Cryopreservation of insulin-producing cells microencapsulated in sodium cellulose sulfate.

INTRODUCTION: Diabetes mellitus may be treated with pancreatic islet cell transplantation. The use of xenogenic islet cells may overcome the shortage of human donor organs. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor-intensive, cryopreservation has emerged as an attractive system for islet banking. In this study sodium cellulose sulfate (NaCS), a novel method for microencapsulation of islet cells, was tested for its capability to protect cells during cryopreservation. METHODS: HIT-T15 cells were microencapsulated in NaCS. Cells were frozen and thawed using three different media containing varying amounts of dimethylsulfoxide (DMSO) and glycerol. Cell viability and cell growth were monitored using 3-(-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide before freezing and 1 week after thawing. RESULTS: NaCS did not show any negative impact on the growth rates of encapsulated HIT-T15 cells compared with nonencapsulated controls. Nonencapsulated cells were adequately cryopreserved by both DMSO- and glycerol-containing freezing media. DMSO was not suitable for cryopreservation of encapsulated HIT-T15 cells, whereas glycerol seemed to produce no considerable cell loss during freezing and thawing. DISCUSSION: Islet banking of cells encapsulated in NaCS was feasible. Microencapsulation did not harm islet cell recovery. As NaCS is less immunogenic and more biocompatible than other materials used for microencapsulation, it may be a promising method for immunoisolation of islet cells to replace the endocrine pancreas in a physiological way.

Morphological and functional characterization of a pancreatic beta-cell line microencapsulated in sodium cellulose sulfate/poly(diallyldimethylammonium chloride).

BACKGROUND: Late diabetic complications cannot be prevented totally by current antidiabetic strategies. Therefore, new therapeutic concepts of insulin replacement such as pancreas transplantation are evolving. Due to the shortage of human donor organs, transplantation of microencapsulated xenogeneic pancreatic islet cells has attracted considerable attention. Sodium cellulose sulfate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC) is a material with favorable biogenic properties that has been used for microencapsulation of various cell types. However, there are no data on the suitability of NaCS/PDADMAC for microencapsulation of pancreatic beta-cells. MATERIAL AND METHODS: Cell growth and viability of NaCS/PDADMAC-microencapsulated HIT-T15 cells, an immortalized hamster pancreatic beta-cell line, were assessed using a dimethylthiazol-diphenyltetrazoliumbromide (MTT)-based cell growth determination kit and apoptosis was detected by antibodies against activated caspase 3. Glucose-dependent insulin secretion was assessed with ELISA and the uptake of glucose was measured using fluorescence-labeled glucose. RESULTS: Statistical analysis revealed no differences in glucose-dependent cell proliferation, insulin secretion and glucose uptake between non-microencapsulated and microencapsulated HIT-T15 cells. Stimulation of HIT-T15 cells with glucose (100 mg/ml) resulted in a biphasic insulin secretion response. CONCLUSION: Microencapsulation of HIT-T15 cells in NaCS/PDADMAC does not influence cell proliferation, insulin secretion and glucose uptake. Our results indicate that NaCS/PDADMAC is well suited for microencapsulation of pancreatic beta-cells.

Porcine islet cells microencapsulated in sodium cellulose sulfate.

One hundred fifty million people suffer from diabetes mellitus worldwide. Modern exogenous insulin therapy cannot prevent late complications. Islet cell transplantation could be a sufficient therapeutic option but the shortage of human organs limits this option. The use of xenogeneic porcine islet cells may also be a viable alternative. One way to manage hyperacute rejection is by the protection of xenogeneic cells from the immune system by microencapsulation. In this study sodium cellulose sulfate (NaCS) was evaluated as a material for encapsulation. An insulin-producing cell line (HIT-T15) was established in our laboratory. Glucose-dependent insulin production and cell growth were monitored. Cells were encapsulated with NaCS by Austrianova, Vienna. The insulin production and mitosis rate were examined. Cell growth and insulin production by HIT-T15 cells affected the glucose levels in the nutrient solution. Cell viability and glucose-dependent insulin production were not influenced by NaCS. Encapsulation with NaCS is feasible and it could be shown that the material is permeable to nutrients and metabolic side products. The encapsulated cells are able to detect the glucose concentration in the nutrient solution and to react in a proper way by producing insulin. Encapsulation with NaCS, which is more biocompatible and less immunogenic than other materials, seems to be a promising method for immunoisolation of porcine beta cells for xenotransplantation to replace the endocrine pancreas in a physiologic way.

