[Diagnosing neuropathic orofacial pain in the general dental practice]. / Diagnosticeren van neuropathische orofaciale pijn in de algemene tandartspraktijk.

Dental pain is a common reason for patients to visit the dentist. This type of pain is usually easy to diagnose and treat. However, diagnosing and treating other forms of orofacial pain remains complicated. One of the most challenging types of orofacial pain to diagnose and treat is neuropathic orofacial pain: pain resulting from damage to nerve tissue. Recognizing this type of pain in a timely manner can prevent unnecessary invasive dental treatments and disappointment for patients who seek help for this type of pain. There are relatively simple tools for dentists to distinguish neuropathic pain from other types of orofacial pain. The treatment of neuropathic pain is primarily focused on symptom relief through medication.

Thiopurine treatment in patients with Crohn's disease leads to a selective reduction of an effector cytotoxic gene expression signature revealed by whole-genome expression profiling.

Crohn's disease (CD) is characterized by chronic inflammation of the gastrointestinal tract, as a result of aberrant activation of the innate immune system through TLR stimulation by bacterial products. The conventional immunosuppressive thiopurine derivatives (azathioprine and mercaptopurine) are used to treat CD. The effects of thiopurines on circulating immune cells and TLR responsiveness are unknown. To obtain a global view of affected gene expression of the immune system in CD patients and the treatment effect of thiopurine derivatives, we performed genome-wide transcriptome analysis on whole blood samples from 20 CD patients in remission, of which 10 patients received thiopurine treatment, compared to 16 healthy controls, before and after TLR4 stimulation with LPS. Several immune abnormalities were observed, including increased baseline interferon activity, while baseline expression of ribosomal genes was reduced. After LPS stimulation, CD patients showed reduced cytokine and chemokine expression. None of these effects were related to treatment. Strikingly, only one highly correlated set of 69 genes was affected by treatment, not influenced by LPS stimulation and consisted of genes reminiscent of effector cytotoxic NK cells. The most reduced cytotoxicity-related gene in CD was the cell surface marker CD160. Concordantly, we could demonstrate an in vivo reduction of circulating CD160(+)CD3(-)CD8(-) cells in CD patients after treatment with thiopurine derivatives in an independent cohort. In conclusion, using genome-wide profiling, we identified a disturbed immune activation status in peripheral blood cells from CD patients and a clear treatment effect of thiopurine derivatives selectively affecting effector cytotoxic CD160-positive cells.

Defective IL-1A expression in patients with Crohn's disease is related to attenuated MAP3K4 signaling.

Crohn's disease (CD) is characterized by an aberrant immune response to bacterial products stimulating TLR, in genetically susceptible hosts. Next to mutations in the TLR signaling molecule NOD2, several other immune response- and autophagy-genes contribute to CD. Since only 10-20% of cases can be explained by a NOD2 defect, we searched for additional TLR-related disease-causing factors. We analyzed the LPS response of peripheral blood mononuclear cells from 23 CD patients in remission, compared to 16 controls in a time course experiment. Individuals with any of the three major contributing NOD2 mutations were excluded. Overall, the LPS-responsive gene transcript levels, determined by low density arrays, were significantly lower in CD patients. In particular IL-1A expression was severely reduced in CD patients (ninefold reduction, p=0.001). Quantification of several important TLR4 signal transducers and cytokines identified MAP3K4 as a candidate signaling molecule with reduced expression in CD patients, which might explain the low IL-1A expression. Silencing of MAP3K4 by lentiviral shRNA transduction indeed showed that the expression of IL-1A was specifically dependent on this kinase. Furthermore, the expression of GSK3ß, an inhibitor of MAP3K4, was increased in CD patients. In conclusion, we identified a novel TLR signaling defect in CD patients involving MAP3K4 and IL-1A. This confirms the hypothesis that CD patients, despite their massive intestinal inflammation, suffer from a relative immune deficiency in TLR-mediated cytokine production.

Molecular subtypes of systemic sclerosis in association with anti-centromere antibodies and digital ulcers.

