Core-binding factor leukemia hijacks the T-cell-prone PU.1 antisense promoter.

The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFß-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.

Phase II trial of single agent amrubicin in patients with previously treated advanced thymic malignancies.

OBJECTIVES: There are limited treatment options for patients with thymic malignancies. Here we present data supporting treatment with single agent amrubicin, a third generation anthracycline and topoisomerase II inhibitor. MATERIALS AND METHODS: This was a phase 2 open-label, single arm trial of amrubicin in patients with thymoma (T) or thymic carcinoma (TC), conducted at two academic institutions. Patients were included if they had received at least one prior chemotherapy regimen. The first 18 patients received amrubicin at 40 mg/m2 IV days 1-3 repeated every 3-weeks. Due to the high incidence of febrile neutropenia, dosing was subsequently amended to 35 mg/m2 for the final 15 patients. RESULTS: A total of 33 patients (14 T/19 TC) were enrolled from 2011 to 2014. Median number of prior therapies was 2. Best response included 6 partial responses, 21 stable disease, and 6 progressive disease (all TC). Objective response rate was 18% (90% exact binomial CI 8.2%-32.8%; T = 4/14 (29%), TC = 2/19 (11%)). Median progression-free survival was 7.7 months (T: 8.3 months; TC: 7.3) and median overall survival was 29.7 months (T: 54.1 months; TC: 18 months). There was a high rate of febrile neutropenia (7 patients) that occurred despite a reduction in amrubicin dose and one related death. Five patients had reduction in LVEF below 50% during the course of treatment resulting in treatment discontinuation in one patient. CONCLUSION: Amrubicin shows promise as a single agent in heavily pre-treated patients with thymic malignancies. Notable side effects include febrile neutropenia and the use of growth factor support is essential. Further investigation of this agent is warranted.

Human lactoferrin attenuates the proinflammatory response of neonatal monocyte-derived macrophages.

Bioactive components of human milk, such as human lactoferrin (hLF), play an essential role in gut microbiome homeostasis and protection against neonatal inflammatory diseases. Neonatal intestinal macrophages display a proinflammatory profile that might contribute to inflammatory mucosal injury. Therefore, the aim of the study was to investigate the immunomodulatory effects of hLF on differentiation and activation of monocyte-derived macrophages (moMϕ). Monocytes isolated from umbilical cord blood of term neonates and peripheral blood of healthy adults were differentiated in the absence or presence of hLF, and differentiation, apoptosis and phagocytosis were evaluated. Cytokine production, Toll-like receptor (TLR) signalling and activation marker expression were investigated upon activation with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge. We demonstrate that hLF-differentiated moMϕ exhibit decreased TLR-4 expression, TLR signalling, proinflammatory cytokine secretion and intracellular tumour necrosis factor (TNF)-α production. Investigation of differentiation markers, morphology and induction of apoptosis showed no alteration in lactoferrin-differentiated moMϕ. Taken together, hLF promote anergic/anti-inflammatory effects by TLR expression and pathway interference, resulting in a diminished proinflammatory moMϕ phenotype. The anergic/anti-inflammatory properties of hLF might contribute to the prevention of harmful TLR-mediated inflammatory disorders in the developing gut of premature infants.

Phase I Study of Lenalidomide and Sorafenib in Patients With Advanced Hepatocellular Carcinoma.

