Effects of diheptyldiselenide (DDS) on human tumor cell lines and on peripheral blood mononuclear cells

In vitro effects of graded concentrations of diheptyldiselenide (DDS) on human tumor cell proliferation, and on the proliferative responses and immunological functions of peripheral blood mononuclear cells (MNC) were investigated. The agent significantly decreased tumor cell proliferation in a dose and time dependent manner. Proliferative responses of MNC to phytohemagglutinin (PHA) and interleukin-2 (IL-2) were also significantly depressed when MNCs were exposed to DDS (250 microM for 18 h) led to a significant increase in NK activity only in MNC samples showing very limited baseline NK function. On the other hand, generation of LAK cells was significantly inhibited by DDS. However, when the agent was added to the effector and target cell mixture during the 4 h 51Cr release cytotoxicity assay, no influence was found on NK and LAK-mediated target cell lysis. These studies show that high concentrations of DDS inhibit tumor cell proliferation and could also impair certain proliferative-dependent immune functions, without directly affecting cell-mediated cytolytic activity of effector cells.     

Hepatitis C virus (HCV) status in recipients transfused with blood from anti-HCV-positive donors

Ten donors positive for antibodies to hepatitis C were discovered in the community of Aarhus after the introduction of screening of blood donors. These donors had donated blood products to 123 recipients. Of these recipients 76 were dead and 21 were not contacted for various reasons. Follow-up of anti-HCV status was performed in the remaining 26 recipients. Twenty-four (92%) of the recipients were positive in the RIBA confirmatory test, one was inconclusive and one was negative. Nine (90%) of the donors were hepatitis C virus RNA positive, while 17 (68%) of the recipients were HCV-RNA positive. Altogether (donors and recipients) 25 (76%) of the HCV-RNA positive patients had abnormal liver enzymes, while all HCV-RNA negative patients had normal enzyme levels. Eight of eleven HCV-RNA positive patients had an abnormal liver biopsy, while one patient in the HCV-RNA negative group had an abnormal liver biopsy. Three have been treated with interferon. In view of the liver damage already found only few years after transfusion, follow-up investigations in order to identify younger persons transfused with hepatitis C positive donations should be carried out and patients offered treatment if necessary. The National Board of Health has decided to recommend this strategy.     

Hepatitis C virus RNA in peripheral blood mononuclear cells: relation with response to interferon treatment

The aim of our study was to compare the short-term efficacy of three different chest physiotherapy (CPT) regimens (PD, postural drainage; PEP, positive expiratory pressure physiotherapy; HFCC, high-frequency chest compression physiotherapy) on patients with cystic fibrosis (CF) hospitalized for an acute pulmonary exacerbation. Sixteen patients with CF, 8 males, 8 females, aged 15-27 years (mean, 20.3 +/- 4), met the inclusion criteria: 1) age over 14 years; 2) mild or moderate airway obstruction; 3) sputum volume > 30 mL/day; 4) being proficient in PD and PEP CPT. Patients at admission had (mean +/- SD) forced volume in 1 second (FEV1) 52.2 +/- 21.9 percent predicted; Shwachman-Kulczycki clinical score 65.1 +/- 11 points; Chrispin-Norman chest radiography score 18.6 +/- 4.3 points. The three CPT regimens and a control-treatment (CONT) were administered in a random sequence, each patient receiving each treatment twice a day (in 50 minute sessions) for 2 consecutive days. During CONT and for 30 minutes after each session only spontaneous coughing was allowed. Wet and dry weight of sputum were recorded during the 50-minute sessions and 30 minutes afterward. Lung function was measured before and 30 minutes after each session. For each treatment a score was given by the patient for efficacy, and by both the patient and the physiotherapist for tolerance. Wet and dry weights of sputum collected during the sessions were greater for all CPT regimens than for CONT (P < 0.001, P < 0.0001). No significant differences between the three CPT regimens for both wet and dry weights were found when the number of coughs was taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells

As part of the evaluation of porcine cells, tissues, and organs intended for transplantation into humans, we investigated the conditions required to induce expression and release of porcine endogenous retrovirus (PoEV) from primary cells. Pigs contain endogenous retroviral sequences encoding infectious retrovirus, yet little is known about the conditions required to activate the expression and release of PoEV from primary cells. We show here that mitogenic activation of peripheral blood mononuclear cells (PBMC) isolated from the National Institutes of Health (NIH) miniature pig and the Yucatan pig resulted in the activation and release of an infectious type C retrovirus. Coculture of activated porcine PBMC with pig or human cell lines resulted in the transfer and expression of PoEV-specific sequences and the establishment of a productive infection. Sequence comparison of portions of the PoEV pol gene expressed in pig cell lines productively infected with virus derived from NIH miniature pig and Yucatan pig PBMC revealed marked similarity, suggesting that one or a few loci may be capable of being activated to yield an infectious virus. These findings demonstrate that the presence of endogenous viruses in source animals needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.     

Hepatitis C virus (HCV) serotype in the asymptomatic HCV-infected patients from selected groups

In asymptomatic hepatitis C virus (HCV)-infected blood donors and persons from high risk groups (intravenous drug users and hemodialyzed patients) HCV serotypes 1, 2, 3, 4 and 6 have been detected. The most frequent was serotype 1. In the group of intravenous drug users (IDU) a coexistence of different serotypes was observed.     

Cytomegalovirus DNA can be detected in peripheral blood mononuclear cells from all seropositive and most seronegative healthy blood donors over time

1. The purpose of this study was to assess the potential of various preservation solutions, orginally designed for solid organs, to protect muscle function during cold storage. 2. The soleus (SOL) and the cutaneous trunci (CT) muscle from the rat were isolated and stored for 2, 4 or 8 h at 10 degrees C. The solutions used, listed in order from an intracellular to an extracellular-like composition, were: University of Wisconsin (UW), Euro-Collins (EC), HTK-Bretschneider (HTK), reversed St. Thomas' Hospital (ST2) and Krebs-Henseleit (KH). After cold storage, the muscles were tested by direct electrical stimulation to obtain the maximum twitch tension (Pt) and the maximum tetanus tension (P0). Subsequently, the muscles were prepared for morphological analysis. 3. In general, storage at 10 degrees C caused a gradual decrease of Pt and P0 with time. After 8 h of storage in the extracellular-like solutions KH and ST2, the P0 was about 50% (SOL) and 35% (CT) of control. Eight hours of storage in intracellular-like solutions resulted in a P0 of 50% of control for HTK, in a P0 of 40% (SOL) and 67% (CT) for UW, but in a P0 of 5% (SOL) and 26% (CT) for EC. These findings corresponded well with the morphological observations. 4. It is concluded that the effects of 10 degrees C storage on skeletal muscle function are not predominantly determined by the intra- or extracellular-like composition of the solutions used. Both UW and HTK were most effective (P0 > 50% of control) in preserving muscle function.     

Quantitation of human immunodeficiency virus type 2 DNA in peripheral blood mononuclear cells by using a quantitative-competitive PCR assay

A new quantitative-competitive PCR-based human immunodeficiency virus type 2 (HIV-2) proviral DNA assay (QC-PCR) was developed and used to determine the proviral load in HIV-2-infected individuals. Proviral load varied considerably, with means of 1,831 copies per 10(6) peripheral blood mononuclear cells for asymptomatic subjects (n = 19) and 2,587 for AIDS patients (n = 2). HIV-2 viral and proviral loads also varied significantly over time in asymptomatic patients. These data suggest that a high level of virus replication occurs throughout the asymptomatic phase of HIV-2 infection.     

Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg)

The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3a(desArg) on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1beta. C3a or C3a(desArg) alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1beta, both C3a and C3a(desArg) were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1beta-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3a(desArg) enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3a(desArg), indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3a(desArg) on induction of NF-kappaB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-kappaB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro.     

