Adenosine production in mesenchymal stromal cells in relation to their developmental status.

BACKGROUND: Mesenchymal stromal cells (MSC) are multipotent progenitor cells found in the tumor microenvironment. They have an innate and regulatory immune activity, and they are able to produce immunosuppressive adenosine (ADO) via their ectonucleotidases CD39 and CD73. The present study explores ADO metabolism of MSC in relation to their developmental status. METHODS: We analyzed MSC (n = 6), chondrogenic progenitor cells (CPC, n = 8), and chondrocytes (n = 8) for surface markers by flow cytometry. The ability to hydrolyze ATP and to produce ADO was tested by luminescence assays and mass spectrometry. RESULTS: Significant differences in the surface marker expression of MSC, CPC, and chondrocytes were seen. While the expression of CD73 was observed to be the same on all cell types, the expression of the ectonucleotidase CD39 was significantly increased on MSC. Consequently, production of ADO was most abundant in MSC as compared with chondrocytes and CPC. CONCLUSION: Mesenchymal stromal cells are potent producers of ADO and are, therefore, able to increase immunosuppression. As MSC differentiate into chondrocytes, they lose this ability and may take on other functions.

In vivo results for interstitial laser application in thyroid gland.

OBJECTIVE: Aim of this study was to evaluate the potential of denaturation of hormone active tissue in the thyroid gland by laser induced interstitial thermotherapy (LITT) as a treatment of autonomous hyperthyroidism. MATERIALS AND METHODS: An interstitial thyroid laser application (Nd:YAG 1064 nm, 5W, 2 min) was performed in 5 pigs. During laser application, the laryngeal recurrent nerve was controlled electro-physiologically. Postoperatively, TSH, total T(3) (TT(3)) and free T(4) (FT(4)) were measured regularly. After a follow-up period of up to 6 weeks, pigs were sacrificed and the thyroid glands were evaluated histological. RESULTS: A malfunction of the nerve due to laser treatment was not detected. During the first postoperative week there was a decrease of both FT(4) and TSH whereas TT(3) showed an extreme decline of its plasma levels reaching nearly the detection limit. All values showed a recovery to their initial levels during an interval of 10 days and than increased to levels sometimes higher than baseline. The coagulation zones were demarcated clearly towards normal tissue with increasing fibrosis of the treated areas. CONCLUSION: Interstitial thyroid ablation using a Nd:YAG laser is a minimal invasive, safe and effective procedure. Further evaluation including long term follow-up in humans is needed to confirm these results.

Interdisciplinary treatment of primary hepatic angiosarcoma: emergency tumor embolization followed by elective surgery.

Among primary hepatic malignancies, sarcomas represent a minority of 2 %. Of those, primary hepatic angiosarcoma is the most common one. In the past its incidence has been related to the exposure of certain chemicals like thorotrast, vinyl-chloride or arsenic. - Patients suffering from this aggressive, highly vascular tumor have a poor prognosis in general. Without treatment most of them die after rapid tumor progression with multifocal dissemination. In case of tumor perforation, fatal abdominal hemorrhage has been observed. - We herein report the successful interdisciplinary treatment of an 81 year-old woman with a perforated primary hepatic angiosarcoma of the left hepatic lobe. Initially, tumor bleeding was stopped by emergency interventional coil embolization. After stabilization of the patient, we performed an elective tumor resection. The patient could eventually be discharged in a good clinical condition. - So far, no standard therapy has established for patients with primary hepatic angiosarcoma. Surgery seems to be the treatment of choice. In addition, preoperative interventional embolization of the tumor supplying vessels reduces the risk of pre- and intraoperative bleeding. The value of adjuvant chemotherapy is not yet clarified. - The outcome of most patients with primary hepatic angiosarcoma remains poor and there is a need for clinical studies.

G-CSF during large field radiotherapy reduces bone marrow recovery capacity.