Lipid peroxidation and antioxidant status in professional American football players during competition.

BACKGROUND: Oxidative stress occurs during strenuous physical exercise, perhaps as a result of increased consumption of oxygen. MATERIALS AND METHODS: In this study, different markers of oxidative stress were determined in eight national league American football players. Before (March) and at three time-points during the competition season (May, June, July) serum total peroxide concentrations, auto-antibody titres against oxidized low-density lipoprotein (oLab), and lag time of reactive oxygen species-induced degradation of the fluorophore 1-palmitoyl-2-((2-(4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl)ethyl)- carbonyl)-sn-glycero-3-phosphocholine (DPHPC) were measured along with serum ascorbate, alpha- and gamma-tocopherol, and beta-carotene concentrations. RESULTS: Before the competition season, serum antioxidant concentrations were within the lower normal range; ascorbate concentrations increased significantly during the competition period (P < 0.05). Serum peroxide concentrations were within the normal range and increased significantly during the competition period (P < 0.05); in four of the eight subjects the increase was several times the baseline values, while four athletes did not show any increase. The oLab titres increased significantly at the mid-competition period time-point (P < 0.01), but levelled off thereafter. DISCUSSION: Given that it could not be predicted from the baseline oxidative stress and antioxidant status which subject would respond to strenuous exercise with an increase in oxidative stress status, it is concluded that oxidative stress should be monitored in all athletes.

Increased neopterin in patients with chronic and acute coronary syndromes.

OBJECTIVES: The aim of our study was to determine neopterin levels in patients with chronic and acute coronary syndromes. BACKGROUND: In chronic and acute coronary syndromes the release of different cytokines activates cellular defense. Infiltration of neutrophils and monocytes/macrophages is detected in the vessel wall as well as in the myocardium. Neopterin, which is a by-product of the guanosine triphosphate-biopterin pathway, is a marker for those activated macrophages. METHODS: We studied 123 subjects: 1) 21 consecutive patients (17 men, 4 women; mean age +/- SD 66 +/- 15 years, range 31 to 87) with acute myocardial infarction (AMI); 2) 62 consecutive patients (50 men, 12 women; mean age 61 +/- 8 years, range 43 to 81) with signs and symptoms of clinically stable coronary artery disease (CAD); and 3) 40 healthy blood donors (28 men, 12 women; mean age 35 +/- 13 years). Neopterin levels were determined with a commercially available enzyme-linked immunosorbent assay method. RESULTS: In patients with AMI before thrombolytic therapy, neopterin levels were significantly higher than levels in patients with CAD and control subjects (13.7 vs. 8.6 and vs. 6.8 nmol/liter, p < 0.0001). Values also differed significantly between patients with CAD and control subjects (p < 0.0001). Neopterin levels in patients with AMI were measured seven times during a 72-h period. Within-group comparison showed significant differences over this period (p < 0.00001). The lowest value (11.4 nmol/liter) was observed after 4 h and differed significantly from the initial value and values after 24 and 72 h (p < 0.05). After 72 h, neopterin increased to 14.9 nmol/liter, a value significantly different from all values other than the initial one. There was no correlation between neopterin and creatine kinase (CK); CK, MB isoenzyme; or lactate dehydrogenase as markers for the extent of the myocardial infarction during the observation period. CONCLUSIONS: Our data support the hypothesis of an activation of monocytes and macrophages in patients with an acute or chronic coronary syndrome. Neopterin as a marker for macrophage activation is significantly increased in patients with chronic CAD and more pronounced in patients with AMI shortly after the onset of symptoms.

Carbohydrate-deficient transferrin in healthy women: relation to estrogens and iron status.

The determination of carbohydrate-deficient transferrin (CDT) in serum has been found useful as a marker of increased alcohol consumption of > 60 g/day. It is not clear why the reference range is different for women (0 to 26 units/liter) and men (0 to 20 units/liter). We evaluated serum CDT in 286 healthy subjects (209 women, 77 men) using a commercially available radioimmunoassay. Premenopausal women had higher CDT levels than postmenopausal women, whereas no age-related difference of CDT levels was found in men. In postmenopausal women, higher CDT levels were associated with estrogen replacement therapy. In premenopausal women, however, neither the phase of the menstrual cycle nor contraceptive steroid use showed a significant association with the increase in CDT levels. No significant correlations were found between CDT and either serum estradiol or serum iron. In conclusion, both premenopausal state and postmenopausal estrogen replacement therapy seem to increase serum levels of CDT. Therefore, menopausal status and exogenous estrogens should be considered when interpreting CDT values in women.