The objective of this study was to identify molecular profiles that may distinguish clinical subtypes in systemic sclerosis (SSc). Large-scale gene expression profiling was performed on peripheral blood (PB) from 12 SSc patients and 6 healthy individuals. Significance analysis of microarrays, two-way hierarchical cluster analysis and PANTHER (Protein ANalysis THrough Evolutionary Relationships) ontology classification were used to analyze the data. Quantitative PCR was applied for validation in a cohort of 43 SSc patients. The results show that the expression of genes involved in immune defense, cell cycle and signal transduction was significantly elevated in PB of SSc patients (n=12) compared with healthy individuals (n=6). SSc patients could be stratified into subgroups based on differential expression of genes induced by type I interferon (IFN) and genes involved in antimicrobial (AM) activity. Differential expression of type I IFN or AM signature genes was validated and extended in an independent cohort of 31 patients by quantitative PCR. Low expression of IFN response genes was associated with the presence of anti-centromere antibodies, whereas increased expression was associated with the appearance of digital ulcers. In conclusion, patients with SSc can be classified on the basis of differential expression of immune defense genes. Differences in the activity of the type I IFN response program stratify patients into two clinically relevant subgroups.

Expression of a pathogen-response program in peripheral blood cells defines a subgroup of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a heterogeneous disease with unknown etiology. Here we aimed to distinguish RA subtypes based on peripheral blood (PB) gene expression profiles in comparison with a pathogen-response transcriptional program. PB was obtained from 35 RA patients and 15 healthy individuals. For expression profiling we used DNA microarrays. A combined cluster analysis of RA and control samples together with samples from a viral infection model revealed that the gene expression profile of a subgroup of RA patients (RA(A)) was reminiscent to that of poxvirus-infected macaques. Statistical analysis, followed by Gene Ontology analysis of the RA(A) patients confirmed that these patients form a distinct group, with activation of several host defense mechanisms that resemble a common host-pathogen response. Analysis of the promoter region of genes that were overexpressed in the RA(A) patients, revealed an enrichment of transcription factor binding sites for NF kappaB and interferon-activated transcription factors. Moreover, this subgroup of RA patients expressed significantly increased titers of anti-cyclic citrullinated peptide antibodies. We conclude that activation of a host-pathogen response defines a subgroup of RA patients characterized by increased autoreactivity against citrullinated proteins.

Responsiveness to anti-tumour necrosis factor alpha therapy is related to pre-treatment tissue inflammation levels in rheumatoid arthritis patients.

OBJECTIVE: The response of rheumatoid arthritis (RA) patients to treatment with neutralising antibodies to tumour necrosis factor alpha (TNFalpha) is highly variable. The underlying mechanism for therapy responsiveness is currently unknown. We therefore evaluated the relationship between baseline molecular profiles of synovial tissues from RA patients and the clinical response to treatment with infliximab. METHODS: Synovial biopsies were obtained by arthroscopy from 18 RA patients with active disease (28 joint count Disease Activity Score (DAS28) > or = 3.2) before initiation of treatment with infliximab. All patients were on stable methotrexate treatment. Clinical response at 16 weeks was defined as a reduction in DAS28 of > or = 1.2, non-response as reduction in DAS28 < 1.2. Large-scale gene expression profiling using microarrays was performed on synovial tissue samples. To identify biological processes in synovial biopsies that could discriminate between responders and non-responders, we performed pathway analysis on the expression profiles. RESULTS: A total of 12 patients responded to therapy, while 6 patients failed to fulfil the response criteria. We identified several biological processes, related to inflammation, which were up-regulated in patients who responded to therapy, compared to those who did not show clinical improvement. CONCLUSION: These results indicate that patients with a high level of tissue inflammation are more likely to benefit from anti-TNFalpha treatment.

Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients.

BACKGROUND: Rheumatoid arthritis (RA) is a heterogeneous disease with unknown cause. AIM: To identify peripheral blood (PB) gene expression profiles that may distinguish RA subtypes. METHODS: Large-scale expression profiling by cDNA microarrays was performed on PB from 35 patients and 15 healthy individuals. Differential gene expression was analysed by significance analysis of microarrays (SAM), followed by gene ontology analysis of the significant genes. Gene set enrichment analysis was applied to identify pathways relevant to disease. RESULTS: A substantially raised expression of a spectrum of genes involved in immune defence was found in the PB of patients with RA compared with healthy individuals. SAM analysis revealed a highly significant elevated expression of interferon (IFN) type I regulated genes in patients with RA compared with healthy individuals, which was confirmed by gene ontology and pathway analysis, suggesting that this pathway was activated systemically in RA. A quantitative analysis revealed that increased expression of IFN-response genes was characteristic of approximately half of the patients (IFN(high) patients). Application of pathway analysis revealed that the IFN(high) group was largely different from the controls, with evidence for upregulated pathways involved in coagulation and complement cascades, and fatty acid metabolism, while the IFN(low) group was similar to the controls. CONCLUSION: The IFN type I signature defines a subgroup of patients with RA, with a distinct biomolecular phenotype, characterised by increased activity of the innate defence system, coagulation and complement cascades, and fatty acid metabolism.

