Three cases with chronic obsessive compulsive disorder report gains in wellbeing and function following rituximab treatment.

Immunological aetiology is supported for a subgroup with obsessive compulsive disorder (OCD) and conceptualized as autoimmune OCD. The longitudinal clinical course is detailed for three severely ill cases with OCD and indications of immunological involvement with off-label rituximab treatment every six months. All cases showed clear and sustained gains regarding symptom burden and function for over 2.5 years. Brief Psychiatric Rating Scale and Yale-Brown Obsessive-Compulsive Inventory Scale scores decreased 67-100% and 44-92%, respectively. These complex cases, prior to rituximab, had very low functioning and disease duration has been eight, nine and 16 years respectively. All three patients had been unsuccessfully treated with at least two antidepressants or anxiolytics, one neuroleptic and cognitive behavioural therapy. Clinical phenotypes and findings were suggestive of possible autoimmune OCD. Indirect immunohistochemistry detected cerebral spinal fluid (CSF) antibodies in all three cases including a novel anti-neuronal staining pattern against mouse thalamic cells. Exploratory analyses of CSF markers and proteomics identified elevated levels of sCD27 and markers indicative of complement pathway activation when compared to CSF from healthy controls. Multidisciplinary collaboration, advanced clinical investigations and rituximab treatment are feasible in a psychiatric setting. The case histories provide a proof of principle for the newly proposed criteria for autoimmune OCD. The findings suggest that clinical red flags and biological measures may predict rituximab response in chronic treatment-resistant OCD. The report provides orientation that may inform the hypotheses and design of future treatment trials.

An exploratory study of the damage markers NfL, GFAP, and t-Tau, in cerebrospinal fluid and other findings from a patient cohort enriched for suspected autoimmune psychiatric disease.

There is growing evidence suggesting that immunological mechanisms play a significant role in the development of psychiatric symptoms in certain patient subgroups. However, the relationship between clinical red flags for suspected autoimmune psychiatric disease and signs of central nervous system (CNS) pathology (e.g., routine cerebrospinal fluid (CSF) alterations, CNS damage markers, neurophysiological or neuroimaging findings) has received limited attention. Here, we aimed to describe the prevalence and distribution of potential CNS pathologies in psychiatric patients in relation to clinical red flags for autoimmune psychiatric disease and psychiatric symptoms. CSF routine findings and CNS damage markers; neurofilament light chain protein (NfL), glial fibrillary acidic protein (GFAP) and total Tau (t-Tau), in CSF from 127 patients with psychiatric disease preselected for suspected immunological involvement were related to recently proposed clinical red flags, psychiatric features, and MRI and EEG findings. Twenty-one percent had abnormal routine CSF findings and 27% had elevated levels of CNS damage markers. Six percent had anti-neuronal antibodies in serum and 2% had these antibodies in the CSF. Sixty-six percent of patients examined with MRI (n = 88) had alterations, mostly atrophy or nonspecific white matter lesions. Twenty-seven percent of patients with EEG recordings (n = 70) had abnormal findings. Elevated NfL levels were associated with comorbid autoimmunity and affective dysregulation symptoms. Elevated t-Tau was associated with catatonia and higher ratings of agitation/hyperactivity. Elevated GFAP was associated with acute onset, atypical presentation, infectious prodrome, tics, depressive/anxiety symptom ratings and overall greater psychiatric symptom burden. In conclusion, preselection based on suspected autoimmune psychiatric disease identifies a population with a high prevalence of CSF alterations suggesting CNS pathology. Future studies should examine the value of these markers in predicting treatment responses.

Cerebrospinal fluid and plasma concentrations of the inflammatory marker soluble CD27 in a large surgical population.

Soluble CD27 (sCD27) is a potential biomarker for diseases involving immune dysfunction. As there is currently little data on cerebrospinal fluid (CSF) sCD27 concentrations in the general population we measured CSF and plasma concentrations in 486 patients (age range 18-92 years, 57% male) undergoing spinal anesthesia for elective surgery. Across the complete cohort the median [range] sCD27 concentrations were 163 [<50 to 7474] pg/mL in CSF and 4624 [1830 to >400,000] pg/mL in plasma. Plasma sCD27, age and Qalb were the factors most strongly associated with CSF sCD27 levels. Reference sCD27 concentration intervals (central 95% of values) in a sub-group without the indication of neuropsychiatric, inflammatory or systemic disease (158 patients) were <50 pg/mL - 419 pg/mL for CSF and 2344-36422 pg/mL for plasma. These data provide preliminary reference ranges that could inform future studies of the validity of sCD27 as a biomarker for neuro- and systemic inflammatory disorders.

