Leaky gut biomarkers in depression and suicidal behavior.

OBJECTIVE: Inflammation is associated with major depressive disorder (MDD) and suicidal behavior. According to the 'leaky gut hypothesis', increased intestinal permeability may contribute to this relationship via bacterial translocation across enterocytes. We measured plasma levels of gut permeability markers, in patients with a recent suicide attempt (rSA), MDD subjects with no history of a suicide attempt (nsMDD), and healthy controls (HC), and related these markers to symptom severity and inflammation. METHOD: We enrolled rSA (n = 54), nsMDD (n = 13), and HC (n = 17). Zonulin, intestinal fatty acid binding protein (I-FABP), soluble CD14, and interleukin-6 (IL-6) were quantified in plasma. Montgomery-Åsberg Depression Rating Scale (MADRS) and Suicide Assessment Scale (SUAS) were used for symptom assessments. RESULTS: The rSA group displayed higher I-FABP and lower zonulin levels compared with both the nsMDD and the HC groups (all P < 0.001). IL-6 correlated positively with I-FABP (r = 0.24, P < 0.05) and negatively with zonulin (r = -0.25, P < 0.05). In all subjects, I-FABP levels correlated positively with MADRS (r = 0.25, P < 0.05) and SUAS scores (r = 0.38, P < 0.001), and the latter correlation was significant also in the nsMDD group (r = 0.60, P < 0.05). CONCLUSION: The 'leaky gut hypothesis' may improve our understanding of the link between inflammation and suicidal behavior. These findings should be considered preliminary until replicated in larger cohorts.

Increased plasma levels of circulating cell-free mitochondrial DNA in suicide attempters: associations with HPA-axis hyperactivity.

Preclinical data suggest that chronic stress may cause cellular damage and mitochondrial dysfunction, potentially leading to the release of mitochondrial DNA (mtDNA) into the bloodstream. Major depressive disorder has been associated with an increased amount of mtDNA in leukocytes from saliva samples and blood; however, no previous studies have measured plasma levels of free-circulating mtDNA in a clinical psychiatric sample. In this study, free circulating mtDNA was quantified in plasma samples from 37 suicide attempters, who had undergone a dexamethasone suppression test (DST), and 37 healthy controls. We hypothesized that free circulating mtDNA would be elevated in the suicide attempters and would be associated with hypothalamic-pituitary-adrenal (HPA)-axis hyperactivity. Suicide attempters had significantly higher plasma levels of free-circulating mtDNA compared with healthy controls at different time points (pre- and post-DST; all P-values<2.98E-12, Cohen's d ranging from 2.55 to 4.01). Pre-DST plasma levels of mtDNA were positively correlated with post-DST cortisol levels (rho=0.49, P<0.003). Suicide attempters may have elevated plasma levels of free-circulating mtDNA, which are related to impaired HPA-axis negative feedback. This peripheral index is consistent with an increased cellular or mitochondrial damage. The specific cells and tissues contributing to plasma levels of free-circulating mtDNA are not known, as is the specificity of this finding for suicide attempters. Future studies are needed in order to better understand the relevance of increased free-circulating mtDNA in relation to the pathophysiology underlying suicidal behavior and depression.

Glucose-lowering effect of the DPP-4 inhibitor sitagliptin after glucose and non-glucose macronutrient ingestion in non-diabetic subjects.