Positive and negative hepatitis C virus RNA strands in serum, liver and peripheral blood mononuclear cells in anti-HCV patients: relation with the liver lesion

To investigate the replicative hepatitis C virus status and its relation to liver damage, serum, peripheral blood mononuclear cells and liver-paired samples from 45 untreated hepatitis C virus infected patients (38 with chronic hepatitis, three with minimal changes, and four with normal liver) were studied by nested polymerase chain reaction, using primers from the 5' untranslated region. Positive HCV-RNA strand was detected in serum (69%), peripheral blood mononuclear cells (100%) and liver samples (100%). The presence of negative HCV-RNA strand was confirmed using specificity controls assays and was only detected in liver and peripheral blood mononuclear cells samples, (95% and 82%, respectively). No correlation between the presence of negative HCV-RNA strand in peripheral blood mononuclear cells and positive HCV-RNA strand in serum was found, whereas serum HCV-RNA was not detected in patients without negative HCV-RNA strand in the liver. Both positive and negative HCV-RNA strands were found in liver and peripheral blood mononuclear cells of four patients with normal liver histology, and three with minimal changes. Furthermore, the presence of HCV-RNA in serum did not correlate with the alanine aminotransferase values and the histological activity index. These data confirm the existence of replicative intermediates in the liver, not only from patients with histologically proven chronic hepatitis, but also from those with normal liver, suggesting the existence of hepatitis C virus in true healthy carriers.     

GB virus C RNA in serum, liver, and peripheral blood mononuclear cells from patients with chronic hepatitis B, C, and D

We examined fiber density, compound muscle action potential (CMAP) amplitude, and motor unit number estimate (MUNE) of the abductor digiti minimi and grip strength longitudinally. We sought to determine the effects of ALS on these measurements and to evaluate which of these tests may be more sensitive in evaluating progression of ALS and possibly predicting survival. Ten patients were examined at months 0, 3, and 6. A significant decrease in MUNE and increase in fiber density were observed at months 3 and 6 (p < 0.02) compared with baseline (month 0). Mean CMAP and grip strength declined, but not significantly. The decrease in MUNE over 6 months was significantly greater than that of CMAP and grip strength (p < 0.025). The significant changes in MUNE and fiber density over time suggest that they are more sensitive in measuring the rate of progression of ALS. To evaluate further the utility of these tests, we arbitrarily divided the patients into equal groups based on length of survival. MUNE declined significantly in the group with shorter survival (p < 0.01). Conversely, fiber density increased significantly in patients with longer survival (p < 0.01). With similar statistical analysis there were no significant differences in decline of CMAP or grip strength in either subgroup over 6 months. Our study suggests that MUNE and fiber density are more sensitive than CMAP and grip strength in detecting progression of ALS. Furthermore, we raise the hypotheses that a greater increase in fiber density identifies a group of patients with ALS who will have longer survival, and that a greater decline in MUNE identifies a group with a worse prognosis.     

Characterization of lymphokine-activated killing by human peripheral blood mononuclear cells stimulated with interleukin 2 (IL-2) analogs specific for the intermediate affinity IL-2 receptor

Interleukin 2-stimulated human peripheral blood mononuclear cells (PBMC) generate lymphokine-activated killing (LAK). Using the IL-2 analogs R38A and F42K, which interact primarily with the beta and gamma subunits of the IL-2 receptor, we assessed the roles of IL-2R beta gamma and the high-affinity IL-2 receptor complex in LAK activation. Although the kinetics of LAK activation were identical, lytic activity was approximately 30% lower and proliferation was up to 55% lower in those PBMC stimulated by R38A or F42K than in those exposed to wild-type IL-2. The percentage of cells expressing cell-surface markers such as CD3, CD4, CD8, and CD16 was not significantly different after treatment with wild-type IL-2, R38A, or F42K; however, the proportion of cells expressing IL-2R alpha increased dramatically in response to stimulation by F42K (30%) compared to stimulation by either rIL-2 or R38A (15%). In addition, by Day 7 the concentration of soluble IL-2R alpha in analog-stimulated LAK culture supernatants was 50-75% less than that from wild-type IL-2-cultured cells. These findings suggest that interaction of IL-2 with IL-2R beta gamma alone is sufficient for both proliferation and the generation of LAK, and that stimulation with subunit-specific IL-2 analogs results in differential regulation of the IL-2R alpha on human LAK cells.     