Fecal Short-Chain Fatty Acid Ratios as Related to Gastrointestinal and Depressive Symptoms in Young Adults.

OBJECTIVE: Short-chain fatty acids (SCFAs) are produced by the gut microbiota and may reflect health. Gut symptoms are common in individuals with depressive disorders, and recent data indicate relationships between gut microbiota and psychiatric health. We aimed to investigate potential associations between SCFAs and self-reported depressive and gut symptoms in young adults. METHODS: Fecal samples from 164 individuals (125 were patients with psychiatric disorders: mean [standard deviation] age = 21.9 [2.6] years, 14% men; 39 nonpsychiatric controls: age = 28.5 [9.5] years, 38% men) were analyzed for the SCFA acetate, butyrate, and propionate by nuclear magnetic resonance spectroscopy. We then compared SCFA ratios with dimensional measures of self-reported depressive and gut symptoms. RESULTS: Depressive symptoms showed a positive association with acetate levels (ρ = 0.235, p = .003) and negative associations with both butyrate (ρ = -0.195, p = .014) and propionate levels (ρ = -0.201, p = .009) in relation to total SCFA levels. Furthermore, symptoms of diarrhea showed positive associations with acetate (ρ = 0.217, p = .010) and negative associations with propionate in relation to total SCFA levels (ρ = 0.229, p = 0-007). Cluster analysis revealed a heterogeneous pattern where shifts in SCFA ratios were observed in individuals with elevated levels of depressive symptoms, elevated levels of gut symptoms, or both. CONCLUSIONS: Shifts in SCFAs are associated with both depressive symptoms and gut symptoms in young adults and may have of relevance for treatment.

Toll-like receptor 4 methylation grade is linked to depressive symptom severity.

This study explores potential associations between the methylation of promoter-associated CpG sites of the toll-like receptor (TLR)-family, plasma levels of pro-inflammatory proteins and depressive symptoms in young female psychiatric patients. Ratings of depressive symptoms and blood samples were obtained from 92 young women seeking psychiatric care. Methylation of 32 promoter-associated CpG sites in TLR1 to TLR10 was analysed using the Illumina Infinium Methylation EPIC BeadChip. Expression levels of 91 inflammatory proteins were determined by proximity extension assay. Statistical correlations between depressive state, TLR1-10 methylation and inflammatory proteins were investigated. Four additional cohorts were studied to evaluate the generalizability of the findings. In the discovery cohort, methylation grade of cg05429895 (TLR4) in blood was inversely correlated with depressive symptoms score in young adults. After correction for multiple testing, plasma levels of macrophage inflammatory protein 1ß (MIP-1ß/CCL4) were associated with both TLR4 methylation and depressive symptom severity. A similar inverse association between TLR4 methylation in blood and affective symptoms score was also found in a cohort of 148 both males and females (<40 years of age) from the Danish Twin Registry. These findings were not, however, replicated in three other external cohorts; which differed from the first two cohorts by a higher age and mixed ethnicities, thus limiting the generalizability of our findings. However, TLR4 methylation inversely correlated with TLR4 mRNA expression in the Danish Twin Study indicating a functional significance of methylation at this particular CpG. Higher depression scores in young Scandinavian adults was associated with decreased methylation of TLR4 in blood.

Melphalan flufenamide inhibits osteoclastogenesis by suppressing proliferation of monocytes.

Myeloma bone disease is a major complication in multiple myeloma affecting quality of life and survival. It is characterized by increased activity of osteoclasts, bone resorbing cells. Myeloma microenvironment promotes excessive osteoclastogenesis, a process of production of osteoclasts from their precursors, monocytes. The effects of two anti-myeloma drugs, melphalan flufenamide (melflufen) and melphalan, on the activity and proliferation of osteoclasts and their progenitors, monocytes, were assessed in this study. In line with previous research, differentiation of monocytes was associated with increased expression of genes encoding DNA damage repair proteins. Hence monocytes were more sensitive to DNA damage-causing alkylating agents than their differentiated progeny, osteoclasts. In addition, differentiated progeny of monocytes showed increased gene expression of immune checkpoint ligands which may potentially create an immunosuppressive microenvironment. Melflufen was ten-fold more active than melphalan in inhibiting proliferation of osteoclast progenitors. Furthermore, melflufen was also superior to melphalan in inhibition of osteoclastogenesis and bone resorption. These results demonstrate that melflufen may exert beneficial effects in patients with multiple myeloma such as reducing bone resorption and immunosuppressive milieu by inhibiting osteoclastogenesis.