AIM: Recent studies suggest that the incretin concept is not restricted to glucose ingestion but relevant also after non-glucose macronutrient administration. We therefore hypothesized that raising incretin hormones reduces circulating glucose after both glucose and non-glucose macronutrient ingestion in healthy subjects. METHODS: Twelve healthy subjects received the dipeptidyl peptidase-4 inhibitor sitagliptin (100 mg) or placebo before ingestion of glucose, fat (olive oil) or protein mix in equicaloric amounts (8 kcal/kg) plus paracetamol (1.5 g). The 120-min areas under curve (AUC) of intact glucagon-like peptide-1 (GLP-1), glucose, insulin, C-peptide, glucagon and paracetamol, and model-derived insulin secretion rate (ISR), insulin sensitivity, insulin clearance and glucose absorption were measured. RESULTS: The increased plasma intact GLP-1 levels after each macronutrient was augmented by sitagliptin. This was associated with a robust lowering of glucose: glucose excursion after oral glucose was diminished, and glucose fell below baseline after oral fat and protein. In spite of lower glucose, AUCC -peptide and ISR did not differ significantly between sitagliptin and placebo after any macronutrient. AUCglucagon , insulin sensitivity and insulin clearance were also not different between sitagliptin and placebo. Glucose absorption after oral glucose was reduced by sitagliptin, whereas AUCparacetamol was not statistically different between sitagliptin and placebo. CONCLUSIONS: Physiological elevation of intact GLP-1 levels after ingestion of glucose and non-glucose macronutrients is robustly glucose-lowering in healthy subjects. Hence, the incretin concept is not restricted to glucose ingestion in normal physiology. The glucose-lowering action of sitagliptin at these low glucose levels in healthy subjects may have complex mechanisms, involving both islet-dependent and islet-independent mechanisms.

Lymph node tissue kallikrein-related peptidase 6 mRNA: a progression marker for colorectal cancer.

BACKGROUND: A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery. METHODS: Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT-PCR. RESULTS: Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8). CONCLUSION: In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence.

Effects of a sphingolipid-enriched dairy formulation on postprandial lipid concentrations.

BACKGROUND: The digestion of sphingolipids (SL) is slow and is catalyzed by mucosal enzymes. Dietary SL was shown to inhibit cholesterol absorption and to lower plasma cholesterol, triglycerides (TG) and hepatic fat accumulation in animal models. AIM: A dairy formulation based on fractionation of buttermilk, which is enriched in milk polar lipids of which SL account for a large part is now available. In this study, we examined whether this formulation, when ingested with a standard breakfast, exerted a different influence on postprandial lipids than an equivalent control formulation lacking the polar milk lipids. METHODS: A total of 18 healthy male volunteers aged 22-65 years ingested a high-fat (40 g) standard breakfast together with a milk-like formulation containing 975 mg of milk SL (A) or the control formulation (B). Postprandial levels of TG, total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, apolipoprotein AI (ApoAI), ApoB, glucose and insulin were measured 1 to 7 h after the meal. RESULTS: No difference was seen between experimental and control groups in postprandial levels of TG, insulin, ApoA1 or ApoB. After 1 hour there was a trend of lower cholesterol concentrations in large TG-rich lipoproteins after formulation A. CONCLUSION: The SL-rich buttermilk drink may affect cholesterol concentrations in TG-rich lipoproteins, but has no effect on postprandial TG after a breakfast with butter fat as the major lipid.

Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT-PCR.

Accurate identification of lymph node involvement is critical for successful treatment of patients with colorectal carcinoma (CRC). Real-time quantitative RT-PCR with a specific probe and RNA copy standard for biomarker mRNA has proven very powerful for detection of disseminated tumour cells. Which properties of biomarker mRNAs are important for identification of disseminated CRC cells? Seven biomarker candidates, CEA, CEACAM1-S/L, CEACAM6, CEACAM7-1/2, MUC2, MMP7 and CK20, were compared in a test-set of lymph nodes from 51 CRC patients (Dukes' A-D) and 10 controls. Normal colon epithelial cells, primary tumours, and different immune cells were also analysed. The biomarkers were ranked according to: (1) detection of haematoxylin/eosin positive nodes, (2) detection of Dukes' A and B patients, who developed metastases during a 54 months follow-up period and (3) identification of patients with Dukes' C and D tumours using the highest value of control nodes as cutoff. The following properties appear to be of importance; (a) no expression in immune cells, (b) relatively high and constant expression in tumour tissue irrespective of Dukes' stage and (c) no or weak downregulation in tumours compared to normal tissue. CEA fulfilled these criteria best, followed by CK20 and MUC2.

Acute antithrombotic effects of ximelagatran, an oral direct thrombin inhibitor, and r-hirudin in a human ex vivo model of arterial thrombosis.