Troponin T as a marker for posttransplantation adaptational problems of the donor heart.

BACKGROUND: Troponin T is used as a marker for myocardial cell damage (e.g., in aiding diagnosis and follow-up of myocardial infarction). Elevated troponin T levels are also observed after heart transplantation, although until now no explanation could be found for this phenomenon. METHODS AND RESULTS: Serum samples of 15 patients who underwent orthotopic heart transplantation were tested for troponin T with a one-step enzyme immunoassay. The highest concentrations of troponin T were seen between day 3 and 14 after transplantation (3.05 +/- 1.30 micrograms/L) and remained elevated up to 3 months. A correlation (r = 0.61, p < 0.02) was found between pretransplantation systolic pulmonary artery pressure and the cumulative troponin T release after transplantation. No association was found with rejection, and no correlation was found with ischemic time of the donor heart. CONDITIONS: These findings support the hypothesis that the acute exposure of the donor heart to the preexisting elevated right ventricular afterload in the recipient represents a strong mechanical stress for the transplanted heart. Measurement of troponin T may therefore be helpful in the posttransplantation monitoring and management of ventricular function after orthotopic heart transplantation.

Antibiotics and increased temperature against Borrelia burgdorferi in vitro.

In 1917, spirochaetal neurosyphilis was treated successfully with malariotherapy in combination with salvarsan or bismuth. Malariotherapy for spirochaetal Lyme disease has been discussed, but the mechanism of an antispirochaetal effect remains unclear. We cultured Borrelia burgdorferi at different temperatures, alone and in combination with antibiotics. Our data demonstrate that growth of the strains PKo and ATCC 35210 (B31) was impaired at temperatures of 37 degrees C and inhibited at 39 degrees C and 40 degrees C, respectively. Strain ATCC 35211, however, grew well up to 39 degrees C but did not multiply at 40 degrees C. A bactericidal effect was seen at 41 degrees C for the strains B31 and PKo and at 42 degrees C for all strains. The susceptibility of all strains to penicillin and ceftriaxone was increased up to 16-fold by an elevation of temperature from 36 degrees C to 38 degrees C. These in vitro data suggest that elevated body temperature may be beneficial during antimicrobial treatment of Lyme disease. This may be particularly important in tissues where high concentrations of antibiotics are difficult to achieve.

Definitions of cytomegalovirus disease after heart transplantation: antigenemia as a marker for antiviral therapy.

In this prospective study, cytomegalovirus (CMV) antigenemia was defined as the marker for initiation and episodes of antigenemia as the indicator for the duration of antiviral therapy (CMV hyperimmune globulin and ganciclovir). The CMV antigenemia assay and CMV-specific IgM and IgG antibody tests were used to monitor CMV infection in 22 heart transplant recipients who, between October 1992 and July 1994, were followed up for 6 months. A total of 178 out of 627 antigenemia assays tested positive. The highest number of positive cells was greater after primary infection than after either reactivation (43.3 vs 0.3; P < 0.01) or reinfection (43.3 vs 9.3; P = NS). Sixty episodes of antigenemia were observed. More episodes of antigenemia were seen after primary infection than after either reactivation (4.6 vs 0.2; P < 0.01) or reinfection (4.6 vs 2.2; P = NS). The detection of antigenemia indicated the initiation of antiviral therapy within 24 h after the blood sample was harvested. Therapy was stopped immediately after a subsequent negative result became available. Our experience indicates that antigenemia directed antiviral therapy prevents CMV disease after primary and secondary infection in heart transplant recipients.

CA 15-3: a multicentre evaluation of automated and manual tests.