Neuroticism is positively associated with leptin/adiponectin ratio, leptin and IL-6 in young adults.

High neuroticism is related to cardiovascular morbidity. Early detection of metabolic and cardiovascular risk is important in high-risk groups to enable preventive measures. The aim of this study was therefore to explore if neuroticism is associated with early biomarkers for cardiovascular and metabolic disease in young adults from a psychiatry cohort. Blood samples and self-ratings on neuroticism with the Swedish universities Scales of Personality (SSP) questionnaire were collected from 172 psychiatric outpatients and 46 healthy controls. The blood samples were analysed for plasma leptin, adiponectin, CRP, IL-6 and TNF-α. Associations between neuroticism and biomarkers were assessed using Spearman's correlation coefficients and generalized linear models adjusting for confounders. In the adjusted generalized linear models, neuroticism predicted the leptin/adiponectin ratio (p = 0.003), leptin (p = 0.004) and IL-6 (p = 0.001). These associations were not better explained by current major depressive disorder and/or anxiety disorder. Adiponectin, CRP and TNF-α were not associated with neuroticism. In conclusion, the findings suggest that high neuroticism is related to elevated levels of plasma leptin/adiponectin ratio, leptin and IL-6 in young adults. Young adults with high neuroticism may therefore benefit from preventive interventions to decrease the risk for future metabolic and cardiovascular morbidity, but more research is required to test this hypothesis.

Autoantibodies against the C-terminus of Lipopolysaccharide binding protein are elevated in young adults with psychiatric disease.

Growing evidence implies interactions between infections, the immune system and vulnerability for psychiatric disease. This study applies an affinity proteomic-based method to investigate potential disease associated autoantibody signatures in serum from patients from the "Young Adults" section of the Department of General Psychiatry at Uppsala University Hospital (n = 395) and population-based controls (n = 102). We found serum levels of antibodies against Lipopolysaccharide Binding Protein (LBP), a protein that is important for mediating innate immune responses involving the toll-like receptor-4 (TLR-4), to be higher in patients compared to controls (Mann Whitney U-test p = 5.248 × 10-10). The patients were divided into three groups based on their relative levels of autoantibodies against LBP. The distribution of autism spectra disorders (p = 2.0 × 10-4) and hospital care for an infection as adults (p = 0.036) differed between the anti-LBP groups, with low incidence in the group of patients with the highest levels of anti-LBP who were diagnosed with primarily affective and anxiety disorders. In a sub-group analysis, the controls who screened positive for current or previous psychiatric diagnosis (n = 20) had higher anti-LBP compared to non-psychiatric controls with negative screening for psychiatric disorders (Mann Whitney U-test p = 0.006). Inflammatory markers were found to differ across anti-LBP groups and several pro-inflammatory markers, including IL-1ß, were low in patients with high anti-LBP and serum LBP levels were lowest in patients with the highest levels of antibodies against LBP (p = 3.5 × 10-5). A cell-based model showed that polyclonal rabbit anti-LBP, obtained through purification via the same protein fragment used in the initial autoantibody analysis, could interfere with LBP signaling since addition of anti-LBP to the assay reduced both IL-1ß and IL-6 release from activated monocytes in response to LBP and LPS (p = 0.0001 and p = 0.02). This novel finding of antibodies against LBP, where high levels were only found in young adults with psychiatric disease, merits further study. Our results suggest that these antibodies may have relevance for TLR4 based immune responses and vulnerability for both infection and psychiatric disorders.

A cross-sectional study of gastrointestinal symptoms, depressive symptoms and trait anxiety in young adults.

