In questo articolo dimostro che un test LTT per malattia di Lyme che utilizzi come uno degli antigeni la OspC (proteina integra) di B. burgdorferi sensu stricto può teoricamente risultare positivo (falso positivo) in soggetti con aumentata permeabilità intestinale.
Some lymphocyte transformation tests (LTT) popular in Europe for the diagnosis of Lyme disease, use full length OspC of B. burgdorferi as one of their antigens and request a positive stimulation index against only one or two antigens, in order to be considered positive. In what follows, we demonstrate that, in case of patients with gut bacteria translocation, such a test has a theoretical risk of false positive results.
Lymphocyte transformation test
Lymphocyte transformation test (LTT) is an assay which allows to measure the activity of peripheral blood Th cells against specific antigens. T cell activation starts shortly after infection, with T cells proliferation and the production of cytokines (such as INF-γ) which regulate the adaptive immune response (Sompayrac, 2012). As T cell response vanishes after the resolution of the infection (Kaech, et al., 2007)↑, LTT may be useful in providing a proof of active infection. When a LTT assay is performed, Th cells from peripheral blood of a patient are exposed to proteins from a particular pathogen. If a significant reaction is noted, which could be either Th cells proliferation or INF-γ expression, the assay is considered positive and suggestive of an active infection by that particular pathogen. The response is expressed through a number, often referred to as stimulatory index (SI). In Lyme disease, several attempts have been made in order to obtain such a tool, either by T cells proliferation assays or by INF-γ measures (Dressler, et al., 1991)↑, (Chen, et al., 1999)↑, (Valentine-Thon, et al., 2007)↑, (von Baehr, et al., 2012)↑, (Callister, et al., 2016 May)↑. Nevertheless, this procedure has not been fully recognized as useful at present and neither the European guidelines (Stanek, et al., 2011)↑ nor the CDC (Centers for disease control and prevention, 2015)↑ recommend the use of this kind of test.
Th cells activation and cross-reactive T cell epitopes
Th cells are activated when their T cell receptors (TCR) recognize a complementary antigen presented by MHC II molecules (see Figure 1) (Sompayrac, 2012). Peptides presented by MHC II to T helper cells are exclusively linear epitopes, and they have a length between 13 and 17 amino acids (Rudensky, et al., 1991)↑. Various experiments have demonstrated that peptides with 5 identical amino acids in a sequence of 10 have good chances to represent cross-reactive T cell epitopes (Root-Bernstein, 2014)↑. That said, the algorithm described above for the LTT test is not free from the risk of false positive results, as on the surface of an antigen there might be one or more linear epitopes of 10 amino acids which share at least 5 amino acids with some epitope of 10 amino acids from another pathogen. This risk is particularly high when the assay uses complete proteins as antigens, and when a high SI for only a few antigens is required in order to have a positive result of the test.
OspC and Pseudomonas aeruginosa
We have used BLAST from NCBI (National Library of Medicine), with OspC from Borrelia burgdorferi (strain ATCC 35210 / B31 / CIP 102532 / DSM 4680) identified by the swiss-prot ID Q07337 (↑) as query sequence, settings being as follows: expected threshold of 10, BLOSUM62 as substitution matrix, and a word of 3 amino acids. We have built a custom database with the main Phyla of the human gut microbiome observed in a healthy population, which are Bacteroides, Firmicutes, Proteobacteria, Verrucomicrobia, Actinobacteria, Tenericutes, and Euryarchaeota (Giloteaux, et al., 2016)↑. One of the possible matches that BLAST gives back is the following alignment between the query sequence and the outer membrane protein G (OprG) of Pseudomonas aeruginosa (PDB ID: 2X27):
As you can see, we have 6 identical amino acids in a peptide 10 amino acids long. This means that this peptide from Borrelia burgdorferi could theoretically binds a Th cell previously activated by P. aeruginosa. As peptides presented by antigen presenting cells through MHC II are digested in the cytoplasm of these leukocytes, they do not need to be surface exposed in order to elicit a response in Th cells, and don’t need to belong to a surface protein, either. But in an LTT assay, T cells are simply exposed to antigens, this means that the peptide 111-120 of OspC has to be surface exposed, otherwise it will not engage the T cell receptor. As you can see from Figure 2 this is actually the case. The 3D structure of OspC from B. burgdorferi strain B31 used for that picture has been experimentally determined with X rays and a resolution of 2,51 Å in 2001 (Kumaran, et al., 2001)↑ and its MMDB ID is 15958 (↑). The conclusion from this data is that Th cells from a patient with an active infection by P. aeruginosa could proliferate and produce INF-γ when exposed to OspC from B. burgdorferi. In other words, a patient with an active P. aeruginosa infection would come out to have a positive LTT test for OspC.
Gut bacteria translocation
A disrupted mucosal barrier of the bowel, with consequent translocation of bacteria from the gut to the peripheral blood, has been described in patients with liver diseases (Zhu, et al., 2013)↑, chronic HIV infection (Openshaw, 2009)↑, Crohn’s disease (Wyatt, et al., 1993)↑, and in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) (Giloteaux, et al., 2016)↑. In ME/CFS it has been possible, in particular, to demonstrate the translocation of Pseudomonas aeruginosa, among other gram-negative enterobacteria. In fact serum concentration of IgA against lipopolysaccharides from P. aeruginosa and other enterobacteria has been found to be significantly greater in ME/CFS patients than in normal volunteers (Maes, et al., 2007)↑. Thus in ME/CFS patients the adaptive immune system usually reacts against pathogens which exit from the gut, and in particular we know that it reacts against P. auruginosa.
Lyme disease LTT test and ME/CFS
We will now discuss the theoretical risk of false positive results, in the ME/CFS population, of a specific LTT test for Lyme disease, widely used in Europe. This test provides results for the reaction of Th cells against three sets of antigens:
- Borrelia burgdorferi ‘Fully Antigen’, which are antigens from burgdorferi B31;
- Borrelia burgdorferi ‘OSP-mix’, which are OspA, OspC, and DbpA from the three main European pathogenic species of Borrelia (B. burgdorferi, B. garinii and B. afzelii);
- Borrelia burgdorferi LFA-1, which is a linear epitope shared by OspA (B. burgdorferi ss) and by some human leukocytes.
As you can see, OspC from B.burgdorferi sensu stricto is present in both the first and the second set of antigens. Based on what discussed above, we are now able to predict that Th cells from the peripheral blood of a patient with P. aeruginosa translocation due to gut increased permeability, might react in both these assays, thus leading to a false positive LTT test for Lyme disease.
ME/CFS patients are among the main users of this kind of tests, because of the similarities between Lyme disease and the clinical picture of ME/CFS (Gaudino, et al., 1997)↑. ME/CFS patients have a high prevalence of increased gut permeability and gut microbiome translocation (Giloteaux, et al., 2016)↑, and their immune system reacts against P. aeruginosa in many cases (Maes, et al., 2007)↑. Thus, each LTT test for Lyme disease which uses full length OspC from B. burgdorferi ss as antigen, could theoretically lead to a high rate of false positive results in this population of patients. The Lyme disease LTT test discussed above, which is currently popular in Europe, is one of such tests. More researches are warranted in order to confirm or exclude the theoretical danger of cross reaction of Lyme disease LTT tests with gut microbiome. Moreover, on the basis of what here presented, it might be possible to develop an LTT test specific for the diagnosis of gut bacteria translocation.