Cell Lysis Products Research Articles

Hexahydro-1,3,5-trinitro-1,3,5-triazine Transformation by Biologically Reduced Ferrihydrite: Evolution of Fe Mineralogy, Surface Area, and Reaction Rates

Microbial respiration of Fe(III) oxides has been shown to produce reduced Fe phases that are capable of transforming a variety of oxidized contaminants. Little data, however, are available on how these Fe phases evolve over time and how this evolution may affect their ability to reduce contaminants. Here,the evolution and reactivity of biologically reduced ferrihydrite were monitored over a period of 14 months. Solids were collected from a culture of Geobacter metallireducens (GS-15) thatwas incubated with ferrihydrite (as the electron acceptor) for 0, 7, 10, 20, 75, and 400 days. Mineralogical composition and surface area of the biologically reduced solids were characterized using Mössbauer spectroscopy, X-ray diffraction, and BET with N2 adsorption. By day 10, ferrihydrite began to transform, and a nanoparticle magnetite/maghemite phase, as well as two ferrous phases, was observed. One of the ferrous phases was identified as siderite, whereas the other could not be positively identified. Likely candidates, however, include Fe(OH)2(s) or an adsorbed Fe(II) species. Over the next few months, ferrihydrite was completely reduced and evolved into a mixture containing about 70% magnetite/maghemite, 19% siderite, and 11% of the second Fe(II) phase. The effect of incubation time on the reactivity of the biologically reduced solids was evaluated by measuring the kinetics of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transformation. The only products observed were the three reduced nitroso products. Rate coefficients (k) for RDX transformation were dramatically influenced by incubation time with half-lives of about 1 month observed in the presence of solids incubated for 10 and 20 days, 3 months with solids incubated for 75 days, and negligible removal with solids incubated for 400 days. The loss of reactivity was not directly correlated to any one mineralogical variable but may be due to particle size or surface chemistry changes in the reactive Fe phase or to cell die-off and the accumulation of cell lysis products after consumption of the electron acceptor. The dramatic effect of incubation time on the rate of RDX removal highlights a potential limitation of studying complex systems, as we have here, in batch reactors and suggests that incubation time is an important variable to consider when measuring and comparing rates of contaminant reduction.

Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds

Anaerobic chemostats fed on glucose (≈10 g chemical oxygen demand (COD)/L) were used to investigate the effects of toxicity on soluble microbial product (SMP) formation. Addition of the toxic compounds chloroform and chromium increased the net accumulation of SMP, despite reducing the percentage of SMP in the effluent due to the overwhelming production of volatile fatty acids (VFAs). In the reactor spiked with chloroform the normalized accumulation of SMP (SMP/ S o) increased from 2% to 8%, whereas in the reactor spiked with Cr (VI) the SMP/ S o ratio reached as high as 20% after the spike, and in both cases SMP net accumulation was proportional to the concentration of toxicant. After the chloroform and chromium spikes biomass seemed to produce more extra cellular polymeric substances (EPS) suggesting that this might have helped them to cope with the stress. Chromatography results indicate that some of the high MW compounds present in the SMP might have been due to EPS release into the bulk solution, and that other compounds, probably released as a result of cell lysis, were also present. Hydrolysis of EPS did not seem to contribute to SMP accumulation in the presence of toxic compounds, and DNA analysis suggested that cell lysis products was an important contribution to SMP accumulation, in the presence of chromium.

Microbial composition and structure of a multispecies biofilm from a trickle‐bed reactor used for the removal of volatile aromatic hydrocarbons from a waste gas

AbstractThe microbial composition and structure of a multispecies biofilm of a laboratory‐scale trickle‐bed bioreactor for the treatment of waste gas was examined. The model pollutant was a volatile organic compound‐mixture of polyalkylated benzenes called Solvesso 100®. Fluorescent in‐situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) were applied. Two new Solvesso 100®‐degrading Pseudomonas sp strains were isolated from the multispecies biofilm. Corresponding isolate‐specific oligonucleotide probes were designed and applied successfully. A major finding was that the fraction of Solvesso 100®‐degrading bacteria in the biofilm was low (about 3–6% during long‐term operation). The majority of the active cells were saprophytes which utilized intermediates and cell lysis products. The measured fraction of extracellular polymeric substances of the mature biofilm was 89–93% of the total biomass. The CLSM examinations of a 3‐days‐old approx 10 µm thick biofilm revealed highly heterogeneous structures with distinguished three‐dimensional matrix‐enclosed microcolony bodies spread across the substratum surface. The 28‐days‐old 80–960 µm thick biofilm exhibited voids, cell‐free channels, and pores of variable sizes. In both cases, an even distribution of active cells and pollutant‐degrading bacteria throughout the biofilm cross‐section as well as through the biofilm depth was observed. Copyright © 2003 Society of Chemical Industry