LESSONS LEARNED: Combination therapies in patients with hepatocellular carcinoma can be associated with overlapping toxicity and are therefore poorly tolerated.Using sorafenib at the maximum tolerated dose can lead to a higher incidence of toxicities. Consequently, combination studies might evaluate sorafenib at alternative schedules or doses to improve tolerance, recognizing this could affect sorafenib efficacy.Although this combination was poorly tolerated, it does not exclude further evaluation of new-generation immunomodulator drugs or immune checkpoint inhibitors in the hope of optimizing tolerance and safety. BACKGROUND: Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC), and to date, no combination therapy has demonstrated superior survival compared with sorafenib alone. The immunosuppressive microenvironment in HCC is a negative predictor for survival. Lenalidomide is an immunomodulator and antiangiogenic agent, with limited single-agent efficacy in HCC. Based on these data, we designed a phase I study of sorafenib plus lenalidomide to determine the safety and preliminary antitumor activity of this combination. METHODS: This was an open-label, phase I study with a 3+3 dose escalation/de-escalation design. The starting dose of sorafenib was 400 mg p.o. b.i.d. and of lenalidomide was 15 mg p.o. daily with a planned dose escalation by 5 mg per cohort up to 25 mg daily. Dose de-escalation was planned to a sorafenib dose of 400 mg p.o. daily combined with two doses of lenalidomide: 10 mg p.o. daily for a 28-day cycle (cohort 1) and 10 mg p.o. daily for a 21- or 28-day cycle (cohort 2). Patients with cirrhosis, a Child-Pugh score of A-B7, and no previous systemic therapy were eligible. RESULTS: Five patients were enrolled. Their median age was 56 years (range 39-61), and the ECOG status was 0-2. Four patients were treated at dose level (DL) 1. Because of the poor tolerance to the combination associated with grade 2 toxicities, one more patient was treated at DL -1. No dose-limiting toxicity was observed as specified per protocol. The most common toxicities were nausea, anorexia, pruritus, elevated liver enzymes, and elevated bilirubin. Three patients experienced one or more of the following grade 3 toxicities: fatigue (DL 1), increased bilirubin (DL 1), skin desquamation (DL -1), and elevated transaminase levels (DL 1). The median duration of therapy was 1 cycle (range 1-3). All patients discontinued the study, 4 because of progressive disease and 1 by patient preference. The best confirmed response was progressive disease. The median progression-free survival was 1.0 month (95% confidence interval 0.9-2.8), and the median overall survival was 5.9 months (95% confidence interval 3.68-23.4). CONCLUSION: In our small study, the combination of lenalidomide and sorafenib was poorly tolerated and showed no clinical activity. Although the study was closed early because of toxicity concerns, future studies assessing combinations of sorafenib with new-generation immunomodulator drugs or other immunomodulatory agents, should consider lower starting doses of sorafenib to avoid excessive toxicity.

Ocular metastasis of colorectal cancer: An uncommon presentation of a common malignancy.

Approximately 20% of patients with colorectal cancer have metastatic disease at time of diagnosis, and another 25-35% develop metastases during the course of their disease. Liver, peritoneum, and lungs are the most common sites of metastases. We report the case of a 60-year-old female who presented with ocular metastasis 4 years after her initial curative-intent treatment for T3N1M0 rectal adenocarcinoma. After local eye radiation therapy, she received palliative systemic chemotherapy and enjoyed a good quality of life for 32 months before succumbing to progressive disease. Ocular metastasis of colorectal cancer is rare. When present, it usually occurs in the setting of diffuse hematogenous spread. In addition to local therapy, systemic chemotherapy should also be considered.

Serum-dependent processing of late apoptotic cells for enhanced efferocytosis.

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.

Phase 1 pharmacogenetic and pharmacodynamic study of sorafenib with concurrent radiation therapy and gemcitabine in locally advanced unresectable pancreatic cancer.

PURPOSE: To define the safety, efficacy, and pharmacogenetic and pharmacodynamic effects of sorafenib with gemcitabine-based chemoradiotherapy (CRT) in locally advanced pancreatic cancer. METHODS AND MATERIALS: Patients received gemcitabine 1000 mg/m(2) intravenously weekly × 3 every 4 weeks per cycle for 1 cycle before CRT and continued for up to 4 cycles after CRT. Weekly gemcitabine 600 mg/m(2) intravenously was given during concurrent intensity modulated radiation therapy of 50 Gy to gross tumor volume in 25 fractions. Sorafenib was dosed orally 400 mg twice daily until progression, except during CRT when it was escalated from 200 mg to 400 mg daily, and 400 mg twice daily. The maximum tolerated dose cohort was expanded to 15 patients. Correlative studies included dynamic contrast-enhanced MRI and angiogenesis genes polymorphisms (VEGF-A and VEGF-R2 single nucleotide polymorphisms). RESULTS: Twenty-seven patients were enrolled. No dose-limiting toxicity occurred during induction gemcitabine/sorafenib followed by concurrent CRT. The most common grade 3/4 toxicities were fatigue, hematologic, and gastrointestinal. The maximum tolerated dose was sorafenib 400 mg twice daily. The median progression-free survival and overall survival for 25 evaluable patients were 10.6 and 12.6 months, respectively. The median overall survival for patients with VEGF-A -2578 AA, -1498 CC, and -1154 AA versus alternate genotypes was 21.6 versus 14.7 months. Dynamic contrast-enhanced MRI demonstrated higher baseline K(trans) in responding patients. CONCLUSIONS: Concurrent sorafenib with CRT had modest clinical activity with increased gastrointestinal toxicity in localized unresectable pancreatic cancer. Select VEGF-A/VEGF-R2 genotypes were associated with favorable survival.

