T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.

We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-gamma secreting cells were detected by ELISPOT. The peptides OspA(17-36), OspA(49-68), OspA(105-124), OspA(137-156), OspA(193-212) and OspA(233-252) showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls (P = 0.004). CSF cells more often showed positive responses to these peptides than blood cells (P = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.

In vitro Th2 deviation of myelin-specific peripheral blood lymphocytes from patients with multiple sclerosis.

This study aimed at investigating if selective ex vivo immune deviation of myelin-specific cytokine secretion towards Th2 is possible in blood cells from patients with multiple sclerosis (MS). Interleukin (IL)-4 (Th2) and interferon-gamma (Th1) secreting cells were recorded by ELISPOT in 13 MS patients. Deviation was successful in 10 patients. Interleukin-4 alone was most effective in inducing myelin-specific immune deviation in MS patients whereas IL-1 or IL-15 in combination with IL-4 did not improve the results. Further studies and improvements are needed before ex vivo immune deviation can be considered a potential treatment in patients with MS.

Cytokine secretion in decidual mononuclear cells from term human pregnancy with or without labour: ELISPOT detection of IFN-gamma, IL-4, IL-10, TGF-beta and TNF-alpha.

Cytokines are believed to be important in maintaining pregnancy and in the process of labour induction in humans. The aim of this study was to investigate the secretion of the cytokines interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-10, transforming growth factor-beta (TGF-beta) and tumour necrosis factor-alpha (TNF-alpha) in decidual tissue with or without labour. Decidual tissue was collected from 32 healthy women undergoing elective caesarean sections before the onset of labour (n=17) or after normal vaginal delivery (n=15). Mononuclear cells were analysed for cytokine secretion with ELISPOT. To validate the widely used method of tissue collected at caesarean sections and after vaginal deliveries as a representative of before and after labour, respectively, placenta biopsies were collected from 12 healthy women to study the expression of the prostaglandin pathway enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E(2) synthase (mPGES). Decidual mononuclear cells from term human pregnancy spontaneously secrete IFN-gamma, IL-4, IL-10, TGF-beta and TNF-alpha. No difference was seen in cytokine secretion with or without labour, indicating that decidual leukocytes are not the main cell population responsible for plausible cytokine regulation in the process of termination of pregnancy. Placental tissues obtained after vaginal delivery showed a higher mRNA expression of the prostaglandin regulating molecules COX-2 and mPGES than tissues from caesarean sections before the onset of labour, validating that the model can be used as a representative of the state before and after labour.

The use of cell products for treatment of autoimmune neuroinflammatory diseases.

Cell products are live cells that are given to patients in order to replace or modify the function of missing or dysfunctional cells. Progress in technology and in the understanding of pathobiology may lead to the use of cell products in many areas. This review outlines the use of cell products in the treatment of autoimmune diseases, with focus on neuroinflammatory diseases like multiple sclerosis. Treatment of autoimmune diseases should be selective and specific in order to avoid serious side effects. To achieve this, T lymphocyte regulation has been in focus for several immunomodulatory regimens. One area of great interest is the use of T cell vaccination, when autologous attenuated auto-reactive T cells are given to patients in order to initiate a specific immune response to the pathogenic T cell populations. Phopheresis may be an immunomodulatory treatment related to T cell vaccination. Another promising area involves ex-vivo alteration of the cytokine profile of harmful auto-reactive T cells. This can be achieved by genetic manipulation or by certain cytokine stimulations. A subsequent adoptive cell transfer will, by homing mechanisms, lead to at site specific delivery of the cells, which will have a local down-regulatory effect on the inflammatory process. Although unsolved questions regarding doses, timing, optimal preparing conditions and mechanisms still remain, both T cell vaccination and adoptive transfer of ex-vivo manipulated cytokine secreting cells have proven successful for treatment of neuroinflammation in experimental models. T cell vaccination was shown to be feasible in patients with multiple sclerosis, however, otherwise the experience in humans so far is limited.