BACKGROUND: Thrombin plays a major role in thrombus formation through activation of platelets and conversion of fibrinogen to fibrin. OBJECTIVES: To investigate the antithrombotic effects of the oral direct thrombin inhibitor (DTI) ximelagatran and the parenteral DTI r-hirudin in humans. SUBJECTS AND METHODS: Healthy male volunteers randomized into four parallel groups each with 15 subjects received either ximelagatran (20, 40 or 80 mg orally) or r-hirudin (0.4 mg kg-1 intravenous bolus + infusion of 0.15 mg kg-1 h-1 for 2 h and 0.075 mg kg-1 h-1 for 3 h). Antithrombotic effects were assessed as changes in total thrombus area (TTA) and total fibrin area (TFA) from baseline, using the Badimon perfusion chamber model at baseline and 2 h and 5 h after drug administration. RESULTS: Two hours postdosing, ximelagatran showed antithrombotic effects at both high and low shear rates (TTA% of mean baseline value +/- SEM was 76 +/- 13% and 71 +/- 17% [both P < 0.05] for the 20-mg dose, 85 +/- 11% [P > 0.05] and 62 +/- 15% [P < 0.05] for the 40-mg dose and 60 +/- 11% and 26 +/- 7% [both P < 0.05] for the 80-mg dose, respectively). r-Hirudin also showed a significant antithrombotic effect at high and low shear rates (76 +/- 11% [P = 0.05] and 57 +/- 17% [P < 0.05] of baseline values, 2 h postdosing, respectively). The inhibitory effects on TFA were similar to those on TTA. CONCLUSIONS: The oral DTI ximelagatran shows antithrombotic effects under both high and low shear conditions. The antithrombotic effect of 40-80 mg ximelagatran appeared comparable to that of parenterally administered r-hirudin, which has been previously demonstrated to be clinically effective in acute coronary syndromes.

Evidence for specific ceramidase present in the intestinal contents of rats and humans.

A neutral ceramidase activity stimulated by bile salt was previously identified in the intestinal content. Recently, bile salt stimulated lipase (BSSL) was found to have ceramidase activity. It is unknown whether the ceramidase activity previously found is attributable to BSSL. To address this question, we compared the behaviors of high quaternary aminoethyl (HQ) anion exchange chromatography, the distributions, the stability, and the responses to lipase inhibitor between ceramidase and pancreatic BSSL. The proteins from whole small intestinal contents of humans and rats were precipitated by acetone and dissolved in 20 mM Tris buffer pH 8.2. These proteins had neutral ceramidase activity but not BSSL activity against p-nitrophenyl acetate. When the proteins were subject to HQ chromatography, two peaks of ceramidase activity were identified, which had acid and neutral pH optima, respectively. Neither of them had BSSL activity against p-nitrophenyl acetate. Western blot using BSSL antiserum failed to identify BSSL protein in the fractions with high neutral ceramidase activity. In rat intestinal tract, pancreatic BSSL activity was high in the duodenum and declined rapidly in the small intestine, whereas neutral ceramidase activity was low in the duodenum and maintained a high level until the distal part of the small intestine. In addition, orlistat, the inhibitor of lipase, abolished human BSSL activity against p-nitrophenyl acetate and slightly reduced its activity against ceramide but had no inhibitory effect on ceramidase activity isolated by HQ chromatography. In conclusion, we provide the evidence for a specific ceramidase other than pancreatic BSSL present in the intestinal content. The enzyme may play important roles in digestion of dietary sphingolipids.

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen.

Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA(D227A)bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a Kd of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA(D227A)tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA(D227A)induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10(-10)M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA(D227A)against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA(D227A). Thus, 5T4FabV13-SEA(D227A)has highly attractive properties for therapy of human NSCLC.

Rapid and specific detection of PCR products using light-up probes.