Preterm neonates display altered plasmacytoid dendritic cell function and morphology.

Bacterial and viral infections cause high rates of morbidity and mortality in premature newborns. In the setting of viral infection, pDCs play a key role as strong producers of IFN-α upon TLR9 activation. We analyzed pDC frequency, phenotype, morphology, and function in CB of preterm and term newborns in comparison with adults. Whereas all age groups show similar pDC numbers, BDCA-2, CD123, and TLR9 levels, the expression of BDCA-4 and capacity to produce IFN-α upon TLR9 challenge were decreased significantly in preterm neonates. Furthermore, we show by means of electron microscopy that pDCs from preterm newborns exhibit a distinct, "immature" morphology. Taken together, these findings suggest decreased functionality of pDCs in the premature newborn. The reduced capacity to produce IFN-α is likely to render such infants more susceptible to viral infections.

Effect of preoperative fever-range whole-body hyperthermia on immunological markers in patients undergoing colorectal cancer surgery.

BACKGROUND: Previous studies have demonstrated beneficial immunological effects of fever-range whole-body hyperthermia (FR-WBH) as an adjunct to non-surgical cancer therapy. We conducted a study of preoperative FR-WBH in patients undergoing colorectal cancer surgery to evaluate perioperative, hyperthermia-induced immunomodulation. METHODS: The trial was conducted as a subject-blinded, controlled, randomized study. Subjects in the FR-WBH group (n=9) were treated with FR-WBH before operation under propofol sedation; the target core temperature was 39 (0.5)°C with 1 h warming and 2 h plateau phase. Subjects in the control group (n=9) were treated with propofol sedation only. Blood samples were acquired before and after treatment, after operation, and 24, 48 h, and 5 days after the end of surgery. The following parameters were measured: lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α, procalcitonin (PCT), interleukin (IL)-6/10, heat shock proteins (HSPs) 60, 70, and 90, human leucocyte antigen-DR (HLA-DR), and LPS-binding protein (LBP). RESULTS: HSPs were increased in the FR-WBH group after treatment [HSP60, 48 h postop: 143 (41)% vs 89 (42)%, P=0.04; HSP90, postop: 111 (33)% vs 64 (31)%, P=0.04; HSP70: P=0.40; FR-WBH vs control, P-values for area under the level/time curve]. TNF-α levels were elevated after surgery in the control group and remained near baseline in the FR-WBH group [24 h postop: 73 (68)% vs 151 (72)%, P=0.04]. PCT increased in both groups 24 h after surgery; in the control group, this increase was significantly higher (P=0.02). There were no significant differences for IL, HLA-DR, or LBP. CONCLUSIONS: The immune system to react to surgical stress, as measured by a panel of laboratory indicators, might be improved by preoperative FR-WBH.

Serum phenylalanine in patients post trauma and with sepsis correlate to neopterin concentrations.