Hepatitis in used syringes: the limits of sensitivity of techniques to detect hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and antibodies to HBV core and HCV antigens

Hepatitis virus infections are common among injecting drug users. Syringes containing hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were identified by polymerase chain reaction (PCR); syringes containing antibodies to HBV core antigen and HCV were identified by EIA. Syringe use was simulated to determine the sensitivity of these assays. The mean limits for PCR were 0.082 microliter of blood for HBV and 0.185 microliter for HCV; the mean limits for EIA were 0.185 microliter for HBV and 0.023 microliter for HCV. HBV PCR testing of 681 syringes returned to the needle exchange program in New Haven, Connecticut, revealed a decline from 7.8% HBV-positive at the program's outset to 2.6%. HCV antibodies were found in 12.1% of 207 syringes tested. Syringe testing can help estimate the prevalence and incidence of hepatitis virus infections when standard seroepidemiologic analyses cannot be applied.     

Detection of the t(2;5)-associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals

The translocation t(2;5), which leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the receptor kinase ALK on chromosome 2p23, is found in CD30+ anaplastic large cell lymphomas and some cases of B-cell lymphoma. Hodgkin's disease (HD) is a malignant lymphoma characterized by large multinucleated tumour cells, Hodgkin and Reed-Sternberg (H&RS) cells, surrounded by a dense lymphohistiocytic infiltrate. Our group recently demonstrated NPM/ALK fusion cDNAs by single-cell RT-PCR in < 3% of CD30+ tumour cells in 2/9 cases of HD. To further delineate the relevance of this finding for HD, we studied the occurrence of NPM/ALK fusion genes in peripheral blood cells of healthy donors by RT-PCR. NPM/ALK fusion cDNAs were found by RT-PCR in 14/29 healthy individuals and confirmed by hybridization with a breakpoint-specific oligonucleotide. Due to the low rate of NPM/ALK-positive cells in the peripheral blood of positive individuals, an assignment to a defined cellular subpopulation was not possible. We conclude that NPM/ALK fusion genes are present in peripheral blood cells of healthy donors. After t(14;18) and t(9;22), t(2;5) represents the third example of tumour-associated translocation products in blood cells of apparently healthy donors. The implications of this finding are discussed.     

Flow cytometric analyses of the specific activation of peripheral blood mononuclear cells from healthy donors after in vitro stimulation with a fermented mistletoe extract and mistletoe lectins

Immunostimulatory properties of mistletoe extracts derived from Viscum album L. (VAL) are well described, demonstrating activation especially of T, T-helper cells and monocytes/macrophages. In order to characterise in detail the communication between different cell populations, we studied mistletoe-induced expression of co-stimulatory signals and their ligands by flow cytometry. Peripheral blood mononuclear cells (PBMC) from 15 healthy controls were incubated for 7 days with a fermented VAL extract. VAL significantly upregulated the expression of the co-stimulatory molecule B7.1 (CD80) on monocytes/macrophages, but not B7.2 (CD86). No significant changes in the expression of either molecules on B cells could be found, suggesting that only monocytes/macrophages act as antigen presenting cells (APCs) in this in vitro system. Purified mistletoe lectins, components of most VAL extracts were also analysed, but did not induce similar responses of monocytes/macrophages. The receptor for B7 molecules, CD28, but not CTLA-4 (CD152), was also found to be significantly enhanced on CD4+ cells after VAL simulation. There was no evidence for activation of a B cell response via the CD40/CD40L pathway. Our data support the concept that stimulation by VAL extracts induces a specific T-helper cell reaction with monocytes/macrophages acting as APCs and purified lectins do not exert the same effects.     

Detection of proliferating cell nuclear antigen (PCNA) in peripheral blood mononuclear cells and sera of patients with malignant lymphoma