Increased expression of the Th1-inducing cytokines interleukin-12 and interleukin-18 in cerebrospinal fluid but not in sera from patients with Lyme neuroborreliosis.

Lyme neuroborreliosis is a complex disease with different clinical outcomes and where immunopathological mechanisms are probably involved. In this study, sera and cerebrospinal fluid (CSF) from 21 neuroborreliosis patients and 26 control patients were analyzed for the Th1-inducing cytokines, interleukin (IL)-12 and IL-18, and the Th2 associated, soluble CD30 (sCD30) by ELISA. The results showed an increased number of neuroborreliosis patients expressing IL-12 (p<0.05) and IL-18 (p<0.05) in the CSF when compared with the controls, but no indication of increased levels in the sera. Nor were there any differences regarding levels of sCD30 in the sera or the CSF, indicating a local Th1-generating milieu in the target organ of neuroborreliosis.

Augmented intrathecal secretion of interferon-gamma in response to Borrelia garinii in neuroborreliosis.

The Lyme disease agent Borrelia garinii has been suggested to be neurotrop, preferentially affecting the nervous system. We compared the secretion of interferon-gamma in response to outer surface proteins from Borrelia garinii and Borrelia afzelii in 10 patients with neuroborreliosis. In cerebrospinal fluid, stimulation with Borrelia garinii revealed higher numbers of interferon-gamma-secreting cells in all patients, whereas in blood, only five displayed higher numbers. This further strengthens the hypothesis of Borrelia garinii being associated with the development of neuroborreliosis.

Compartmentalization of antigen specific cytokine responses to the central nervous system in CNS borreliosis: secretion of IFN-gamma predominates over IL-4 secretion in response to outer surface proteins of Lyme disease Borrelia spirochetes.

The neurological manifestations of Lyme disease have been proposed to be partly due to cytokine-mediated immunopathological mechanisms. In this study, the number of Borrelia-specific cells secreting interferon-gamma and interleukin-4 was determined in blood and cerebrospinal fluid from patients with CNS borreliosis (n = 23), other neurological diseases (n = 20), and in blood from healthy controls (n = 10), utilizing an ELISPOT-assay. Elevated specific secretion of IFN-gamma was found in CNS borreliosis, most pronounced in cerebrospinal fluid, whereas secretion of IL-4 was strikingly low. This may indicate that symptoms are due to side effects of the immune response, since IFN-gamma secretion in the absence of corresponding levels of IL-4 may be associated with tissue destruction.

Phenotypes indicating cytolytic properties of Borrelia-specific interferon-gamma secreting cells in chronic Lyme neuroborreliosis.

The immuno-pathogenetic mechanisms underlying chronic Lyme neuroborreliosis are mainly unknown. Human Borrelia burgdorferi (Bb) infection is associated with Bb-specific secretion of interferon-gamma (IFN-gamma), which may be important for the elimination of Bb, but this may also cause tissue injury. In order to increase the understanding of the pathogenic mechanisms in chronic neuroborreliosis, we investigated which cell types that secrete IFN-gamma. Blood mononuclear cells from 13 patients with neuroborreliosis and/or acrodermatitis chronicum atrophicans were stimulated with Bb antigen and the phenotypes of the induced IFN-gamma-secreting cells were analyzed with three different approaches. Cells expressing CD8 or TCRgammadelta, which both have cytolytic properties, were the main phenotypes of IFN-gamma-secreting cells, indicating that tissue injury in chronic neuroborreliosis may be mediated by cytotoxic cells.

Elevated number of cells secreting transforming growth factor beta in Guillain-Barré syndrome.

We used ELISPOT and cell ELISA to study secretion of IL-4, IFN-gamma, TGF-beta, IL-6, and TNF-alpha by circulating mononuclear cells during the course of Guillain-Barré syndrome (GBS). Compared to healthy controls, patients with GBS had higher numbers of TGF-beta-secreting cells and the number of individuals with myelin-peptide-induced IL-4 and TGF-beta secretion was higher in the GBS group. No significant differences were seen concerning the predominantly pro-inflammatory cytokines IFN-gamma, IL-6 or TNF-alpha. Our findings indicate a down-regulatory role for TGF-beta and IL-4 in GBS.

