An active haemovigilance programme characterizing the safety profile of 7437 platelet transfusions prepared with amotosalen photochemical treatment.

BACKGROUND: An active haemovigilance programme was implemented to survey adverse events (AE) associated with transfusion of platelets photochemically treated with amotosalen and ultraviolet A (PCT-PLT). The results of 5106 transfusions have already been reported. Here we report the results of an additional 7437 PCT-PLT transfusions. METHODS: The focus of this ongoing haemovigilance programme is to document all AEs associated with PCT-PLT transfusion. Data collected for AEs include: time of event after starting transfusion, clinical descriptions, vital signs, results from radiographs and bacterial cultures, event severity (Grade 0-4) and causal relationship to PCT-PLT transfusion. RESULTS: One thousand four hundred patients (mean 60 years, range 1-96) received PCT-PLT transfusions. The majority of the patients (53.4%) had haematology-oncology diseases and required conventional chemotherapy (44.8%) or stem cell transplantation (8.6%). Sixty-eight PCT-PLT transfusions were associated with AE. Acute transfusion reactions (ATR), classified as an AE possibly related, probably related, or related to PCT-PLT transfusions were infrequent (n = 55, 55/7437 = 0.7%) and most were of Grade 1 severity. Thirty-nine patients (39/1400 = 2.8%) experienced one or more ATRs. The most frequently reported signs/symptoms were chills, fever, urticaria, dyspnoea, nausea and vomiting. Five AEs were considered severe (> or = Grade 2); however, no causal relationship to PCT-PLT transfusion was found. Repeated exposure to PCT-PLT did not increase the likelihood of an ATR. No cases of transfusion-related acute lung injury and no deaths due to PCT-PLT transfusions were reported. CONCLUSIONS: Routine transfusion of PCT-PLT is well-tolerated in a wide range of patients. ATRs related to PCT-PLT transfusion were infrequent and most were of mild severity.

Treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a serious condition associated with sepsis. Clinical management of DIC is hampered by lack of clear diagnostic criteria. The International Society on Thrombosis and Haemostasis (ISTH) has proposed a diagnostic scoring algorithm for overt DIC based on routine laboratory tests. The objective was to assess a modified version of the ISTH scoring system and determine the effect of drotrecogin alfa (activated) (DrotAA, recombinant human activated protein C) on patients with DIC. The large database from the PROWESS clinical trial in severe sepsis was retrospectively used to assess a modified ISTH scoring system. Baseline characteristics and treatment effects of DrotAA were evaluated. At baseline, 29% (454/1568) of patients had overt DIC. Overt DIC was a strong predictor of mortality, independent of APACHE II score and age. Placebo-treated patients with overt DIC had higher mortality than patients without (43 vs. 27%). DrotAA-treated patients with overt DIC had a trend towards greater relative risk reduction in mortality than patients without (29 vs. 18%, P = 0.261) but both groups had greater relative risk reduction than placebo-treated patients. Serious bleeding rates during DrotAA infusion in patients with and without overt DIC were slightly increased (P = 0.498), compared with placebo, while clinically overt thrombotic events during the 28-day period were slightly reduced (P = 0.144). Modified ISTH overt DIC scoring may be useful as an independent assessment for identifying severe sepsis patients at high risk of death with a favorable risk/benefit profile for DrotAA treatment. Patients without overt DIC also received significant treatment benefit.

Low-dose daily subcutaneous interleukin-2 in combination with highly active antiretroviral therapy in HIV+ patients: a randomized controlled trial.