Increased blood concentrations of phenylalanine in patients with trauma and sepsis are common but unexplained. We examined the potential relationship between serum concentrations of phenylalanine and the immune activation marker neopterin in 84 specimens of 18 patients (14 males and 4 females) post-trauma during 12-14 days of follow up. Compared to healthy controls, average phenylalanine and neopterin concentrations were elevated in patients, and there existed a positive correlation between concentrations of the two analytes (r (s) = 0.375, p < 0.001). No such association existed between neopterin and tyrosine concentrations (r (s) = -0.018), but neopterin concentrations correlated to the phenylalanine to tyrosine ratio (r (s) = 0.328, p = 0.001). Increased phenylalanine implies insufficient conversion by phenylalanine (4)-hydroxylase (PAH). Oxidative stress due to immune activation and inflammation may destroy cofactor 5,6,7,8-tetrahydrobiopterin and impair PAH activity. This assumption is further supported by the correlation found between higher neopterin concentrations and higher phenylalanine to tyrosine ratio, which estimates efficacy of PAH.

The role of Foxp3+ T cells in long-term efficacy of prophylactic and therapeutic mucosal tolerance induction in mice.

BACKGROUND: Mucosal tolerance induction is suggested as treatment strategy for allergic diseases. Using a murine model of birch pollen (BP) allergy we investigated the long-term efficacy and the underlying mechanisms of mucosal tolerance induction with two structurally different molecules in a prophylactic and in a therapeutic set-up. METHODS: The three-dimensional major BP allergen Bet v 1 or a nonconformational hypoallergenic fragment thereof was intranasally applied before (prophylaxis) or after sensitization (therapy). RESULTS: In the prophylactic application both the Bet v 1 allergen and the fragment prevented allergic sensitization, and this effect lasted for 1 year. In the therapeutic approach established allergic immune responses were also suppressed after treatment with either of the molecules. However, a long-lasting curative effect (6 months) was only achieved with the Bet v 1 allergen but not with the Bet v 1 fragment. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of splenocytes revealed that tolerance induction with the Bet v 1 allergen was associated with enhanced expression of transforming growth factor (TGF)-beta, interleukin (IL)-10, and Foxp3 mRNA in CD4+ T cells, whereas treatment with the fragment led to the induction of either Foxp3 (prophylaxis) or IL-10 (therapy) alone. CONCLUSION: From these data we concluded (i) that the mechanisms underlying peripheral tolerance are linked to the conformation of the antigen, (ii) that mucosal tolerance is mediated by separate regulatory cell subsets, and (iii) that the long-term efficacy of immunosuppression is associated with the presence of Foxp3+ T cells.

Effect of single and combined supply of glutamine, glycine, N-acetylcysteine, and R,S-alpha-lipoic acid on glutathione content of myelomonocytic cells.

BACKGROUND & AIMS: Several diseases are characterised by decreased glutathione (GSH) levels due to an enhanced formation of oxygen radicals. To increase GSH levels, the additional supply of GSH precursors was suggested. In this study we evaluated the potency of a single and combined administration of the GSH modulating substances glutamine (GLN), N-acetylcysteine (NAC), and glycine (GLY) as well as R,S-alpha-lipoic acid (LA) to enhance intracellular GSH content in a well-defined model system. RESULTS: Exposure of myelomonocytic U937 cells for 24 h to GLN revealed a 1.5-fold enhancement of GSH levels with a concomitant decrease in the formation of reactive oxygen species and lipid peroxidation. Addition of NAC stimulated GSH formation only at subphysiological GLN levels. GLY enhanced GSH levels under GLN starvation, but caused a diminution of GSH content under optimal GLN supply. LA in combination with 2 mmol/l GLN evoked a 3.6-fold enhancement of GSH content compared to GLN starved cells. CONCLUSION: These results demonstrate that the GSH content of U937 cells is dependent on the supply of GLN, NAC, LA, and GLY. Combinations of the single substances can enhance but also decrease the intracellular GSH content, which is of clinical importance when supplying GSH-modulating substances to patients.

Administration of proteolytic enzymes bromelain and trypsin diminish the number of CD4+ cells and the interferon-gamma response in Peyer's patches and spleen in endotoxemic balb/c mice.