Newly developed light-up probes offer an attractive tool for PCR product detection. The light-up probe, which consists of a thiazole orange derivative linked to a peptide nucleic acid oligomer, hybridizes specifically to complementary nucleic acids. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly fluorescent. This eliminates the need to separate bound from unbound probes and reduces the risk of cross contamination during sample handling. We demonstrate here the applicability of light-up probes in two different PCR assays, one directed towards the human beta-actin gene and the other towards the invA gene of Salmonella. The probes do not interfere with the PCR reaction and can either be included in the sample mixture or added after completed amplification. The specificity of the probe is found to be excellent: a single-base mismatch in the target sequence is sufficient to prevent probe binding as indicated by the lack of fluorescence increase. Furthermore, a clear correlation is found between the intensity of gel bands and the measured probe fluorescence in solution, which suggests that the amount of PCR products can be quantified using light-up probes.

A3--a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells.

The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.

Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma.

The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted cross-reactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens.

Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors.

C215Fab-IL-2 fusion protein, with full IL-2 and antigen binding activity, was produced in E. coli at high level (>50 mg/l). When co-administered with Fab-superantigen fusion protein (C215Fab-SEA) in mice strong and sustained T cell activation was observed. Combination treatment of mice carrying B16 melanoma transfected with C215 antigen was also more efficient than using C215Fab-SEA (p<0.01) or C215Fab-IL-2 alone (p<0.001). In a long-term survival experiment 5/12 mice having received combination treatment 5 days after i.v. inoculation of B16 cells survived >85 days. Improved therapeutic efficacy correlated with increased tumor infiltration by activated CD25+ T cells, indicating a T cell mediated mechanism.

The distinct role of CD4+ and CD8+ T-cells during the anti-tumour effects of targeted superantigens.

To target T-cells to the tumour area we created a recombinant protein of the bacterial superantigen (SAg) Staphylococcal enterotoxin A (SEA) and the Fab-fragment of a tumour-reactive antibody. This antibody-targeted SAg immunotherapy therapy has been shown to be highly efficient, eliminating > 95% of the pulmonary metastasis in mice carrying established melanoma micrometastases. Earlier studies demonstrated that elimination of the C215-expressing B16-melanoma lung metastasis was dependent on interferon (IFN)-gamma release and expression of perforin. In the present study, therapeutic effector functions were analysed both locally at the tumour site and systemically in the spleen. In order to elucidate the role of each T-cell subset during Fab-SEA therapy, CD4 knock-out (KO) and CD8 KO mice were used. Tumour size reduction was statistically significant in Fab-SEA-based tumour therapy in both types of T-cell-deficient mice compared to wild-type mice. CD4 KO mice displayed a drastic reduction in the number of tumour-infiltrating macrophages and CD8+ T-cells. Therapy-induced accumulation of perforin-containing cells at the tumour site was significantly impaired in CD8 KO mice, and marginally in CD4 KO mice. Moreover, CD4 KO mice failed to produce substantial amounts of the tumour suppressive cytokine IFN-gamma. This is in sharp contrast to normal mice where a massive local release was recorded. CD8 KO mice displayed a spontaneous production of interleukin (IL)-4 and IL-10 locally in the tumour. Neither normal nor CD4 KO mice produced detectable levels of these Th-2-associated cytokines. The high level of IL-10 was demonstrated to inhibit Fab-SEA tumour therapy, since the therapeutic efficacy was significantly higher in IL-10 KO mice. These results illustrate the importance of a finely tuned cellular collaboration to regulate the various phases of an efficient anti-tumour immune response.

Impact of functional dyspepsia on quality of life and health care consumption after cessation of antisecretory treatment. A multicentre 3-month follow-up study.

BACKGROUND: The impact of response to treatment on subsequent symptoms, quality of life, health care consumption, and absence from work in functional dyspepsia is unknown. METHODS: Patients with functional dyspepsia from Denmark, France, Germany, The Netherlands, Hungary, and Poland (n = 567 (215 men), 18-80 years old) were followed up for 3 months after a 4-week treatment trial with omeprazole (20 mg or 10 mg) or placebo. The patients were blinded to the initial treatment. Dyspeptic symptoms and quality of life were assessed, and dyspepsia-related costs were calculated in terms of number of clinic visits, days on medication, and absence from work. RESULTS: Responders had fewer clinic visits than non-responders (1.5 versus 2.0 mean visits) and fewer days on medication (mean, 9 days versus 23 days) over the 3-month period (both, P < 0.001). The quality of life in responders was better at study entry and persisted over 3 months (all, P < 0.001). When analysed country by country, health care costs due to clinic visits and medications were significantly lower in responders in all countries (P < 0.05), except Denmark and The Netherlands. CONCLUSION: Symptom resolution in patients with functional dyspepsia has a positive impact on quality of life and reduces the subsequent costs over a 3-month period after cessation of initial treatment.