The introduction of a new and automated CA 15-3 immunoassay (IMx Abbott) prompted us to compare the analytical performance of this new test with established tests from CIS ELSA, Sorin, and Boehringer Mannheim in a multicentre study. CA 15-3 measurements in blood samples of breast tumour patients, comparison of intra- and inter-assay variation, dilution linearity, and lower limit of detection are described. The study showed improved precision for the automated over the manual test systems (intra-assay variation: IMx < 5%, CIS ELSA 4-9%, ES 300 < 3% and ETI Sorin > 10%; inter-assay variation: IMx < or = 8%, CIS ELSA < or = 19%, ES 300 < or = 9% and ETI Sorin < or = 27%). Results on patients' samples (n = 101 to 184) showed highly comparable results; IMx vs CIS ELSA site 1: r = 0.950; IMx vs CIS ELSA site 2: r = 0.998; IMx vs ES 300: r = 0.980; IMx vs ETI Sorin: r = 0.931. Slopes of regression lines varied from 0.666 for IMx vs ETI Sorin to 0.988 for IMx vs CIS ELSA (site 1, where heparin plasma was used instead of serum as recommended by the manufacturer found to be only slightly dependent on assay ranges analysed by statistical procedures applied. Despite good correlations between methods, it is recommended that samples collected in the follow-up of disease and at higher CA 15-3 concentrations are analysed by the same test; a changeover to another test is not encouraged.

Transient reduction of autoantibodies against oxidized LDL in patients with acute myocardial infarction.

Fifteen consecutive patients (mean age 66 +/- 14, range 31-82) with an acute myocardial infarction (MI) suitable for thrombolytic therapy were included in this study. Autoantibodies against oxidized low-density lipoprotein (LDL) were determined by enzyme-linked immunosorbent assay (ELISA). Patients (n = 10) with marked elevation of the MB isoenzyme of creatinine kinase (CK-MB)-mass had significant decreases of oLDL-Ab during the acute phase, with a minimum after 8 h following the onset of thrombolytic therapy (within-group significance: p < .001; between groups: p = .01). Patients (n = 5) with CK-MB-mass values less than 70 ng/ml did not show this phenomenon. Furthermore, significant correlations existed between CK-MB-mass and oLDL-Ab after 6 and 8 h (n = 15; r = .72; p = .003) and the time of the highest CK-MB-mass values (after 12 h) and the time of the maximal decrease of oLDL-Ab (after 8 h) (r = .74; p = .003). Our observations provide further evidence for the release of free radicals and for increased lipid peroxidation during reperfusion after prolonged ischemia. The decrease of oLDL-Ab appears to be a marker for the severity of MI.

CMV antigenemia (the lower matrix protein PP65), a marker for the guidance of antiviral therapy in cytomegalovirus disease after orthotopic heart transplantation.

During CMV viremia, the CMV specific lower matrix protein CMV pp65 can be detected in the nucleus of polymorphonuclear cells. A relationship has been found between the number of CMV pp65 positive cells, the clinical course and the effect of antiviral treatment on CMV disease. From 1990, heart recipients (triple drug therapy) were screened for CMV pp65 (antigenemia, according to the method described by The et al.), anti-CMV-IgM and -IgG. Tests were repeated at least every 4 weeks. Group 1 consisted of 23 patients who had been transplanted at least one year before the introduction of CMV testing as described. Between 1990 and 1992 26 patients were followed up during the first year after transplantation and represent group 2. In group 1, 1184 antigenemia assays were performed and 13 tested positive. In group 2 (1195 tests, 261 positive results), 20 out of the 26 recipients tested positive for CMV pp65. Without preceding evidence of a positive CMV pp65, no rise of IgM or IgG antibodies was observed. The time until the first antigenemia (time from detection until a subsequent test remains negative); 13 were found in group 1, 84 in group 2. In group 2, 46 episodes of antigenemia (mean duration 24.5 +/- 27.1 days) consisted of more than 1 consecutive positive result of the antigenemia assay (4.8 +/- 4.1). During these episodes the white blood cell count was 3460 +/- 1790/mm3. After the episodes, the mean leucocyte count was 6320 +/- 1870/mm3. The detection of CMV antigenemia indicated the initiation of antiviral treatment (hyperimmune globulin and ganciclovir). Therapy was stopped again when the antigenemia assay tested negative again. Antigenemia disappeared in all patients after initiation of antiviral treatment, CMV disease was not observed. CMV antigenemia mainly cumulates within the first year after heart transplantation. Antigenemia directed antiviral therapy does not prevent infection or repeated antigenemia but prevents CMV disease after heart transplantation.

Cytomegalovirus after heart transplantation: definitions for the guidance of antiviral therapy.