Lyme borreliosis in Sweden--diagnostic performance of five commercial Borrelia serology kits using sera from well-defined patient groups.

Five commercial Borrelia serology kits available in Sweden were evaluated and compared for their diagnostic performance in sera from clinically well-characterized patient groups. With the clinically defined groups as the gold standard, i.e. without knowledge of antibody status in serum and cerebrospinal fluid, the diagnostic performance of the kits was compared and important differences in diagnostic usefulness were found. The kits from Abbot and DAKO, that often predict clinically relevant Borrelia infection and do not detect antibodies in sera from patients without strong suspicion of Borrelia infection, were considered the most useful in the population studied. This kind of validation study is an important part of good laboratory practice and should be performed by laboratories serving patient populations with varying endemicity of Borrelia.

Avidity distribution of antibodies against peripheral nerve myelin in patients with polyneuropathy associated with IgM monoclonal gammopathy and in healthy controls.

To elucidate the role and nature of antibodies against peripheral nerve myelin (PNM) we studied their avidity distribution. Twelve patients with demyelinating polyneuropathy associated with IgM monoclonal gammopathy were compared with 12 healthy blood donors previously found to have anti-PNM antibodies of IgM isotype. For comparison, the avidity distribution of IgM antibodies against the varicella zoster antigen in 10 patients with herpes zoster infection was also studied. Microtitre plates containing antibody bound to antigen were exposed to increasing concentrations of sodium thiocyanate (NaSCN) followed by an ELISA assay. NaSCN changes the ion strength and the pH, and thereby the critical conditions for antibody-antigen binding. Resistance to NaSCN was used as a measure of antibody avidity. Anti-PNM antibodies from patients with monoclonal gammopathy were of predominantly low avidity whereas antibodies from blood donors were of predominantly high avidity. Avidity index, representing the molar concentration of NaSCN required to reduce the initial absorbance values by 50%, was on average 11.7 times higher in blood donors (range 0.24-2.65, mean = 0.82) than in patients with monoclonal gammopathy (range 0.04-0.10, mean = 0.07) (p = 0.002). On the other hand, patients with monoclonal gammopathy had on average a 100-fold higher relative concentration of antibodies against PNM compared to blood donors (range 4.1-392.6 AU, mean 85.0 AU, and range 0.2-1.7 AU, mean 0.85 AU, respectively) (p = 0.002). Antibodies against the varicella zoster antigen from patients with herpes zoster showed a high avidity index (range 0.25-2.6, mean = 1.24). Using Western blot, several 14-30 kDa proteins in PNM were found to be the target antigen for IgM anti-PNM antibodies in both patients with monoclonal gammopathy and polyneuropathy, and in blood donors.(ABSTRACT TRUNCATED AT 250 WORDS)

T helper type 2 like cytokine responses to peptides from P0 and P2 myelin proteins during the recovery phase of Guillain-Barré syndrome.

T-lymphocytes are probably involved in the pathogenesis of Guillain-Barré syndrome (GBS). T-helper-1 (Th1) cytokines activate macrophages and induce a delayed type hypersensitivity (DTH) inflammatory response, consistent with the morphology of the demyelination in GBS. Th2 cytokines encourage antibody production and downregulate Th1 responses. To study the Th1/Th2 cytokines in relation to the clinical course of GBS an ELISPOT method for determination of single cells secreting interferon-gamma, IFN-gamma (Th1) or interleukin-4, IL-4 (Th2) was used. We serially investigated antigen-induced cytokine secretion from circulating T-cells stimulated with human peptides from the P0 and P2 proteins in seven patients and compared to results from seven serially investigated healthy controls. Most patients (five of seven) showed IL-4 responses during the plateau- or recovery-phase as compared to controls. One patient with a prolonged disease course, on the other hand, had an IFN-gamma dominated reactivity. We suggest that the IL-4 responses are beneficial in GBS, and may have a role in terminating the disease process in this self-limiting inflammatory disease.