PURPOSE: Previous studies with intermittent interleukin-2 (IL-2) therapy using intermediate and high levels of IL-2 have demonstrated significant increases in the CD4 + T cell count in HIV-infected patients. Intermittent regimens are amenable to outpatient use, but severe adverse events are frequently experienced with intermediate- and high-dose levels of IL-2. Therefore in this study, the effect of daily, subcutaneous low-dose IL-2 therapy on safety and immunological endpoints was investigated to determine whether immunological benefit could be achieved without toxicity in HIV-infected patients also receiving highly active antiretroviral therapy (HAART). METHOD: A total of 115 patients were enrolled in the trial. Fifty-six asymptomatic HIV-infected patients who had CD4 + T cell counts less than 300 cells/microL at screening and a stable HIV viral load received low-dose IL-2 (1.2 million IU [MIU]/m 2 beginning dose) once daily in conjunction with HAART (IL-2 group). Fifty-nine patients received HAART alone (control group). RESULTS: A dramatic effect of IL-2 on the natural killer (NK) cell population was observed with mean increases of 156 cells/microL in the IL-2 group compared to 19.93 cells/microL in the control group (p <.001). Additionally, IL-2-treated patients experienced a statistically significant increase in the mean percentage of CD4 + T cells (3.52% increase) when compared to control patients (1.33% increase) (p <.001). The expanded CD4 + T cell population was primarily of the naive phenotype, with mean increases of 4.53% for the IL-2 group and 0.31% for the control group (p <.001 for between-group difference). In addition, a higher proportion of IL-2-treated patients (67%) compared to control patients (33%) achieved increases of greater than 50% in the CD4+ T cell count (p =.08). Adverse events of grade 3 or grade 4 toxicity were infrequent in the current study and were substantially lower by comparison to those in studies of intermittent dose IL-2 therapy. Also, negligible changes in the HIV viral load from baseline to final measurement were observed in both groups. A trend toward a reduced number of modifications of antiretroviral therapy was apparent in the IL-2 group when compared to control patients. CONCLUSION: Daily, low-dose subcutaneous IL-2 therapy in conjunction with HAART is safe and well tolerated and is effective in expanding lymphocyte cell types including NK cells and naive T cells in individuals who have <300 CD4+ T cells.

Persistent infection of a lymphoma cell line by herpes simplex virus.

The peripheral blood cells from a patient with a B-cell lymphoma were established in long-term tissue culture. Two years after establishment of the cells in culture they were infected with herpes simplex virus type 2 and the productivity and duration of viral persistence investigated. One week after infection the lymphoblastoid cells were productively infected and have remained so for a period of over 3 years. Expression of a viral glycoprotein antigen was evaluated by using a fluorescein-labeled monoclonal anti-herpes simplex virus type 2 antibody and revealed a spectrum of staining reactions grading from a lightly stippled to very intense pattern. Polymerase chain reaction analysis of the infected cells revealed the presence of the herpes simplex virus type 2 DNA polymerase gene in the infected cells that was absent from the uninfected lymphoblastoid cells. These results taken together with the long-term growth characteristics of both the infected and uninfected lymphoblastoid cells suggest that this cell line may be a good model system for studying viral infection, viral replication, viral latency, and clinical application for the isolation of human herpes virus.

Triazole Seed Treatments Suppress Spore Production by Puccinia recondita, Septoria tritici, and Stagonospora nodorum from Wheat Leaves.

Treatments of winter wheat seed with the systemic triazole fungicides triadimenol (31 g a.i./100 kg = Baytan 30F at 1.5 fl oz/cwt) and difenoconazole (24 g a.i./100 kg = Dividend 3FS at 1.0 fl. oz/cwt) were tested for effect on asexual sporulation by Puccinia recondita, Septoria tritici, and Stagonospora nodorum. Spore production was measured on seedlings grown in a growth chamber (24°C day/15°C night, 12-h photoperiod) and inoculated with the pathogens 3, 5, or 7 weeks after sowing. Spore production was converted to a percentage of the non-treated control and regressed against weeks after planting when plants were inoculated. Linear models fit data for both fungicides against all three pathogens. According to the models, difenoconazole suppressed sporulation levels of P. recondita and Septoria tritici to 10% of the levels on plants from non-treated seed for about 3 weeks after sowing. Spore production for all three fungi was suppressed to 25% of the non-treated level for at least 4.2 weeks and to 50% for at least 6.5 weeks. Similarly, triadimenol suppressed all three pathogens to 50% of the non-treated level for at least 3.2 weeks. The two fungicides showed similar effects against S. tritici; however, difenoconazole showed significantly greater suppression of sporulation by P. recondita and Stagonospora nodorum compared with triadimenol. Responses occurred even though large concentrations of spores were used to inoculate plants and environmental conditions were optimized for spore production. Reduced sporulation should help protect fall-planted wheat seedlings and may significantly delay epidemics in the following spring.

Toxicity management in patients receiving low-dose aldesleukin therapy.