Outcomes of periradicular surgery in cases with apical pathosis and untreated canals.

OBJECTIVE: Surgical management is intended to eliminate or block infection originating in the root canals. The root end is customarily sealed to prevent pathogenic products remaining in the root canal from reaching the periradicular tissues. The purpose of this study was to evaluate the microbiologic and radiographic outcomes of surgical treatment of periradicular pathosis associated with teeth with necrotic pulps. STUDY DESIGN: One tooth from each of 10 patients was root-end resected and root-end filled without prior root canal treatment. One year postoperatively, the outcomes were assessed radiographically and the root canals were sampled for bacteria. RESULTS: Radiographic examination showed complete or incomplete (scar tissue) healing in 5 teeth and uncertain healing in the other 5 teeth. Bacteriologic samples from the root canals were positive in 9 of the 10 cases. CONCLUSIONS: In teeth with necrotic pulps, treatment of periradicular pathosis by surgery and root-end filling may show radiographic evidence of satisfactory healing 1 year postoperatively. However, viable bacteria may persist in the canals, constituting a potential risk factor for recurrence of periradicular pathosis.

Dendritic cells and macrophages are the first and major producers of TNF-alpha in pancreatic islets in the nonobese diabetic mouse.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune insulin-dependent diabetes mellitus (IDDM) and serves as an animal model for human type I diabetes. TNF-alpha is known to be produced by islet-infiltrating mononuclear cells during insulitis and subsequent beta cell destruction and has been implicated in the pathogenesis of IDDM. Previously, T cells have been suggested as the main source of TNF-alpha in the islet infiltrate. However, on immunohistochemical analysis of TNF-alpha expression in islets, we are able to show that the staining pattern of TNF-alpha resembles that of dendritic cells (DC) and macrophages (Mphi) rather than T cells and that TNF-alpha is expressed in islets at the very early stages of insulitis when no T cells are detected. On double staining for TNF-alpha and cell surface markers, we can demonstrate that TNF-alpha staining clearly correlates with DC and Mphi, whereas there is a poor correlation with T cells. This feature was observed at both early and late stages of insulitis. TNF-alpha expression was also seen in NOD-SCID islets, in addition to a peri-islet infiltration consisting of DC and Mphi, indicating that T cells are not required for the early DC and Mphi infiltration and TNF-alpha expression in islets. In conclusion, our results show that DC and Mphi are the major, early source of TNF-alpha in the NOD islet infiltrate and that TNF-alpha can be expressed independently of T cells, indicating that the early DC and Mphi infiltration and expression of TNF-alpha are crucial in initiation of diabetes.

Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth.

Superantigens (SAg) are microbial proteins with the capacity to activate a large proportion of T cells. We have developed a novel approach for cancer immunotherapy by genetically fusing the SAg staphylococcal enterotoxin A (SEA) to a Fab-fragment of a tumor-specific antibody. Repeated exposure to SEA induces a state of unresponsiveness including cell deletion and functional hyporesponsiveness, i.e., anergy. In this study we have developed improved therapeutic schedules to allow repeated injections of Fab-SEA, limit development of immunological unresponsiveness and promote maximal anti-tumor response. Four daily injections of Fab-SEA to mice carrying B 16-C215 lung metastases resulted in 90-95% reduction in the number of metastases. However, the animals did retain a minimal residual tumor disease. The immune system was in a hyporesponsive state after 4 daily Fab-SEA injections, and further injections did not improve therapy. Two repeated cycles, each comprising 4 daily injections of Fab-SEA, significantly prolonged the survival and resulted in complete cure of a fraction of the animals. A rest period of 10 days between the cycles was required to mount an efficient secondary anti-tumor response. This secondary immune response was characterized by partial recovery of cytokine production i.e., interleukin-2, interferon-gamma and tumor necrosis factor-alpha. Strong CTL activity was detected in animals that had rested for 8 weeks between the 2 cycles. Interestingly, irrespective of the resting period, the CD4+ SEA-reactive T cells expanded in response to all 4 additional Fab-SEA injections both locally and in spleen. In contrast, only marginal expansion of CD8+ T cells was seen if restimulation was given within 1 month. Our data show that potent anti-tumor effector functions can be induced after repeated stimulation cycles with a SAg-monoclonal antibody fusion protein resulting in a CD4+ T cell-dependent cytokine release, prolonged survival and induction of complete cures.