Induction of neutralizing autoantibodies to interferon-gamma in patients with polyneuropathy.

We previously demonstrated the induction of IFN-gamma in polyneuropathy patients associated with monoclonal gammopathy, but not in patients with presumably non-immunological types of neuropathy. We herein examined mechanism involving release of neutralizing autoantibodies (Aabs) to IFN-gamma in sera from those patients. In contrast to polyneuropathy patients with monoclonal gammopathy, patients with polyneuropathy of presumably non-immunological types showed increased production of neutralizing Aabs specific for IFN-gamma. These results demonstrate a role for autoimmunity in cytokine regulation. However, their association to the clinical manifestations of the disease requires further investigations, which are necessary for future consideration in therapeutic strategies.

Longitudinal interferon-ß effects in multiple sclerosis: differential regulation of IL-10 and IL-17A, while no sustained effects on IFN-γ, IL-4 or IL-13.

BACKGROUND: Recent studies in experimental models and in vitro indicate lowering of IL-17/Th17 as an important mechanism of interferon-beta (IFN-ß) treatment in multiple sclerosis (MS). MATERIAL AND METHODS: In this longitudinal study of MS patients (n=25), spontaneous and myelin antigen-induced secretion of IL-4, IFN-γ and IL-10 (ELISPOT), mitogen stimulated secretion of IL-13 and IL-17A (ELISA) and circulating cytokine levels (Luminex) were recorded at inclusion and after 1.5, 3, 6 and 12months of IFN-ß treatment. RESULTS: Early changes were noted for IL-4, while after one year of treatment the only recorded significant effects were a decrease in secreted IL-17A levels and an increase in IL-10 secreting cells. While IL-17A levels tended to be higher in non-responders (n=8), the decrease in IL-17A levels seemed to be more pronounced in responders (n=17) showing significantly lower IL-17A levels after one year as compared with non-responders. CONCLUSION: IFN-ß treatment seems to mainly affect IL-17/IL-10-associated pathways rather than the IFN-γ/IL-4 axis.

Mercury exposure as a model for deviation of cytokine responses in experimental Lyme arthritis: HgCl2 treatment decreases T helper cell type 1-like responses and arthritis severity but delays eradication of Borrelia burgdorferi in C3H/HeN mice.

Lyme borreliosis is a complex infection, where some individuals develop so-called 'chronic borreliosis'. The pathogenetic mechanisms are unknown, but the type of immune response is probably important for healing. A strong T helper cell type 1 (Th1)-like response has been suggested as crucial for eradication of Borrelia and for avoiding development of chronic disease. Many studies aimed at altering the Th1/Th2 balance in Lyme arthritis employed mice deficient in cytokine genes, but the outcome has not been clear-cut, due possibly to the high redundancy of cytokines. This study aimed at studying the importance of the Th1/Th2 balance in murine Borrelia arthritis by using the Th2-deviating effect of subtoxic doses of inorganic mercury. Ninety-eight C3H/HeN mice were divided into four groups: Borrelia-infected (Bb), Borrelia-infected exposed to HgCl(2) (BbHg), controls exposed to HgCl(2) alone and normal controls. Mice were killed on days 3, 16, 44 and 65 post-Borrelia inoculation. Arthritis severity was evaluated by histology, spirochaetal load determined by Borrelia culture, IgG2a- and IgE-levels analysed by enzyme-linked immunosorbemt assay (ELISA) and cytokine-secreting cells detected by enzyme-linked immunospot (ELISPOT). BbHg mice showed less severe histological arthritis, but delayed eradication of spirochaetes compared to Bb mice, associated with increased levels of IgE (Th2-induced) and decreased levels of IgG2a (Th1-induced), consistent with a Th2-deviation. Both the numbers of Th1 and Th2 cytokine-secreting cells were reduced in BbHg mice, possibly explained by the fact that numbers of cytokine-secreting cells do not correlate with cytokine concentration. In conclusion, this study supports the hypothesis that a Th1-like response is required for optimal eradication of Borrelia.