OBJECTIVE: To review the pathophysiology and subsequent treatment options for low-dose aldesleukin-induced toxicity when administered via intravenous bolus infusion, continuous intravenous infusion, or subcutaneous injection. BACKGROUND: The adverse events associated with high-dose aldesleukin therapy (600,000 IU per kg i.v. every 8 h for a maximum of 14 doses) are well documented in the literature; however, the adverse event profile of lower doses and alternative administration routes are less well described. An understanding of the adverse event profile associated with these alternative regimens can enhance management of toxicity. DATA SOURCES: English-language clinical studies, abstracts, and review articles pertaining to low-dose intravenous, continuous intravenous infusion, or subcutaneous injection of aldesleukin, as well as aldesleukin-induced adverse events. STUDY SELECTION AND DATA EXTRACTION: Relevant studies were selected that assist with understanding the pathophysiology, clinical management, diagnosis, and management of aldesleukin-induced adverse events. CONCLUSIONS: Aldesleukin therapy initiates a cytokine-mediated proinflammatory process resulting in a toxicity profile that is different from traditional nonbiologic chemotherapeutic agents. The frequency and severity of adverse events associated with aldesleukin administration are dependent upon dose, route, and administration schedule. In addition, most adverse reactions are self-limiting. Alleviation of aldesleukin-induced adverse effects can usually be achieved on an outpatient basis with agents such as antiemetics, antipyretics, and topical creams or lotions, as well as nonmedication interventions. Aggressive and proactive management of aldesleukin associated toxicities can help facilitate completion of therapy.

Cellular mechanism of aminoglycoside tolerance in long-term gentamicin treatment.

In the rat, nephrotoxicity results from uptake of gentamicin at the apical membrane of proximal tubule (PT) cells. However, during continuous gentamicin treatment, the PT epithelium has been shown to recover. The mechanism(s) of cellular recovery and development of tolerance remains unknown. Therefore, we undertook studies designed to characterize cellular adaptations that occur during long-term gentamicin (LTG) treatment. After 19 days of gentamicin treatment, electron microscopy morphological evaluation revealed cellular recovery with an apparent mild decrease in height and number of microvilli. Enzymatic analysis of LTG PT membranes showed that apical and basolateral membranes had essentially returned to normal. Analysis of apical membrane lipid content revealed persistent statistically significant (P < 0.01) elevations in phosphatidylinositol (PI). In vivo immunogold morphological studies and biochemical studies in LTG rats revealed that endocytosis of gentamicin was selectively reduced, whereas the markers of fluid-phase (horseradish peroxidase) and receptor-mediated (beta2-microglobulin) endocytoses were unaffected or increased. Biochemical analysis showed that, although gentamicin binding to apical membranes isolated from LTG rats increased greater than twofold (P < 0.05) over membranes from untreated rats, in vivo cellular uptake, quantified with [3H]gentamicin, was reduced. Western blot analysis of LTG apical membranes and immunofluorescent staining of perfusion-fixed LTG kidneys showed no change in megalin levels or its apical membrane localization. These data imply that recovery of PT cells from and tolerance to LTG treatment involve a selective inhibition of gentamicin uptake across the apical membrane. They indicate that the mediators of gentamicin endocytosis were affected differently: PI levels increased, whereas megalin levels did not change. We conclude that selective inhibition of gentamicin uptake during LTG treatment is not affected by a reduction in PI or megalin levels. We postulate that trafficking of gentamicin and/or gentamicin-containing endocytic structures is reduced in LTG rats, allowing cells to develop tolerance to gentamicin.

Three summertime outbreaks of influenza type A.

In the Northern Hemisphere, sporadic cases of influenza occur during the summer, yet summertime outbreaks are rare. From 12 August through 2 September 1993, three influenza outbreaks in Louisiana were investigated using medical-record review, interviews, viral cultures, serology, and active surveillance for influenza-like illness in Louisiana. Attack rates in the outbreaks were 61% (69/114), 42% (24/57), and 45% (23/51). Viruses isolated were most closely related to influenza A/Beijing/32/92 (H3N2). The identification of influenza A as the cause of the first two outbreaks led to the recommendation for amantadine use in the third outbreak. Active surveillance did not detect any other outbreaks of influenza-like illness during August or September 1993. Out-of-season influenza A outbreaks can therefore occur when little influenza-like illness is present in a community. Evaluation of outbreaks of acute, febrile respiratory illness outside the influenza season should include this possibility, since rapid detection can lead to the timely use of amantadine or rimantadine.