BACKGROUND: >Patients with functional gastrointestinal disorders have a high psychiatric co-morbidity. This study aimed to investigate and characterise gastrointestinal symptoms in relation to depressive symptoms and trait anxiety in a well-defined population of young adult psychiatric outpatients and healthy controls. METHODS: Gastrointestinal symptoms were assessed with the Gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome (GSRS-IBS). Depressive symptoms were assessed with the Montgomery-Åsberg Depression Rating Scale- Self assessment (MADRS-S). Trait anxiety was estimated with three of the Swedish universities of Personality (SSP) scales: Somatic trait anxiety, Psychic trait anxiety and Stress susceptibility. Self-ratings were collected from 491 young adult psychiatric outpatients and 85 healthy controls. Gastrointestinal symptom severity was compared between patients with and without current psychotropic medication and controls. Associations between gastrointestinal symptoms, depressive symptoms and trait anxiety were assessed using Spearman's coefficients and generalized linear models adjusting for possible confounders (sex, body mass index, bulimia nervosa). RESULTS: Patients, with and without current psychotropic medication, reported significantly more gastrointestinal symptoms than controls. In the generalized linear models, total MADRS-S score (p < 0.001), Somatic trait anxiety (p < 0.001), Psychic trait anxiety (p = 0.002) and Stress susceptibility (p = 0.002) were independent predictors of the total GSRS-IBS score. Further exploratory analysis using unsupervised learning revealed a diverse spectrum of symptoms that clustered into six groups. CONCLUSION: Gastrointestinal symptoms are both highly prevalent and diverse in young adult psychiatric outpatients, regardless of current psychotropic medication. Depressive symptom severity and degree of trait anxiety are independently related to the total gastrointestinal symptom burden.

Impact of time and temperature on gut microbiota and SCFA composition in stool samples.

Gut dysbiosis has been implicated in the pathophysiology of a growing number of non-communicable diseases. High through-put sequencing technologies and short chain fatty acid (SCFA) profiling enables surveying of the composition and function of the gut microbiota and provide key insights into host-microbiome interactions. However, a methodological problem with analyzing stool samples is that samples are treated and stored differently prior to submission for analysis potentially influencing the composition of the microbiota and its metabolites. In the present study, we simulated the sample acquisition of a large-scale study, in which stool samples were stored for up to two days in the fridge or at room temperature before being handed over to the hospital. To assess the influence of time and temperature on the microbial community and on SCFA composition in a controlled experimental setting, the stool samples of 10 individuals were exposed to room and fridge temperatures for 24 and 48 hours, respectively, and analyzed using 16S rRNA gene amplicon sequencing, qPCR and nuclear magnetic resonance spectroscopy. To best of our knowledge, this is the first study to investigate the influence of storage time and temperature on the absolute abundance of methanogens, and of Lactobacillus reuteri. The results indicate that values obtained for methanogens, L. reuteri and total bacteria are still representative even after storage for up to 48 hours at RT (20°C) or 4°C. The overall microbial composition and structure appeared to be influenced more by laboratory errors introduced during sample processing than by the actual effects of temperature and time. Although microbial activity was demonstrated by elevated SCFA at both 4°C and RT, SCFAs ratios were more stable over the different conditions and may be considered as long as samples are come from similar storage conditions.

Daytime melatonin levels in saliva are associated with inflammatory markers and anxiety disorders.

BACKGROUND: The bidirectional interaction between melatonin and the immune system has largely gone unexplored in a clinical context and especially in a psychiatric population. This study explored the association between melatonin during the day and inflammatory cytokines in young adult patients seeking psychiatric care. METHODS: Samples and data were collected from 108 young adults (mean age 21, SD = 2) at an outpatient clinic for affective disorders. Daytime saliva melatonin levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in relation to normalized serum expression levels of 72 inflammatory markers in a proximity extension assay (PEA). In a post hoc analysis the markers associated with melatonin were tested in a generalized linear model to see whether there is a relationship to anxiety disorder or depression. RESULTS: After Bonferroni correction for multiple testing, melatonin levels at 11:00 were positively correlated with CD5 (p = 4.2e-4). Melatonin levels after lunch were correlated with CCL2/MCP-1 (p = 4.2e-4), CCL3/MPI-1α (p = 6.5e-4) and VEGF-A (p = 5.3e-6). In the generalized linear model, positive associations were found for the presence of any anxiety disorder with melatonin after lunch (p = 0.046), VEGF-A (p = 0.001) and CCL3/MPI-1α (p = 0.001). CONCLUSION: Daytime saliva levels of melatonin were related to several inflammatory markers in young adults with psychiatric disorders. This observation likely reflects the bidirectional relationship between melatonin production and the immune system. These findings may have relevance for the understanding of psychiatric disorders and other conditions associated with low-grade inflammation.

A sensitive method detecting trace levels of levonorgestrel using LC-HRMS.