Intensified chemotherapy supported by DMSO-free peripheral blood progenitor cells in breast cancer patients

The majority of high-dose chemotherapy (HDC)-related complications results from bone marrow aplasia, but the graft infusion per se may cause adverse reactions due to the injection of both dimethyl sulfoxide (DMSO) and cell lysis products. We evaluated the feasibility of a two-step chemotherapy regimen with peripheral blood progenitor cell (PBPC) support in association with a novel procedure to remove DMSO and products of cell lysis from the cryopreserved cells. Stage III and IV breast cancer patients received induction chemotherapy with three cycles of CEF (cyclophosphamide 600 mg/m2, epirubicin 100 mg/m2, 5-fluorouracil 600 mg/m2) followed by three cycles of HDC consisting of escalating doses of cyclophosphamide (dose range 1200 3000 mg/m2) and carboplatin (dose range 600-1000 mg/m2), supported by DMSO-free PBPC reinfusion. DMSO was removed by a washing/enzymatic digestion procedure. Twenty patients received induction chemotherapy and eighteen completed the entire chemotherapy program; a total of fifty-four cycles of HDC were administered. Dose limiting toxicity of HDC was long-lasting grade 4 neutropenia associated with documented infection. The maximum tolerated dose (MTD) was cyclophosphamide 3000 mg/m2 and carboplatin 600 mg/m2. No side effects related to PBPC reinfusion were observed. The proposed two-step chemotherapy regimen, associated with a novel washing/enzymatic digestion procedure, is feasible in advanced breast cancer patients in the absence of complications related to the specific toxicity of PBPC reinfusion.

Significance of viral lysis and flagellate grazing as factors controlling bacterioplankton production in a eutrophic lake.

The effects of viral lysis and heterotrophic nanoflagellate (HNF) grazing on bacterial mortality were estimated in a eutrophic lake (Lake Plusssee in northern Germany) which was separated by a steep temperature and oxygen gradient into a warm and oxic epilimnion and a cold and anoxic hypolimnion. Two transmission electron microscopy-based methods (whole-cell examination and thin sections) were used to determine the frequency of visibly infected cells, and a model was used to estimate bacterial mortality due to viral lysis. Examination of thin sections also showed that between 20.2 and 29.2% (average, 26.1%) of the bacterial cells were empty (ghosts) and thus could not contribute to viral production. The most important finding was that the mechanism for regulating bacterial production shifted with depth from grazing control in the epilimnion to control due to viral lysis in the hypolimnion. We estimated that in the epilimnion viral lysis accounted on average for 8.4 to 41.8% of the summed mortality (calculated by determining the sum of the mortalities due to lysis and grazing), compared to 51.3 to 91.0% of the summed mortality in the metalimninon and 88.5 to 94.2% of the summed mortality in the hypolimnion. Estimates of summed mortality values indicated that bacterial production was controlled completely or almost completely in the epilimnion (summed mortality, 66.6 to 128.5%) and the hypolimnion (summed mortality, 43.4 to 103.3%), whereas in the metalimnion viral lysis and HNF grazing were not sufficient to control bacterial production (summed mortality, 22.4 to 56.7%). The estimated contribution of organic matter released by viral lysis of cells into the pool of dissolved organic matter (DOM) was low; however, since cell lysis products are very likely labile compared to the bulk DOM, they might stimulate bacterial production. The high mortality of bacterioplankton due to viral lysis in anoxic water indicates that a significant portion of bacterial production in the metalimnion and hypolimnion is cycled in the bacterium-virus-DOM loop. This finding has major implications for the fate and cycling of organic nutrients in lakes.

Comparison between Bone Marrow and G‐CSF‐Mobilized Peripheral Blood Allografts Undergoing Clinical Scale CD34 + Cell Selection