Convergence of apical and basolateral endocytic pathways for beta 2-microglobulin in LLC-PK1 cells.

Polarized epithelial cells internalize molecules from both apical and basolateral (BL) plasma membrane (PM) domains via receptor-mediated endocytosis. In the kidney, low-molecular-weight proteins (LMWP) are internalized across the apical membrane of proximal tubule cells and degraded in lysosomes. Although indirect evidence suggests some uptake may occur at the BL surface, in vivo studies performed in rats suggest little if any LMWP uptake occurs at the BL surface. The studies presented here showed that native human beta 2-microglobulin (beta 2M) was internalized across the apical surface and followed the same intracellular pathway in the proximal tubule-derived cell line LLC-PK1 as that described in vivo. Either 125I- or gold-labeled beta 2M (125I-beta 2M and gold-beta 2M) bound specifically and reversibly to the apical surface of confluent LLC-PK1 cells. These results were qualitatively similar to previously documented in vivo results. Subsequently, using gold-beta 2M and LLC-PK1 cells grown on porous supports, we showed that a functional uptake system for the LMWP beta 2M was present at the BL surface. Finally, using different-size gold particles conjugated to beta 2M applied simultaneously to the apical and BL surfaces, we observed that apical and BL endocytic routes converged in multivesicular acid phosphatase-negative endosomal structures. Taken together, these data imply that beta 2M can be internalized across both apical and BL domains and that the two pathways converge at a multivesicular level within the endosomal pathway.

Characterization of the beta 2-microglobulin endocytic pathway in rat proximal tubule cells.

The uptake mechanism(s) of low-molecular-weight proteins by proximal tubule cells remains incompletely characterized. We utilized a biochemical and semiquantitative morphological approach to better characterize the endocytic pathway of an anionic protein, beta 2-microglobulin (beta 2M), in the rat proximal tubule. Indirect immunogold techniques revealed beta 2M was taken up via a classic receptor-mediated endocytic pathway. In vitro biochemical and morphological characterization of iodinated beta 2M and gold-conjugated beta 2M (gold-beta 2M) binding to isolated brush-border membrane vesicles (BBMV) documented specific and quantitatively similar binding interactions of the modified beta 2M with BBMV. Kinetic characterization of the in vivo endocytic pathway of gold-beta 2M was undertaken using microinfusion of individual tubules. beta 2M initially bound at the apical surface, was internalized into subapical coated vesicles and delivered to endosomal-like structures within 5 min, and, finally, was concentrated in lysosomal-like structures within 15 min. This uptake was inhibited by excess unconjugated beta 2M. In addition, we directly showed that uptake did not occur across the basolateral surface. Finally, by passing solubilized BBMV over beta 2M affinity columns we were able to isolate binding activity.

Isolation of parainfluenza virus type 3 from cerebrospinal fluid associated with aseptic meningitis.

Parainfluenza virus type 3 has been isolated from the cerebral spinal fluid (CSF) from six individuals--four children and two adults--over a 10-year period. All had fever, and four had signs of meningitis. All recovered uneventfully, including one child undergoing chemotherapy for medulloblastoma. The clinical presentation of this child who developed parainfluenza virus type 3 meningitis is described, and the cases of five other individuals with parainfluenza virus type 3 isolated from the CSF are briefly reviewed. The paramyxovirus parainfluenza type 3, in addition to mumps virus, may be considered capable of infecting the central nervous system.

Analysis of the nucleoside moiety of cobalamin and cobalamin analogues using gas chromatography-mass spectrometry.