Orally fed digalactosyldiacylglycerol is degraded during absorption in intact and lymphatic duct cannulated rats.

Membrane lipids of green plants digalactosyldiacylglycerol (DGalDG) and monogalactosyldiacylglycerol (MGalDG) are hydrolyzed in vitro by human duodenal contents, pancreatic juice and bile salt stimulated lipase and guinea pig and rat pancreatic lipase-related protein 2 to free fatty acids, di- and monogalactosylmonoacylglycerols and water soluble galactose-containing compounds. The fate of intermediate products is unknown. We have investigated the digestion and absorption of DGalDG in rats. [3H]- and [14C]-labeled DGalDG in galactolipid dispersions, and 200 g/L soybean triacylglycerol (TG) oil-galactolipid emulsions of different concentrations were fed orally to intact and lymphatic duct cannulated rats. Chyle, gastrointestinal tract, liver and plasma were analyzed for radioactivity in different lipid classes. Recovery of [3H] also was determined in feces. Comparison was made with an emulsion of [14C]dipalmitoyl-phosphatidylcholine ([14C]DPPC), soybean TG oil and soybean phosphatidylcholine (PC). Less than 2% of the radioactivity in chyle was found in DGalDG, >70% of the radioactivity in triacylglycerol (TG), and the remaining part in glycerophospholipids. In intact rats, <1.5% of radioactivity in liver and plasma was identified as DGalDG. In experiments where 120 mg galactolipid-phospholipid mixture or 120 mg PC were given in a soybean TG oil-emulsion, the absorption of galactolipid fatty acids was less complete than PC-fatty acids, as indicated by analysis of feces and intestinal contents. Galactolipids are not absorbed intact or as reacylated monoacyl compounds by rats.

Man-made superantigens: Tumor-selective agents for T-cell-based therapy.

Superantigens (SAgs) are a collection of bacterial and viral proteins with potent immunostimulatory properties. SAgs bind to Major Histocompatibility Complex Class II (MHC II) molecules of antigen presenting cells (APCs) and activate a high frequency of T lymphocytes. To target a T-cell attack against tumor cells we genetically linked tumor-specific antibody Fab fragments to the SAg Staphylococcal enterotoxin A (SEA). Fab-SEA fusion protein efficiently targeted to solid tumors and induced a T-cell-mediated eradication of established metastases in animal models. Successful therapy was T-cell-dependent and required tumor specificity of the Fab moiety of the Fab-SEA fusion protein. Due to the high affinity of SAg for MHC II, a limitation of this approach was retention of Fab-SEA proteins in normal tissues expressing MHC II, which caused systemic immune activation and dose limiting toxicity. We recently solved the structure of SEA and applied structure-based drug design to develop a novel generation of 'man-made' SAg with improved pharmacological and pharmacokinetic properties. Mutation of the major MHC II binding site of SEA substantially reduced retention in MHC II(+) tissues and systemic toxicity, while local immune activation at targeted tumor sites was retained. The Fab-SEA mutants display a 10000-fold higher affinity for tumor tissue compared to normal tissue and the therapeutic window was improved >100-fold compared to native Fab-SEA protein. Thus protein engineering can be applied to convert harmful bacterial toxins into tolerable tumor-specific agents.