Decreased up-regulation of the interleukin-12Rbeta2-chain and interferon-gamma secretion and increased number of forkhead box P3-expressing cells in patients with a history of chronic Lyme borreliosis compared with asymptomatic Borrelia-exposed individuals.

Lyme borreliosis (LB) can, despite adequate antibiotic treatment, develop into a chronic condition with persisting symptoms such as musculoskeletal pain, subjective alteration of cognition and fatigue. The mechanism behind this is unclear, but it has been postulated that an aberrant immunological response might be the cause. In this study we investigated the expression of the T helper 1 (Th1) marker interleukin (IL)-12Rbeta2, the marker for T regulatory cells, forkhead box P3 (FoxP3) and the cytokine profile in patients with a history of chronic LB, subacute LB, previously Borrelia-exposed asymptomatic individuals and healthy controls. Fifty-four individuals (12 chronic LB, 14 subacute LB, 14 asymptomatic individuals and 14 healthy controls) were included in the study and provided a blood sample. Mononuclear cells were separated from the blood and stimulated with antigens. The IL-12Rbeta2 and FoxP3 mRNA expression was analysed with real-time reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of IL-12Rbeta2 on CD3(+), CD4(+), CD8(+) and CD56(+) cells was assessed by flow cytometry. Furthermore, the secretion of interferon (IFN)-gamma, IL-4, IL-5, IL-10, IL-12p70 and IL-13 was analysed by enzyme-linked immunospot (ELISPOT) and/or enzyme-linked immunosorbent assay (ELISA). Chronic LB patients displayed a lower expression of Borrelia-specific IL-12Rbeta2 on CD8(+) cells and also a lower number of Borrelia-specific IFN-gamma-secreting cells compared to asymptomatic individuals. Furthermore, chronic LB patients had higher amounts of Borrelia-specific FoxP3 mRNA than healthy controls. We speculate that this may indicate that a strong Th1 response is of importance for a positive outcome of a Borrelia infection. In addition, regulatory T cells might also play a role, by immunosuppression, in the development of chronic LB.

Different cytokine profiles in patients with a history of gangrenous or phlegmonous appendicitis.

Appendicitis is one of the most common and costly acute abdominal states of illnesses. Previous studies suggest two types of appendicitis which may be different entities, one which may resolve spontaneously and another that progresses to gangrene and perforation. Gangrenous appendicitis has a positive association to states of Th1 mediated immunity whereas Th2 associated immune states are associated with lower risk of appendicitis. This study investigated the inflammatory response pattern in patients previously appendicectomized for gangrenous (n = 7), or phlegmonous appendicitis (n = 8) and those with a non-inflamed appendix (n = 5). Peripheral blood mononuclear cells were analysed with ELISPOT analysis for number of spontaneous or antigen/mitogen stimulated IFN-gamma, IL-4, IL-10 and IL-12 secreting cells or with ELISA for concentration of spontaneous or antigen/mitogen stimulated IFN-gamma, IL-5 and IL-10. Spontaneously IL-10 secreting cells/100,000 lymphocytes were increased in the gangrenous group compared to the phlegmonous group (P = 0.015). The median concentration of IL-10 secreted after Tetanus toxoid (TT)-stimulation were higher in the gangrenous group and the control group, than the phlegmonous group (P = 0.048 and P = 0.027, respectively). The median concentration of TT induced IFN-gamma secretion was higher for the gangrenous group compared to both the phlegmonous group and the control group (P = 0.037 and P = 0.003). Individuals with a history of gangrenous appendicitis demonstrated ability to increased IL-10 and IFN-gamma production. The increased IFN-gamma may support the notion of gangrenous appendicitis as an uncontrolled Th1 mediated inflammatory response and increased IL-10 may speculatively indicate the involvement of cytotoxic cells in the progression to perforation.

