory potential of DCs.

Disordered lymphoid purine metabolism contributes to the pathogenesis of persistent Borrelia garinii infection in mice.

Extracellular ATP and adenosine are important regulators of immune responses; however, contribution of purinergic signaling to host defense during persistent microbial infections remains obscure. Lyme borreliosis is a common arthropod-borne infection caused by Borrelia burgdorferi sensu lato. In this study, we investigated whether lymphoid purinergic signaling contributes to the mechanisms by which borreliae species evade the immune system and trigger joint inflammation. Intracutaneous inoculation of Borrelia garinii to C3H/He mice induced symptomatic infection manifested in elevated levels of borrelia-specific IgG Abs, persistent spirochete dissemination into the tissues and joint swelling, as well as approximately 2- to 2.5-fold enlargement of draining lymph nodes with hyperplasia of B cell follicle area and L-selectin shedding from activated T lymphocytes. Purine catabolism was also activated in lymph nodes but not spleen and blood of infected C3H/He mice within the first 4 postinfection weeks, particularly manifested in transient upregulations of adenosine triphosphatase/ectonucleoside triphosphate diphosphohydrolase and ecto-5'-nucleotidase/CD73 on CD4(+)CD8(+) T lymphocytes and adenosine deaminase activity on B220(+) B lymphocytes. Compared with borrelia-susceptible C3H/He strain, lymphocytes from C57BL/6 mice displayed markedly enhanced adenosine-generating capability due to approximately three times higher ratio of ecto-5'-nucleotidase to adenosine deaminase. Borrelia-infected C57BL/6 mice efficiently eradicated the inoculated spirochetes at more chronic stage without any signs of arthritis. Strikingly, deletion of key adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, was accompanied by significantly enhanced joint swelling in borrelia-infected CD73-deficient C57BL/6 mice. Collectively, these data suggest that insufficient basal adenosine level and/or pathogen-induced disordered lymphoid purine homeostasis may serve as important prerequisite for promotion of inflammatory responses and further host's commitment to persistence of bacterial infection and arthritis development.

Fate of five celiac disease-associated antibodies during normal diet in genetically at-risk children observed from birth in a natural history study.

OBJECTIVES: To explore the natural history of antibodies against tissue transglutaminase (TGA), endomysium (EMA), reticulin (ARA), and gliadin (AGA-IgG and AGA-IgA) in children carrying HLA-conferred risk for celiac disease (CD) and observed frequently from birth. METHODS: TGA was measured in serum samples obtained between years 2000 and 2003 from 1,320 children carrying genetic CD risk. If a sample was TGA positive, all five antibodies were analyzed in all banked and forthcoming samples from that child, and a duodenal biopsy was recommended. At the end of this observation, in August 2004, the age of the children was from 1 to 9.5 yr (mean 4.1 yr). RESULTS: Forty-nine children (3.7%) were TGA positive. In these children, AGA-IgG had emerged at the mean age (+/- SD, range) of 2.0 +/- 1.5, 0.5-6.6 yr, while TGA, EMA, and ARA all emerged concurrently somewhat later (TGA at 3.2 +/- 1.5, 1.0-7.0 yr, P < 0.001 when compared to AGA-IgG). Despite continuing gluten exposure, positive TGA, EMA, ARA, AGA-IgA, and AGA-IgG values were spontaneously lost in 49%, 45%, 43%, 41%, and 32% of the children, respectively. CD was diagnosed by biopsy in 20 of the 26 TGA-positive children who consented to a biopsy. CONCLUSIONS: Potential CD trigger(s) other than only gluten probably function before AGA-IgG emerges, i.e., > or =3 months earlier than the transglutaminase-associated antibodies appear. In a remarkable proportion of the children, antibodies disappear spontaneously suggesting that regulatory immune phenomena under favorable circumstances are able to extinguish incipient CD in genetically at-risk children even without exclusion of gluten from the diet.