A subtype of multiple sclerosis defined by an activated immune defense program.

Given the heterogeneous nature of multiple sclerosis (MS), we applied DNA microarray technology to determine whether variability is reflected in peripheral blood (PB) cells. In this study, we studied whole-blood gene expression profiles of 29 patients with relapsing-remitting MS (RRMS) and 25 age- and sex-matched healthy controls. We used microarrays with a complexity of 43K cDNAs. The data were analyzed using sophisticated pathway-level analysis in order to provide insight into the deregulated peripheral immune response programs in MS. We found a remarkable elevated expression of a spectrum of genes known to be involved in immune defense in the PB of MS patients compared to healthy individuals. Cluster analysis revealed that the increased expression of these genes was characteristic for approximately half of the patients. In addition, the gene signature in this group of patients was comparable with a virus response program. We conclude that the transcriptional signature of the PB cells reflects the heterogeneity of MS and defines a sub-population of RRMS patients, who exhibit an activated immune defense program that resembles a virus response program, which is supportive for a link between viruses and MS.

Association between polymorphisms in the human chemokine receptor genes CCR2 and CX3CR1 and rheumatoid arthritis.

Cell trafficking into the rheumatoid synovium is thought to play an important role in the inflammation seen in rheumatoid arthritis. Chemokine receptors play a central role in this process, and several common variants are known, including the CCR2 variant, CCR2-64I, and two variants of the CX3CR1 gene, V249I and T280M. All three variants result in functional amino acid substitutions. We studied the association of these chemokine receptor variants with susceptibility to and severity of rheumatoid arthritis in two Dutch patient populations; 282 consecutive rheumatoid arthritis patients from a rheumatology outpatient clinic, and a cohort of 101 female rheumatoid arthritis patients, followed closely for a 12-year period, from whom hand and feet X-rays taken at three year intervals were scored and analyzed in this study. Although there was a trend towards increased severity of disease in patients carrying CX3CR1 variants, this was not independent of known risk factors. We found no evidence for a significant independent role for the CCR2 and CX3CR1 variants in the susceptibility to or severity of rheumatoid arthritis.

Chronic obstructive pulmonary disease is associated with the -1055 IL-13 promoter polymorphism.

IL-13 is strongly implicated in the development of asthma and chronic obstructive pulmonary disease (COPD). We previously identified an IL-13 promoter polymorphism (-1055 C to T) that is associated with allergic asthma. We now report an increased frequency of the -1055 T allele in COPD patients compared to healthy controls (P=0.002) and compared to a second control group consisting of smoking individuals with normal lung function (P=0.01). A closely linked IL-13 exon polymorphism is present at normal allelic frequencies (P=0.3 and 0.4, respectively). In addition, we observed a normal distribution of two IL-4 polymorphisms at positions -590 and +33 (P=0.2 and 0.9, respectively). These results could implicate a functional role for the IL-13 promoter polymorphism in the enhanced risk to develop COPD.

Modulation of the activity of the internal defence system of the pond snail Lymnaea stagnalis by the avian schistosome Trichobilharzia ocellata.

Effects of infection with the avian schistosome Trichobilharzia ocellata on the activity of the internal defence system of the intermediate snail host Lymnaea stagnalis were studied, utilizing an in vitro phagocytosis assay for determining haemocyte activity. A distinction was made between plasma- and cell-associated effects. The period immediately after penetration of the parasite into the snail host (1.5-72 h post-exposure (p.e.)) was extensively studied. In addition, several time-points coinciding with the later-successive-stages of parasite development (2, 4, 6, 8 and 10 weeks p.e.) were investigated. Plasma-associated enhancement of defence activity was found between 1.5 and 6 h p.e., followed by plasma-associated suppression between 12 and 72 h p.e. A cell-associated activation was found between 1.5 and 6 h p.e. and also at 8 and 10 weeks p.e. How these effects on the defence system may be related to phenomena observed in infected snails at these time-points is discussed.