Allogeneic transplantation of selected CD34+ cells, rather than conventional transplantation of bone marrow (BM) harvest or peripheral blood (PB) leukapheresis products, has the advantage of reducing volume, facilitating storage and decreasing the amount of dimethylsulfoxide (DMSO) and cell lysis products, as well as reducing the number of T-lymphocytes responsible for graft-versus-host disease (GVHD). Using biotinavidin immunoaffinity columns (Ceprate SC system, CellPro; Bothell, WA), CD34+ cells were selected from each of 20 allografts (12 G-CSF-mobilized PB and 8 BM) collected from 14 HLA-identical normal healthy donors for transplantation. After the clinical-scale selection, the median concentration of CD34+ cells was 44.6% (range, 13% to 91%) in BM and 50.4% (range, 15% to 77%) in PB. Whereas 75% of the PB allografts had a CD34+ cell yield of more than 65%, only 37.5% of the BM allografts achieved such a yield, p < 0.01. The number of T-lymphocytes in the selected CD34+ cell allografts was reduced by two to three logs from a median of 4.2 x 10(9) to 7.8 x 10(5) CD3+ cells. The enrichment in CD34+ cells was 240-fold (range, 24- to 382-fold) in PB versus only 34-fold (range, 14- to 108-fold) in BM. Also, the enrichment in clonogenic cells was significantly more in PB (median value of 38.6-fold) than in BM (median value of 19.2-fold) and more in allografts from younger (< 50 years old) rather than older (> or = 50 years old) adult donors. A correlation was found between the percentage of CD34 or CD3+ cells before and after selection (r = 0.58 or r = 0.60, respectively, p < 0.05). Selective enrichment of the colony forming units-granulocyte-macrophage (CFU-GM) was found in all 20 allografts. The progenitor cell recovery after freezing and thawing was similar in BM and PB allografts, with a mean of about 60% for the CFU-GM and BFU-E. In the same six donors, the CD34+ cell yield was significantly more in the PB after mobilization (median 78.5%, range 50% to 90%) than in the BM before mobilization (median 41.5%, range 25% to 87%), p < 0.01. Ten patients with hematologic malignancies have been allotransplanted with 14 of the 20 selected CD34+ cells either combined BM + PB (n = 4) or single (n = 6) grafts. Seven patients did not develop acute GVHD, and only two patients developed > or = grade II GVHD, one of whom developed only grade II GVHD that resolved after brief treatment with corticosteriods. Only one patient showed chronic GVHD (skin and liver). The low incidence and severity of GVHD seen in the present study (only 30%) could be due to the two- to three-log reduction of T-lymphocytes in the selected CD34+ cell allotransplants. All 10 patients had stable hematological recovery, and seven had full donor hematopoiesis. In conclusion, G-CSF-mobilized PB leukapheresis products undergoing selection of CD34+ cells have a greater yield and enrichment of progenitor cells than BM harvests collected from HLA-identical normal healthy donors for allogeneic transplantation. The low incidence and severity of both acute and chronic GVHD (30%) seen in the present study are very encouraging.

A more potent bystander cytocidal effect elicited by tumor cells expressing the herpes simplex virus-thymidine kinase gene than by fibroblast virus-producer cells in vitro.

Retrovirus-mediated herpes simplex virus-thymidine kinase (HSV-tk) gene therapy is a promising approach in the treatment of brain tumors. Previous in vitro and in vivo studies have demonstrated a bystander effect in which nonmodified tumor cells in proximity to HSV-tk-modified tumor cells are killed with the modified cells in the presence of ganciclovir. In the present study the authors assessed the contribution of infectious HSV-tk retrovirus made by producer cells to the bystander cytocidal effect in tissue culture using Walker 256 rat breast carcinosarcoma cells, which represent an established model for carcinomatous meningitis. The authors observed ganciclovir-dependent growth inhibition even when only one HSV-tk-positive Walker cell was mixed with 1000 HSV-tk-negative Walker cells and showed that the bystander cytocidal effect is not mediated by toxic cell lysis products. Walker cells engineered to produce HSV-tk retrovirus with titers ranging from 10(3) to 10(5) colony-forming units/ml exert no greater cytocidal effect than nonviral producer HSV-tk-positive Walker cells in vitro. Murine fibroblast-producer cells with viral titers ranging from 10(6) to 10(7) colony-forming units/ml exerted a stronger cytocidal effect than nonviral producer HSV-tk-positive murine fibroblasts. Despite the high viral titers of fibroblast producer cells, HSV-tk-modified Walker cells performed better than fibroblast producer cells in their cytotoxic effect on wild-type Walker tumor cells. Given that HSV-tk-modified tumor cells can become ganciclovir resistant, we tested gamma-irradiation as a means to overcome resistance. Lethal gamma-irradiation of the HSV-tk-positive Walker cells did not abolish their bystander effect on Walker HSV-tk-negative cells. One can infer from these results that HSV-tk-modified tumor cells, irradiated or not, may be a better alternative to murine fibroblast producer cells in the treatment of central nervous system neoplasia.