Anti-tumor necrosis factor-alpha treatment activates Borrelia burgdorferi spirochetes 4 weeks after ceftriaxone treatment in C3H/He mice.

BACKGROUND: The effect of anti-tumor necrosis factor (TNF)-alpha treatment in Borrelia burgdorferi-infected and ceftriaxone-treated C3H/He mice was evaluated. METHODS: Mice were infected with B. garinii A218 or B. burgdorferi sensu stricto N40. At 2 weeks of infection, one group was treated simultaneously with ceftriaxone and anti-TNF-alpha, whereas another received ceftriaxone at 2 weeks and anti-TNF-alpha 4 weeks later. One group received ceftriaxone treatment only. Infected and noninfected control groups were sham treated. RESULTS: At 14 weeks of infection, B. burgdorferi could not be detected by cultivation or by polymerase chain reaction in tissue samples of any mouse treated with ceftriaxone only. However, spirochetes grew from the tissue samples of one-third of the mice treated with anti-TNF-alpha simultaneously or 4 weeks after ceftriaxone. These activated spirochetes showed ceftriaxone sensitivity rates, plasmid profiles, and virulence rates similar to those of bacteria used to infect the mice. All infected control mice and mice given anti-TNF-alpha only were culture positive. CONCLUSIONS: This report shows that, after ceftriaxone treatment for 5 days, a portion of B. burgdorferi-infected mice still have live spirochetes in their body, which are activated by anti-TNF-alpha treatment.

Persistent joint swelling and Borrelia-specific antibodies in Borrelia garinii-infected mice after eradication of vegetative spirochetes with antibiotic treatment.

We wanted to study the pathogenesis and the long-term manifestations of Borrelia garinii infection in SJL and C3H/He mice. We report here that B. garinii A218 causes a persisting infection in these mouse strains. Mice infected with intracutaneous inoculation of B. garinii at 4-5 weeks of age developed a disseminated infection and joint swelling within 2 weeks of inoculation and remained infected with joint symptoms until the end of follow-ups of up to 52 weeks. Treatment with ceftriaxone or ampicillin at 18 or 44 weeks of infection did not affect the joint swelling during the follow-ups of 19 and 8 weeks, respectively. However, B. garinii could not be cultured from any of the post mortem tissue samples of the treated mice, whereas the spirochete grew from samples of all untreated infected animals. Borrelia-specific IgG antibodies were detectable after 2 weeks of infection, and in late infection, all mice had high anti-borrelia IgG levels. Antibiotic treatment had no effect on antibody levels. Histology showed only slight changes in the joints of the infected mice with occasional lymphocyte infiltration, synovial proliferation and slight involvement of the Achilles' tendon. No difference was seen in the findings between ceftriaxone-treated and untreated mice. The results suggest that the presence of vegetative spirochetes is no prerequisite for persisting joint symptoms and elevated anti-borrelia IgG levels in these B. garinii-infected mice.

Species-specific serodiagnosis of Lyme arthritis and neuroborreliosis due to Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii by using decorin binding protein A.

The antigenic potential of decorin binding protein A (DbpA) was evaluated in serodiagnosis of human Lyme borreliosis (LB). The dbpA was cloned and sequenced from the three pathogenic Borrelia species common in Europe. Sequence analysis revealed high interspecies heterogeneity. The identity of the predicted amino acid sequences was 43 to 62% among Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii. The respective recombinant DbpAs (rDbpAs) were produced and tested as antigens by Western blotting and enzyme-linked immunosorbent assay (ELISA). One hundred percent of patients with neuroborreliosis (NB) and 93% of patients with Lyme arthritis (LA) reacted positively. Sera from the majority of patients reacted with one rDbpA only and had no or low cross-reactivity to other two variant proteins. In patients with culture-positive erythema migrans (EM), the sensitivity of rDbpA immunoglobulin G (IgG) or IgM ELISA was low. The DbpA seems to be a sensitive and specific antigen for the serodiagnosis of LA or NB, but not of EM, provided that variants from all three pathogenic borrelial species are included in the combined set of antigens.