OBJECTIVE: To develop a high resolution mass spectrometry (HRMS) method to quantify levonorgestrel (LNG) in serum. STUDY DESIGN: Levonorgestrel was extracted using solid phase extraction and measured using liquid chromatography (LC) HRMS. RESULTS: Low limit of quantification (LLOQ) was 25 pg/mL and low limit of detection (LLOD) was 12.5 pg/mL. Precision and accuracy bias were <10%. LNG in serum samples from Mirena® users ranged between 37 to 219 pg/mL (n=12). In eight out of 22 patients with suspected intrauterine device (IUD) expulsion LNG was detected (26-1272 pg/mL). CONCLUSION: A sensitive, fast and simple LC-HRMS method was developed to detect trace levels of LNG.

The Free Hormone Hypothesis: Is Free Serum 25-Hydroxyvitamin D a Better Marker for Bone Mineral Density in Older Women?

It is presently unclear whether free serum 25-hydroxyvitamin D (S-25(OH)D) better reflects bone health than total S-25(OH)D. We have previously shown that summer total S-25(OH)D values are more useful to predict bone mineral density (BMD) than winter values. Our objective was therefore to compare the relative importance of free and total S-25(OH)D for BMD by season. BMD was measured by dual-energy X-ray absorptiometry (DXA) in 5002 Swedish women (mean age 68 years) randomly selected from a large population-based longitudinal cohort study. Free S-25(OH)D was analyzed by a commercial ELISA and total S-25(OH)D by HPLC-tandem mass spectrometry (MS/MS). Free and total S-25(OH)D co-varied with season, with 26% and 29% higher values in August compared with those in January-March (nadir). There were no differences in mean BMD between categories of free or total S-25(OH)D in samples collected during winter. Women with higher total S-25(OH)D measured during summer had higher BMD at the total hip. Compared with women who had total S-25(OH)D values above 80 nmol/L during summer, adjusted BMD at the total hip was 6% (95% CI, 1% to 11%) lower for S-25(OH)D concentrations between 30 and 40 mmol/L, and 11% (95% CI, 3% to 19%) lower for those with total S-25(OH)D <30 nmol/L. In contrast, free S-25(OH)D measured during summer was not associated with BMD. Compared with women who had highest free S-25(OH)D measured during summer (>8.8 pmol/L), those with intermediate (2.4-3.5 pmol/L) and lowest (<2.4 pmol/L) free S-25(OH)D during summer did not have lower total hip BMD values (3% [95% CI, -2% to 7%] and -2% [95% CI, -8% to 4%]). In addition, we found no added value for the prediction of BMD with the combined measurement of total and free S-25(OH)D during summer or winter. We conclude that vitamin D status assessed by direct measurements of free S-25(OH)D does not reflect BMD better than total S-25(OH)D. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Low-dose developmental exposure to bisphenol A induces sex-specific effects in bone of Fischer 344 rat offspring.

BACKGROUND: Bisphenol A (BPA) is a component of polycarbonate plastics to which humans are regularly exposed at low levels, and an endocrine disruptor with effects on several hormonal systems. Bone is a sensitive hormone target tissue, and we have recently shown that in utero and lactational exposure to 25µg BPA/kg BW/day alters femoral geometry in rat offspring. OBJECTIVE: To investigate bone effects in rat offspring after developmental exposure to a BPA dose in the range of human daily exposure (0.1-1.5µg/kg BW/day) as well as a dose to corroborate previous findings. METHODS: Pregnant Fischer 344 rats were exposed to BPA via drinking water corresponding to 0.5µg/kg BW/day: [0.5], (n=21) or 50µg/kg BW/day: [50], (n = 16) from gestational day 3.5 until postnatal day 22, while controls were given only vehicle (n = 25). The offspring was sacrificed at 5 weeks of age. Bone effects were analyzed using peripheral quantitative computed tomography (pQCT), the 3-point bending test, plasma markers of bone turnover, and gene expression in cortical bone and bone marrow. RESULTS: Compared to controls, male offspring developmentally exposed to BPA had shorter femurs. pQCT analysis revealed effects in the [0.5] group, but not in the [50] group; BPA reduced both trabecular area (-3.9%, p < 0.01) and total cross sectional area (-4.1%, p < 0.01) of femurs in the [0.5] group, whereas no effects were seen on bone density. Conversely, bone length and size were not affected in female offspring. However, the procollagen type I N-terminal propeptide (P1NP), a peptide formed during type 1 collagen synthesis, was increased in plasma (42%: p < 0.01) in female offspring exposed to [0.5] of BPA, although collagen gene expression was not increased in bone. The biomechanical properties of the bones were not altered in either sex. Bone marrow mRNA expression was only affected in male offspring. CONCLUSIONS: Developmental low-dose exposure to BPA resulted in sex-specific bone effects in rat offspring. A dose approximately eight times lower than the current temporary EFSA human tolerable daily intake of 4µg/kg BW/day, reduced bone length and size in male rat offspring. Long-term studies are needed to clarify whether the increased plasma levels of P1NP in female offspring reflect development of fibrosis.