OBJECTIVE: Side effects of chemo- and radiotherapy are granulo- and thrombocytopenia. However, the long-term effects of in vivo granulocyte-colony-stimulating factor (G-CSF) stimulation of the hematopoietic system during radiotherapy are not yet completely understood. In the present study, we sought to determine the bone marrow effect of G-CSF during radiotherapy. MATERIAL AND METHODS: In a prospective, randomized clinical trial 10 patients (6 m, 4 f, 30-64 yrs, mean 50.6 yrs) were assigned to large field radiotherapy (RT). 7 patients (pat.) with non-Hodgkin lymphoma, one patient with Hodgkin's disease and 2 patients with small-cell carcinoma of the lung were included. The patients were randomized to either radiotherapy alone (group A) or radiotherapy with simultaneous G-CSF (group B) treatment and assessed for acute and late toxicity. Blood samples were drawn and analyzed before and after G-CSF stimulation. The mobilization effectivity of G-CSF on CD34 superset+ progenitor cells was measured using flow cytometry and colony forming units (CFU) testing on admission and during the complete follow-up period (1, 3 and 18 months post RTx). RESULTS: Overall, 50 pat. were intended to be included to the protocol. However, the preliminary analysis revealed a significant decrease of thrombocytes and CD34 superset+ progenitor cells in the G-CSF treatment group. According to the study protocol further treatment was stopped. Peripheral leukocyte counts ranged between 2800 - 4375 /mul in 9/10 pat. In group B mean thrombocyte levels dropped below 30.000 mg/l and CD34 superset+ progenitor cells to 50% (interruption criteria, p<0.02, Student's t-test). Hemoglobin values did not vary. Differential blood smears showed differences in granulocyte counts and a higher proportion of neutrophils in group B. Lymphocyte counts of patients randomized to group A were significantly decreased when compared to group B. In group A, 3/5 pat. developed an overshooting reaction (4,7 x increase) after G-CSF-stimulation. In arm B circulating CD34 superset+ progenitor cells dropped. In arm A, 3/5 pat. had an initial overshoot reaction when compared to none in group B. CFU (> 40 cells) and cluster (4 -39 cells) showed considerable variations. CONCLUSION: Our results demonstrate that simultaneous treatment with G-CSF during radiotherapy reduces the mobilization of CD34+ progenitor cells and exhaust the bone marrow capacity while peripheral leukocyte counts remain at baseline levels.

Evaluation of endotoxiuria for diagnosis of urinary tract infection after major surgical procedures.

In 148 patients after major surgical procedures urinary endotoxin levels were determined and compared with bacteriological results. The study was designed as a screening study. Urine samples were collected once by suprapubic or transurethral catheters. In a first series of 49 patients urine bacteriology was positive (mainly, Gram-negative rods were found) in 3 cases. However, endotoxin determination was positive in these 3 patients and in a further 10 patients receiving antibiotic therapy for other reasons. Therefore, the following 99 patients were studied also by urinalysis by reagent strips for leukocytes and nitrite. In the second series, 12 urine cultures positive for bacteria were observed. Eleven samples were also endotoxin positive. Five more patients were endotoxin positive and had pathological but unspecific reagent strip results. These patients were treated with antibiotics for other reasons. Patients with candida found in the urine culture (n = 5) were endotoxin negative. Thus, endotoxin determination in urine obtained by suprapubic or transurethral catheters proved to be a very sensitive method for diagnosis of bacterial contamination, even during antibiotic treatment.

Endotoxemia and mediator release during cardiac surgery.

Endotoxemia in man is a controversial issue. However, endotoxin is a potent trigger of the inflammatory response. Therefore, endotoxin translocation and mediator release was investigated in patients undergoing cardiac surgery. In 40 patients (13 women and 27 men, ages ranging from 30 to 73 years with a median of 60 years), plasma concentrations of endotoxin, interleukin-6 (IL-6), and C-reactive protein (CRP) were determined during and after cardiovascular bypass. In a subgroup of 10 patients, myeloid-related proteins: MRP8, MRP14, and the soluble heterocomplex (MRP8/MRP14) levels were additionally studied. A significant increase (p < 0.01) of plasma endotoxin concentrations was found during surgery, culminating in a peak (median value of 0.82 EU/mL) during reperfusion. Plasma levels of endotoxin continued to be slightly raised until the 5th postoperative day, whereas those of interleukin-6 rose at the end of the operation and were at their highest level 6 hours postoperatively (median value of 218 pg/mL). CRP levels were increased 24 hours postoperatively with a median value of 114 mg/L and peaked on day 2 (191 mg/L). A statistically significant correlation between the intraoperative endotoxin plasma concentrations and IL-6 concentrations was established (p < 0.05). The MRP8/MRP14 heterocomplex increased until day 2 after surgery, except MRP14, which showed the highest level at day 1 (55 ng/mL). Cardiac surgery is associated with endotoxemia and a marked acute-phase response. Therefore, endotoxin must be regarded as a pathophysiologic mediator. The role of the gut as a source of endotoxemia following cardiac surgery deserves further attention.

Hepatic metastasectomy for soft-tissue sarcomas: is it justified?