Reduced number of interleukin-12 secreting cells in patients with Lyme borreliosis previously exposed to Anaplasma phagocytophilum.

Lyme borreliosis and human granulocytic ehrlichiosis are tick-borne diseases caused by Borrelia burgdorferi and Anaplasma phagocytophilum, respectively. Infection with A. phagocytophilum has been observed to induce immunosuppression and animal studies suggest that the bacteria might also have prolonged inhibitory effects on immune cells. The aim of this study was to investigate the cytokine secretion in patients exposed previously to A. phagocytophilum and currently infected with B. burgdorferi compared with patients infected with B. burgdorferi and seronegative for A. phagocytophilum. Eight patients with erythema migrans and antibodies against A. phagocytophilum, 15 patients with erythema migrans and negative A. phagocytophilum serology and 15 non-exposed healthy individuals were included in the study. Blood mononuclear cells were stimulated with Borrelia-antigen and the number of cytokine [interleukin (IL)-4, IL-5, IL-12, IL-13 and interferon (IFN)-gamma]-secreting cells was detected by enzyme-linked immunospot (ELISPOT). This study shows that patients with a previous exposure to A. phagocytophilum and a current infection with B. burgdorferi have a lower number of Borrelia-specific cells secreting IL-12 compared to Ap seronegative patients infected with B. burgdorferi (P < 0.001), indicating impairment in the ability to mount strong Th1-responses. We suggest that this mirrors a reduced Th1 response caused by A. phagocytophilum which could influence the outcome of the Borrelia infection and, speculatively, may also have implications in other conditions.

Innate immune responses in Lyme borreliosis: enhanced tumour necrosis factor-alpha and interleukin-12 in asymptomatic individuals in response to live spirochetes.

Innate immunity is important for early defence against borrelia spirochetes and should play a role in the clinical outcome of the infection. In order to study early cytokine responses, in vitro differentiated dendritic cells (DCs) and whole blood cells from 21 patients with different clinical outcomes of Lyme neuroborreliosis were stimulated with live borrelia spirochetes. The borrelia-induced secretion of interleukin (IL)-4, IL-10, IL-12p70, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in DCs and IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNF-alpha, regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and eotaxin in whole blood cells was measured by enzyme-linked immunospot (ELISPOT) and multiplex arrays, respectively. We found increased numbers of TNF-alpha-secreting DCs (P = 0.018) in asymptomatic seropositive individuals compared to patients with subacute neuroborreliosis and seronegative controls. Asymptomatic individuals were also found to have elevated levels of IL-12p70 (P = 0.031) in whole blood cell supernatants compared to seronegative controls. These results are in line with previous experiments using cells of the adaptive immune response, indicating that strong T helper type 1 (Th1) proinflammatory responses might be associated with a successful resolution of Lyme disease.

Th1-like responses to peptides from peripheral nerve myelin proteins in patients with polyneuropathy associated with monoclonal gammopathy.

Polyneuropathy associated with monoclonal gammopathy is usually regarded as an immune-mediated disorder with autoantibody activity against myelin glycoproteins. The pathogenic mechanisms are, however, not fully understood. Several reports have indicated an additional involvement of T cells. We investigated the occurrence of myelin-specific T cells as well as their functional characteristics. Cytokine responses generated after stimulation of peripheral blood mononuclear cells with selected peptides of myelin proteins P0 and P2 were investigated with an ELISPOT method. Three P0 peptides caused significantly elevated levels of interferon-gamma (IFN-gamma)-secreting cells in patients with polyneuropathy associated with monoclonal gammopathy (n = 8) compared with controls (n = 8), whereas none of the peptides caused elevated levels of IL-4-secreting cells. Patients with non-immunological types of neuropathy (n = 4) did not reveal any cytokine responses to myelin peptides. Our results indicate that a Th1-like response against myelin proteins occurs in polyneuropathy associated with monoclonal gammopathy.