Existing techniques for identification of cobalamin and cobalamin analogues generally use the intact molecule during characterization with somewhat ambiguous results. In this study a method is described for the identification of the nucleoside in the lower axial ligand of cobalamin and a variety of naturally occurring cobalamin analogues that differ from cobalamin in the base that is present in the nucleoside. Cobalamin and cobalamin analogues were isolated from biological samples by affinity chromatography using R-protein-Sepharose columns. The nucleosides of the lower axial ligand were then hydrolyzed and isolated by column chromatography using a mixed bed column. Nucleosides were oxidized with periodate and reduced with borohydride. After reisolation, the t-butyldimethylsilyl derivatives were prepared and analyzed using gas chromatography/mass spectrometry with selected ion monitoring. A stable isotope internal standard of cobalamin was biosynthetically produced and used to quantitate cobalamin in rabbit kidney. Cobalamin analogues were also shown to be present in rabbit kidney, but they contain the 5,6-dimethylbenzimidazole nucleoside (alpha-ribazole) in the lower axial ligand, indicating that these analogues differ from cobalamin in the corrin ring region of the molecule.

Development, use, and availability of a job exposure matrix based on national occupational hazard survey data.

A job exposure matrix has been developed based on potential exposure data collected during the 1972-1974 National Occupational Hazard Survey (NOHS). The survey sample was representative of all U.S. non-agricultural businesses covered under the Occupational Safety and Health Act of 1970 and employing eight or more employees. Potential worker exposure to all chemical, physical, or biological agents was recorded during the field survey if certain minimum guidelines for exposure were met. The job exposure matrix (JEM) itself is a computerized database that assists the user in determining potential chemical or physical exposures in occupational settings. We describe the structure and possible uses of the job exposure matrix. In one example, potential occupational exposures to elemental lead were grouped by industry and occupation. In a second example, the matrix was used to determine exposure classifications in a hypothetical case-control study. Present availability as well as future enhancements of the job exposure matrix are described.

National estimates of occupational exposure to animal bladder tumorigens.

A prevention program for occupational bladder cancer should be based on an estimate of the number of workers previously and currently exposed to bladder carcinogens. The National Occupational Exposure Survey (NOES), which identified potential occupational exposures in approximately 5000 private sector firms in 1981 to 1983, is the best available source for recent hazard estimates; the National Occupational Hazard Survey (NOHS), conducted in 1972 and 1974, for past exposure estimates. The National Institute for Occupational Safety and Health Registry of Toxic Effects of Chemical Substances (RTECS) identified nearly 200 substances associated with animal bladder tumors. From NOES and NOHS, the numbers of workers with full time (greater than or equal to 4 hours/day) or any potential occupational exposure were estimated for the United States. About 60,000 workers were potentially exposed in the early 1970s and about 700,000 in the early 1980s on a full-time basis to the compounds on the RTECS list also appearing in NOES, and about 1.8 million workers in the 1970s and almost 3.5 million in the 1980s had some occupational exposure. Because matches were not found for many compounds and because NOES covers only part of the US work force, these are probably underestimates. The estimates for the number of exposed workers do not imply that these workers all have increased risk of developing bladder cancer, because some animal tumorigens may not be human carcinogens and our estimates are based on potential rather than measured exposures. The risk would depend on the potency, duration, and intensity of the actual exposures. Nevertheless these estimates are useful in estimating the approximate magnitude of the potential occupational exposure to animal bladder tumorigens.

Enhanced infectivity of bluetongue virus in cell culture by centrifugation.

The effects of centrifugation of the infection of cell culture with bluetongue virus (BTV) were investigated. Baby hamster kidney cells were infected with BTV with or without centrifugation. Viral antigen was detected by immunofluorescence at 24 h in both centrifuged and noncentrifuged cultures. However, after 24 h of infection, the production of PFU in centrifuged cell cultures was 10- to 20-fold greater than that seen in cultures not centrifuged. In addition, centrifugation enhanced the direct detection of PFU from blood samples collected from a sheep experimentally infected with BTV.

In vitro neutralization of antigenic variants of bluetongue virus is related to in vivo protection.

Neutralization resistant variants of bluetongue virus serotype 10 were selected with a neutralizing monoclonal antibody. Three variants were selected for further characterization. One of these variants was completely resistant to neutralization, while the other two variants showed intermediate resistance to neutralization as compared to the parent virus. The completely resistant variant failed to bind the selecting monoclonal antibody as determined by immune precipitation and enzyme linked immunosorbent assay; whereas, the other two variants bound antibody to a similar extent as the parent virus as determined by these tests. The ability of the variants to bind monoclonal antibody correlated with passive protection in the newborn mouse model. These results indicate that the ability of the virus to bind antibody is directly related to in vivo protection and that in vitro neutralization and in vivo protection are also related.