Excessive dietary intake of vitamin A reduces skull bone thickness in mice.

Calvarial thinning and skull bone defects have been reported in infants with hypervitaminosis A. These findings have also been described in humans, mice and zebrafish with loss-of-function mutations in the enzyme CYP26B1 that degrades retinoic acid (RA), the active metabolite of vitamin A, indicating that these effects are indeed caused by too high levels of vitamin A and that evolutionary conserved mechanisms are involved. To explore these mechanisms, we have fed young mice excessive doses of vitamin A for one week and then analyzed the skull bones using micro computed tomography, histomorphometry, histology and immunohistochemistry. In addition, we have examined the effect of RA on gene expression in osteoblasts in vitro. Compared to a standard diet, a high dietary intake of vitamin A resulted in a rapid and significant reduction in calvarial bone density and suture diastasis. The bone formation rate was almost halved. There was also increased staining of tartrate resistant acid phosphatase in osteocytes and an increased perilacunar matrix area, indicating osteocytic osteolysis. Consistent with this, RA induced genes associated with bone degradation in osteoblasts in vitro. Moreover, and in contrast to other known bone resorption stimulators, vitamin A induced osteoclastic bone resorption on the endocranial surfaces.

Increased Bone Mass in Female Mice Lacking Mast Cell Chymase.

Here we addressed the potential impact of chymase, a mast-cell restricted protease, on mouse bone phenotype. We show that female mice lacking the chymase Mcpt4 acquired a persistent expansion of diaphyseal bone in comparison with wild type controls, reaching a 15% larger diaphyseal cross sectional area at 12 months of age. Mcpt4-/- mice also showed increased levels of a bone anabolic serum marker and higher periosteal bone formation rate. However, they were not protected from experimental osteoporosis, suggesting that chymase regulates normal bone homeostasis rather than the course of osteoporosis. Further, the absence of Mcpt4 resulted in age-dependent upregulation of numerous genes important for bone formation but no effects on osteoclast activity. In spite of the latter, Mcpt4-/- bones had increased cortical porosity and reduced endocortical mineralization. Mast cells were found periosteally and, notably, bone-proximal mast cells in Mcpt4-/- mice were degranulated to a larger extent than in wild type mice. Hence, chymase regulates degranulation of bone mast cells, which could affect the release of mast cell-derived factors influencing bone remodelling. Together, these findings reveal a functional impact of mast cell chymase on bone. Further studies exploring the possibility of using chymase inhibitors as a strategy to increase bone volume may be warranted.

Melatonin Immunoreactivity in Malignant Small Intestinal Neuroendocrine Tumours.

BACKGROUND/AIMS: Small intestinal neuroendocrine tumours (SI-NETs) are derived from enterochromaffin cells. After demonstrating melatonin in enterochromaffin cells, we hypothesized that SI-NETs may express and secrete melatonin, which may have an impact on clinical factors and treatment response. METHODS: Tumour tissue from 26 patients with SI-NETs, representing paired sections of primary tumour and metastasis, were immunohistochemically stained for melatonin and its receptors, MT1 and MT2. Plasma melatonin and immunoreactivity (IR) for melatonin, MT1 and MT2 in tumour cells were compared to other tumour markers and clinical parameters. Melatonin was measured at two time points in fasting morning plasma from 43 patients with SI-NETs. RESULTS: Melatonin IR was found in all SI-NETS. Melatonin IR intensity in primary tumours correlated inversely to proliferation index (p = 0.022) and patients reported less diarrhoea when melatonin IR was high (p = 0.012). MT1 IR was low or absent in tumours. MT2 expression was medium to high in primary tumours and generally reduced in metastases (p = 0.007). Plasma-melatonin ranged from 4.5 to 220.0 pg/L. Higher levels were associated with nausea at both time points (p = 0.027 and p = 0.006) and flush at the second sampling. In cases with disease stabilization or remission (n = 34), circulating melatonin levels were reduced in the second sample (p = 0.038). CONCLUSION: Immunoreactive melatonin is present in SI-NETs. Circulating levels of melatonin in patients with SI-NETs are reduced after treatment. Our results are congruent with recent understanding of melatonin's endocrine and paracrine functions and SI-NETs may provide a model for further studies of melatonin function.