Recent publications revealed that bromelain exerts a marked effect on T-cell response by inhibiting T-cell signal transduction. These experimental studies may help to explain former clinical investigations showing that Phlogenzym (PHL), a preparation consisting of the proteases bromelain and trypsin and the antioxidant rutosid, ameliorate certain diseases with an underlying inflammatory process. In this study, we showed that orally administered PHL significantly reduced lymphocyte subpopulations in Peyer's patches (PPs) of healthy and endotoxemic mice. Similarly, the number of splenic lymphocytes in endotoxin-boostered mice was significantly lowered by PHL. The effect of PHL was more pronounced on T cells than on B cells leading especially to a diminution of CD4+ cells. Moreover, PHL pretreatment decreased IFN-gamma mRNA in PPs and spleen of endotoxemic mice. These results reveal that PHL may ameliorate inflammatory process by reducing the number of CD4+ cells and by diminishing INF-gamma mRNA levels.

Oral feeding with glutamine prevents lymphocyte and glutathione depletion of Peyer's patches in endotoxemic mice.

OBJECTIVE: To determine the effect of oral glutamine feeding on lymphocyte subpopulations and glutathione metabolism in Peyer's patches (PPs) of healthy and endotoxemic mice. SUMMARY BACKGROUND DATA: Recent data indicate that nutrients both maintain nitrogen and energy balances and modulate cell and organ function. In particular, glutamine has an impact on gut and immune function. This is of special importance in the perioperative phase. METHODS: Female Balb/c mice were fed a glutamine-enriched diet or a control diet for 10 days. On day 7 25 microg lipopolysaccharide (LPS) or saline was injected. On day 3 after the challenge, mice were killed, total cell yield was determined, and lymphocyte subpopulations (total T cells, CD4+, CD8+ cells, and B cells) were analyzed by flow cytometry. One experimental group was treated with buthionine sulfoximine, a specific inhibitor of glutathione synthesis. The glutathione content in PPs was measured by high-performance liquid chromatography. RESULTS: Glutamine administration led to a significant increase in total cell yield, including T and B cells, in PPs. The LPS-induced reduction of T cells (-45%) and of B cells (-30%) was significantly lower in glutamine-treated mice. Endotoxemia caused a 42% decrease of glutathione in control animals, but not in glutamine-treated animals. As with LPS, buthionine sulfoximine also lowered lymphocyte numbers and glutathione content of the PPs. CONCLUSIONS: Administration of glutamine prevents LPS-stimulated lymphocyte atrophy in PPs, possibly by increasing the glutathione content in the PPs. Therefore, oral glutamine supply seems to be a suitable approach for improving intestinal immunity in immunocompromised patients.

Postoperative glycyl-glutamine infusion reduces immunosuppression: partial prevention of the surgery induced decrease in HLA-DR expression on monocytes.

BACKGROUND AND AIMS: Surgery, trauma and inflammation reduce HLA-DR expression on monocytes, which is associated with an increased susceptibility to infection and sepsis. Furthermore, surgery decreases plasma glutamine (GLN) levels. The expression of HLA-DR on human monocytes in vitro is dependent on the concentration of GLN in the culture medium. We therefore hypothesized that postoperative infusions of glutamine-dipeptides would prevent the decreased HLA-DR expression on monocytes. METHODS: Thirty patients undergoing major abdominal surgery were randomly allocated to receive either 1500 ml Vamin (control) or an isonitrogenic formulation containing Vamin and 500 ml glycyl-glutamine (35 g GLN; 0.5g/kg BW) (GLY-GLN), or Vamin and 500 ml alanyl-glutamine (35 g GLN; 0.5 g/kg BW) (ALA-GLN) as a continuous infusion over 48 h post-operatively. Immediately and 48 h after surgery blood samples were collected to determine HLA-DR expression on monocytes by flow cytometry. RESULTS: The groups were comparable with respect to age, gender distribution and operation time. In patients receiving GLY-GLN mean HLA-DR expression on monocytes at 48 h was significantly better preserved than in controls (65.0 %+/-7 % vs 42.5 %+/-4 %;P<0.05), whereas HLA-DR expression on monocytes in patients receiving ALA-GLN was not significantly different. CONCLUSION: This is the first study comparing the dipeptides GLY-GLN and ALA-GLN in the postoperative setting. The GLY-GLN induced preservation of HLA-DR on monocytes following surgery may prevent infectious complications in these patients.