The proximity of farms to badger setts was compared between farms that had experienced a tuberculosis breakdown and those that had not, over the 6 year period from 1988 to 1993. The data were derived from a badger removal study conducted in East Offaly County in the Republic of Ireland. Badger removal began in 1989 and continued through 1993; by the end of 1990, approximately 80% of all badgers caught in the 6 year period had been removed. All badgers were examined, grossly, for evidence of tuberculosis. Tuberculosis status of the approximately 900 study herds was based on the results of the single intradermal comparative skin test and/or lesions of bovine tuberculosis. All herds were tested at least once annually. The number of herds experiencing bovine tuberculosis declined over the period, particularly in the years 1992 and 1993. The data on farm and badger sett location were stored and analysed, initially, in a geographical information system. Owing to the badger removal programme, the distance between the barn yard of a typical farm and the nearest occupied badger sett increased, by about 300 m year-1, and by about 600 m year-1 to the closest infected sett. In bivariate analyses, in the years 1988 and 1989, the risk of tuberculosis declined with increasing distance to a badger sett containing one or more tuberculous badgers. In multivariable logistic regression analyses, year and the average number of cattle tested per farm per year were controlled. A second identical analysis was conducted to control for the repeated observations on the same herds using generalised estimating equations. In both analyses, the risk of a multiple reactor tuberculosis breakdown decreased for herds at least 1000 m away from an infected badger sett, and increased as the number of infected badgers per infected sett increased. Despite the significantly reduced risk of a breakdown with increasing distance to infected badger setts, the relationship was not strong (sensitivity and specificity of the model in the low 70% range) and explained only 9-19% of tuberculosis breakdowns.     

Membrane and soluble forms of Fas (CD95) and Fas ligand in peripheral blood mononuclear cells and in plasma from human immunodeficiency virus-infected persons

The expression of membrane-bound Fas ligand (FasL) and Fas in lymphocytes and monocytes and levels of soluble forms of FasL (sFasL) and Fas (sFas) in plasma from human immunodeficiency virus (HIV)-positive and -negative subjects was evaluated. Surface FasL was detectable on monocytes, but poorly so on lymphocytes, even in the presence of KB8301, a metalloproteinase inhibitor. Unexpectedly, monocytes of HIV-positive subjects expressed less FasL than those of HIV-negative volunteers. sFasL levels in plasma of HIV-positive persons were elevated and correlated with levels in plasma and with HIV RNA burden. sFas levels in plasma of HIV-positive subjects were also elevated and correlated with Fas expression in apoptotic lymphocytes. Finally, culture-induced lymphocyte apoptosis of HIV-positive subjects was enhanced by anti-Fas agonistic antibody but was not inhibited by anti-FasL blocking antibodies. These results suggest that significant dysregulation of both Fas and FasL occurs in HIV infection and contributes to increased sensitivity of lymphocytes to apoptosis.     

Allergen-specific increase in interleukin (IL)-4 and IL-5 secretion from peripheral blood mononuclear cells during birch-pollen immunotherapy

BACKGROUND: In the multicenter European Intergroup Cooperative Ewing's Sarcoma Studies, localized Ewing tumors of bone were treated by combination chemotherapy with surgery and/or radiotherapy. Patients with primary metastases (pm-pts) were treated in high risk protocols. PATIENTS AND METHODS: One hundred seventy-seven pm-pts were registered from January 1990 to December 1995, 171 were evaluable for survival analyses. Thirty-six pm-pts received myeloablative megatherapy with stem cell rescue following conventional treatment. Bilateral whole lung irradiation (WLI) was administered in 57 pm-pts with pulmonary involvement. Event-free survival (EFS) rates were estimated by Kaplan-Meier analysis. Prognostic factors were identified by log-rank statistics, Cox procedures and logistic regression. RESULTS: Eighty-nine deaths were recorded by 1 February 1997, EFS four years after diagnosis for all 171 pm-pts was 0.27. EFS for isolated lung metastases was 0.34, for bone/bone marrow (BM) metastases, 0.28, and for combined lung plus bone/BM metastases, 0.14 (P < 0.005). WLI improved outcome in case of isolated pulmonary involvement (0.40 vs. 0.19, P < 0.05). In pm-pts with combined pulmonary/skeletal metastases, intensification by megatherapy and/or WLI improved EFS from 0.00 to 0.27 (P = 0.0001). CONCLUSIONS: EFS four years after diagnosis in patients with disseminated Ewing tumors is 0.27. Whole lung irradiation and megatherapy improve outcome in subgroups of patients with disseminated Ewing tumors is 0.27. Whole lung irradiation and megatherapy improve outcome in subgroups of patients with disseminated Ewing disease.