Trichobilharzia ocellata in Lymnaea stagnalis: a flow cytometric approach to study its effects on hemocytes.

Flow cytometric analysis was performed on hemocytes in suspension derived from individual Lymnaea stagnalis. The distribution of cell sizes within the hemocyte population was comparable in all 40 specimens studied. The size distribution of circulating hemocytes is unimodal and continuous, with no discrete subpopulations, and is not affected by age or by infection with Trichobilharzia ocellata. Flow cytometry proved to be a very useful technique in analysis of hemocyte populations in snails and anti-hemocyte monoclonal antibodies can be employed in these studies. The use of individual instead of pooled hemolymph samples in studying hemocyte populations of molluscs is stressed.

Trichobilharzia ocellata: Quantification of effects on haemocytes of the pond snail Lymnaea stagnalis by morphometric means.

To identify functionally different subpopulations, we quantified by morphometric means the spreading activity of circulating haemocytes of the pond snail Lymnaea stagnalis, recognized by monoclonal antibodies (5 surface and 5 cytoplasmic). The influence of snail age and of the different intramolluscan stages of the compatible avian schistosome Trichobilharzia ocellata on this activity were studied. The antibody-recognized cells could be separated into two groups, differing in their spreading activities. The probes detecting cytoplasmic markers recognized the majority of cells (78-95%). These were active (well spreading), differentiated cells. The surface probes recognized a smaller part (12-38%) of the total haemocyte population. These harmocytes were less active (less spreading), and were predominantly immature cells. The relative sizes of the antibody-detected subpopulations were not affected by snail age or infection with T. ocellata. The maximal size (planimetric area) attained after attaching to glass of all cells increased between day 0 (juvenile snails) and about 6 weeks post (sham) exposure and then decreased. Infection had little effect on the spreading activity of the more differentiated cells. The less differentiated cells showed a larger spreading activity when the parasite was present as mother sporocyst and also when the digestive gland area became colonized by growing daughter sporocysts.

Analysis of surface carbohydrates of Trichobilharzia ocellata miracidia and sporocysts using lectin binding techniques.

Miracidia and in vitro-derived primary sporocysts of the avian schistosome Trichobilharzia ocellata were studied for the expression and the characteristics of glycoconjugate moieties comprising the surface coat. Using a panel of 9 peroxidase labelled lectins, several different lectin binding sites were demonstrated on the larvae. Fixed miracidia have binding sites for 7 of the lectins; wheat-germ agglutinin binds to both the ciliated plates and the tegumental ridges between them; the other 6 lectins bind to the plates only. Three of the miracidia-binding lectins, wheat-germ agglutinin, concanavalin A and peanut agglutinin, also bind to fixed sporocysts. Since the miracidial ridges are devoid of binding sites for concanavalin A and peanut agglutinin, whereas the sporocyst tegument binds these lectins, it appears that these sites become exposed during or shortly after transformation. In saturation experiments, low concentrations of peanut agglutinin and concanavalin A are bound more avidly by sporocysts than by miracidia, indicating a higher binding affinity of the former. The two larval forms do not differ in affinity for wheat-germ agglutinin but they have different binding capacities; when offered in high concentrations, more of this lectin is bound by sporocysts than by miracidia. Lectin binding was competitively inhibited by adding the appropriate free saccharides. Live larvae showed the same lectin binding pattern as did fixed specimens. Proteinase treatment reduced lectin binding to living and, to a lesser extent, to fixed larvae, suggesting that binding sites are constituents of proteoglycoconjugates. After SDS-PAGE of extracts from miracidia and sporocysts and subsequent Western blotting, some of the lectins failed to bind glycoproteins, others reacted with an array of bands. The patterns differed among the lectins and each lectin gave different patterns for miracidia and sporocysts. The results obtained with these two lectin-binding techniques support the conclusion that stage-specific proteoglycoconjugates occur at the surface of T. ocellata larvae.