Relationship between interleukin-6 plasma concentration in patients with sepsis, monocyte phenotype, monocyte phagocytic properties, and cytokine production.

Monocyte phenotype, their phagocytic capacity as well as the cytokine production from 10 patients with sepsis with low interleukin-6 (IL-6) serum concentrations (<1000 pg/mL) and 8 patients with sepsis with high IL-6 (> or = 1000 pg/mL) plasma concentrations were investigated within 24 hours of fulfilling the criteria for sepsis. Monocytes from patients with high IL-6 levels had higher levels of human leukocyte antigen (HLA)-DR, HLA-ABC, CD64, and CD71, and the production of tumor necrosis factor-alpha (TNF-alpha) and IL-8, as well as the capacity of monocytes to phagocytose, was significantly elevated. Of 8 patients with high levels of plasma IL-6, 4 patients died. In contrast, all 10 patients with low plasma IL-6 concentrations survived until day 28. Patients who died had constant high IL-6 concentrations during the first 3 days, whereas IL-6 levels in patients who survived decreased by 88%. Our data indicate that IL-6 levels are a better prognostic parameter in the early phase of sepsis than the monocyte HLA-DR expression.

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands.

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.

Severe acute pancreatitis causes alterations in HLA-DR and CD14 expression on peripheral blood monocytes independently of surgical treatment.

OBJECTIVE: To find out if the severity of acute pancreatitis or the surgical treatment of severe acute pancreatitis influences HLA-DR and CD14 expression on peripheral blood monocytes. DESIGN: Prospective open study. SETTING: University hospital, Austria. SUBJECTS: 9 consecutive patients with severe acute pancreatitis in need of operative treatment, 5 patients with mild acute pancreatitis, and 7 healthy volunteers. INTERVENTIONS: Samples of 5 ml blood were taken daily into endotoxin free tubes at same time points. Surgical treatment for severe acute pancreatitis consisted of blunt necrosectomy, operative lavage, laparostomy, and open drainage. MAIN OUTCOME MEASURES: Correlation between HLA-DR and CD14 expression on peripheral blood monocytes on the one hand and the severity of acute pancreatitis and operative treatment of severe acute pancreatitis, on the other. RESULTS: In patients with severe acute pancreatitis expression of HLA-DR and CD14 was significantly downregulated both before and after operation (p < 0.0001; ANOVA), compared with patients with mild acute pancreatitis or healthy controls. However the expression of the two cell surface markers was not affected either by the first operation, or by the reoperations. CONCLUSION: These findings suggest that in acute pancreatitis the expression of cell surface markers on peripheral blood monocytes is related to the severity of disease but is not influenced by operative treatment.

Influence of enteral diets supplemented with key nutrients on lymphocyte subpopulations in Peyer's patches of endotoxin-boostered mice.

BACKGROUND AND AIMS: This study was undertaken to compare the effect of different key nutrients on lymphocyte subsets of Peyer's patches (PP) and spleen in endotoxemic mice. METHODS: Female Balb/c mice were fed over a period of 10 days either with an isocaloric and isonitrogenous control diet (Control), a glutamine enriched diet (Diet I) or a diet containing glutamine, arginine, glycine, and n-3 fatty acids (Diet II). On day 7 the mice were challenged intraperitoneally with 25 microg LPS. The lymphocyte subpopulations (B cells, T cells, CD4+ and CD8+) of PP and spleen were analysed by flow cytometry. Glutathione content of small intestinal mucosa and spleen was determined by HPLC and luminal small intestinal IgA by ELISA. RESULTS: Both experimental diets increased the number of B and T cells in the PP and that of T cells in the spleen (P<0.01). Glutathione content in PP and spleen was higher under administration of key nutrients (P<0.05). Diet II reduced luminal small intestinal IgA content in comparison to the two other groups. CONCLUSION: The addition of arginine, glycine and n-3 fatty acids to a glutamine supplemented diet does not enhance lymphocyte numbers in PP and spleen, but reduces intestinal IgA content.