BACKGROUND: Except for patients with gastrointestinal stromal tumors (GIST), systemic chemotherapy in patients with liver metastasis of soft-tissue sarcoma (STS) is not effective. Therefore, all patients with resectable liver metastases underwent surgical therapy. We present our experience with this approach during the last 13 years. METHODS: All patients (n=45) with liver metastasis of STS undergoing surgical therapy were prospectively analyzed. Clinical and histopathological parameters as well as the postoperative course were recorded. Survival data were analyzed by using the Kaplan-Meier method and the log-rank test. RESULTS: Twenty-seven of 45 patients with liver metastasis underwent hepatic resection; 59% of these patients had a solitary metastasis, 22% had two metastases, and 18% had three or more metastatic nodules. The surgical perioperative mortality was 7%. The median survival was 44 (range, 1-123) months, and the 5-year survival was 49%. Repeated resection for recurrent tumor was performed in eight patients, which yielded a median survival of 76 months. CONCLUSIONS: Patients who have hepatic metastases that are functionally and technically resectable should be considered for surgery because this treatment offers the chance for long-term survival (>5 years).

Urinary endotoxin excretion and urinary tract infection following kidney transplantation.

Following kidney transplantation, urine endotoxin levels were measured among 44 patients and compared to bacterial cultures. Urine samples were collected either via transurethral catheters or - after removal of the catheter on postoperative day 4 - by midstream void. In a control group of ten healthy volunteers, urine endotoxin levels were measured daily for 10 days. Urinary endotoxin concentration was measured by means of a chromogenically modified Limulus amebocyte lysate (LAL) test. The levels among patients with positive bacteriological findings (n = 21) were always elevated ( > 0.7 EU/ml). Furthermore, there was a marked, statistically significant difference in endotoxin values between samples with bacterial growth and samples with fungal or without any growth (P < 0.001). All 21 of the 44 patients with urinary tract infection (UTI) were endotoxin-positive. Seven more patients who received antibiotics had elevated urinary endotoxin levels, but no bacterial growth in the urine culture. No bacterial infection or significant urinary endotoxin was found in the control group. In summary, the detection of urinary endotoxin in samples obtained by either suprapubic/transurethral catheters or midstream void is an early, sensitive, and specific means of diagnosis that can be carried out even during antibiotic treatment.

Endotoxemia and specific antibody behavior against different endotoxins following multiple injuries.

The aim of this study was to establish the incidence of endotoxemia and the influence of endotoxin on specific antibody response after multiple injury. Blood samples were collected from 39 patients (median Injury Severity Score: 20.5) at 0-3 and 6-12 hours, and 1, 3, 5, and 10 days after admission. The endotoxin plasma levels were high at the first time point (mean = 0.421 endotoxin units/mL) and decreased in the later course. Total immunoglobulin levels of IgM, IgG, or IgA were low and increased throughout the observation period. Specific antibodies of the IgM class against two lipid A and four lipopolysaccharide preparations increased transiently but significantly on day 3 and/or day 5. No changes of specific antibody content against endotoxin or lipid A was seen in the IgG or IgA class. The specific antibody content of the different classes against alpha-hemolysin of Staphylococcus aureus did not differ during 10 days after trauma. The specific antibodies of the IgM class reacted with all lipid A and LPS lipopolysaccharide preparations demonstrating cross-reactivity. These results suggest that endotoxin may be a specific stimulator of IgM antiendotoxin antibody secretion following trauma.

Endotoxemia and mediator release during colonoscopy.

BACKGROUND AND STUDY AIMS: Previous clinical and experimental studies have shown evidence of a leakage of whole bacteria and bacterial products after major trauma through the gut barrier. By determining plasma endotoxin levels, products of the arachidonic pathway, interleukin-6, and the endotoxin-neutralizing capacity (ENC) of plasma during colonoscopy, we studied the gut barrier function and the pathogenetic sequelae of mediator release during a minimally invasive procedure. PATIENTS AND METHODS: Thirty-two patients were enrolled in a controlled prospective study. Endotoxin and ENC were determined by a chromogenic modification of the limulus amebocyte lysate test. Prostanoids and interleukin-6 were measured using commercially available ELISA tests. C-reactive protein levels were checked by nephelometry. RESULTS: Twenty-one of the 32 patients had elevated endotoxin plasma levels during colonoscopy. In one patient, gut-derived bacteria were detected in plasma. ENC decreased after 5 min, and thromboxane B2 levels also started to increase at that time. No acute-phase response took place after 24 h. CONCLUSION: During colonoscopy, endotoxin can be detected in blood. ENC measurement was shown to be even more sensitive. The pathogenetic sequelae leading to gut barrier failure remain unclear, because mediator release and endotoxemia, as checked by ENC, took place simultaneously.

Endotoxin binding and elimination by monocytes: secretion of soluble CD14 represents an inducible mechanism counteracting reduced expression of membrane CD14 in patients with sepsis and in a patient with paroxysmal nocturnal hemoglobinuria.

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% +/- 0.8% in healthy subjects to 50.5% +/- 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-alpha) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-alpha release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 +/- 0.4 and 20.1 +/- 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-alpha.