Besides the current classification of cytomegalovirus (CMV) infection and disease we defined "CMV antigenaemia" as the marker for initiation of antiviral therapy (CMV hyperimmune globulin 2 ml/kg/d and ganciclovir 1000 mg/d), and "episodes of CMV antigenaemia"(the time from detection of antigenaemia until a subsequent antigenaemia assay tested negative again) indicated the time period of antiviral treatment. Patients were at highest risk for antigenaemia at day 38.2 +/- 20.9 after heart transplantation. We observed 50 episodes of antigenaemia in 18 patients. The mean duration was 7.3 +/- 6.4 days. No antigenaemia associated symptoms and no anti-CMV IgM was observed without preceding evidence of antigenaemia. Antigenaemia-associated symptoms and antigenaemia disappeared after antiviral therapy was initiated. Our therapy did not prevent CMV infection, but despite the repeated evidence of active CMV infection, no patient suffered CMV disease.

Serum levels of the soluble receptor for tumor necrosis factor in patients with renal disease.

Tumor necrosis factor-alpha (TNF-alpha) has been found to be elevated in patients during hemodialysis and is thought to mediate some of the immune and metabolic dysfunctions in these patients. It has been speculated that infusions of soluble TNF receptor (sTNF-R) may prevent some of the cytotoxic effects of TNF. However, little is still known about preexisting serum TNF-R levels in patients with chronic renal failure, with or without hemodialysis. Therefore we analyzed serum samples of sTNF-R in 26 patients with chronic renal failure (group I), 61 hemodialysis patients (group II), 9 renal transplant recipients with acute renal failure requiring posttransplant dialysis (group III), 13 renal transplant patients with rejection and moderate kidney dysfunction (group IV), and 21 renal transplant recipients with borderline kidney dysfunction and diverse infectious complications (group V). Control groups consisted of 34 blood donors and diseased controls (11 renal transplant recipients with normal kidney function without complications). All patient groups showed significantly higher sTNF-R levels compared to the control groups. In groups I, IV, and V comparable levels were observed. In group I there was a clear correlation between sTNF-R levels and serum creatinine. The highest sTNF-R serum levels were seen in groups II and III, but there was no correlation with creatinine. In the posttransplant cases (group III and diseased controls) there was a decrease in sTNF-R with improvement of kidney function. These data strongly suggest that sTNF-R serum levels are dependent on kidney function.

A rapid immunocytochemical assay for CMV detection in peripheral blood of organ-transplanted patients in clinical practice.

This study describes clinical experience with a rapid method for diagnosis of cytomegalovirus infection in organ-transplanted patients, based on the detection of CMV-specific antigens in peripheral polymorphonuclear cells with a mixture of monoclonal antibodies. This CMV-pp65 assay was formerly called the "CMV immediate early antigen assay." A group of 180 organ-transplanted patients were examined with this assay; 75 of them could be observed from the date of transplantation. These 75 patients consisted of two groups: 59 kidney transplant patients receiving no CMV hyperimmunoglobulin prophylaxis (group I), 13 heart-transplanted patients, and 3 liver transplanted patients receiving prophylaxis (group II). Group III consisted of 105 patients who had been transplanted ca. 2 years before starting this study. In group I, 26 (44%) were CMV-pp65-positive (13 primary and 13 secondary infections). Fifteen of these 26 (58%) positive patients showed clinical symptoms of CMV infection. Eleven of these 15 (73%) were primary infections. Symptomatic patients had significantly more CMV-pp65-positive cells than asymptomatic patients; 12 patients showed a high number of positive cells and 11 of them developed severe CMV illness. Thirty-three patients were CMV-pp-65-negative (22 CMV IgG-sero-positive, 11 CMV IgG-seronegative). None of them had symptoms of CMV infection. In all patients of group I there were 36 periods of graft dysfunction in which CMV infection had to be differentiated from transplant rejection. In 10 out of 36 there was a CMV-pp65-positive test result and subsequent seroconversion. Treatment of viral infection resulted in improvement of clinical problems. In the remaining 26 episodes no CMV-pp65-positive cells were detected: in 17 cases graft dysfunction was caused by rejection, in 9 cases by other complications. In group II, 13 of 16 patients (81%) were positive in the CMV-pp65 assay (6 primary infections, 7 secondary infections). However, none of them showed clinical signs of CMV infection, regardless of the number of positive cells. No CMV-related graft dysfunction was observed. In group III, CMV infections did not play an important role. The experiences described suggest that this test is a valuable tool in early CMV diagnosis and in differentiating CMV-dependent graft dysfunction from other graft dysfunctions. It allows prompt therapeutic intervention.