Decreased sludge production strategy for domestic wastewater treatment

With new EEC regulations, alternative treatment and disposal techniques of the excess sludge produced by Activated Sludge (AS) wastewater treatment plants have to be performed. In order to reduce the excess sludge produced, experiments have been carried out with a Membrane BioReactor (MBR) to study the maintenance and cryptic growth phenomena of Pseudomonas fluorescens culture taken as a model when grown on a limiting substrate complex medium similar to a synthetic urban wastewater. Experiments with various imposed wasting rates showed that viability and sludge production yield decreased when sludge age increased. Same variations were observed on the cell content ratio protein/polysaccharide by analysis of the cell lysis products released after discontinuous thermal treatment. Biomass growth on these cell lysis products was achieved to characterize cryptic growth and its impact on sludge production yield. Finally, a continuous sludge thermal treatment system was operating with MBR to amplify sludge breakage and consequently biomass growth on the lysis products. With the promising results obtained, this work gives a new outlook on the AS process and leads to the development of processes with control and reduction of sludge production.

Algal bloom episodes and the formation of bituminite and micrinite in hydrocarbon source rocks: evidence from the Devonian and Mississippian, northern Williston Basin, Canada

Organic petrology (incident light microscopy) of Middle Devonian inter-reef laminates and Devonian-Mississippian epicontinental black shales, Williston Basin, Canada, indicates that algal bloom episodes and consequential bacterial activity played a significant role in the accumulation of amorphous, bituminite III-rich organic microfacies. Corpohuminite-like algal akinete cells produced by filamentous algae during algal bloom periods are persistent maceral inclusions within the potential hydrocarbon source rock intervals. These cells (% R o mean range 0.24-0.90) are regarded as positive indicators of stressful palaeoenvironmental conditions. Unicellular Tasmanites and Leiosphaeridia marine alginite and variably degraded alginite remnants (“ghosts”) within the amorphous kerogen may be products of cell lysis, photo-oxidation and microbial alteration; these processes are characteristic of algal bloom periods. Minute (ca. 1 μm) spheroidal and coccoidal bacteria-like macerals are dispersed throughout the bituminite III network, attesting to the importance of microbial activity within the water column and sediment during and after organic matter accumulation. Dispersed granules, laminations and replacement textures of micrinite-like macerals within bituminite III are interpreted as remnants of microbial alteration rather than a residual product of thermal maturation and hydrocarbon generation.

Quality of Blood Prepared for Autotransfusion in Primary Cemented Total Hip Replacement

We have measured levels of contaminants (products of cell lysis and other substances) in blood salvaged from patients undergoing primary cemented total hip replacement. Washing of this blood removed an average of 91.4% of the free haemoglobin mass, reduced white cell lysozyme concentrations by 86% and almost totally eliminated fats and particulate matter. Osmotic fragility was significantly improved by washing although not to control levels.

Clinical Toxicity of Cryopreserved Bone Marrow Graft Infusion

We prospectively evaluated infusion-related toxicities in 82 recipients of autologous bone marrow grafts. The grafts were cryopreserved in 10% dimethylsulfoxide and stored in liquid nitrogen. All grafts were concentrated and buffy-coat cells were collected. Forty-seven grafts were treated ex vivo with 4-hydroperoxycyclophosphamide (4-HC) at 100 micrograms/mL; 26 grafts were further processed using density-gradient separation and treated with 4-HC at 60 micrograms/mL. Nine buffy-coat concentrates were frozen without drug treatment. Before infusion, patients were medicated with mannitol, hydrocortisone, and diphenhydramine. Grafts were rapidly thawed and immediately infused without further manipulation. During the infusions, 33 (70%) recipients of treated buffy-coat, 5 (56%) recipients of untreated buffy-coat, and 6 (23%) recipients of density-gradient separated grafts experienced varying symptoms including nausea, abdominal cramping, and flushing. Forced vital capacities for 83% of the recipients of treated buffy-coat concentrates decreased after the graft infusion; six of these patients complained of dyspnea and one patient experienced an acute episode of respiratory decompensation. Decreased heart rates were observed in 98% of the recipients of treated buffy-coat cells with asymptomatic bradycardia occurring in 45%. Forty-five patients (96%) in this group experienced transient hypertension, with 18 (38%) requiring additional medications within 6 hours after the infusion for control of blood pressure. Similar cardiovascular changes were observed in the recipients of untreated buffy-coat concentrates. One recipient of an untreated buffy-coat concentrate had 2 degrees heart block after the graft infusion. Twenty-three (88%) recipients of density-gradient separated grafts had decreased heart rates and 21 (81%) had increased blood pressure. However, the degrees of change were less than those experienced by the recipients of treated buffy-coat cells (P less than .01). Forced vital capacities were not affected by the infusion of the density-gradient separated grafts. No renal failure or obvious hemolytic episodes occurred for any patient group. Minor to moderate toxicities were associated with cryopreserved graft infusions. Recipients of buffy-coat separated grafts, both treated and untreated, experienced more complications than the recipients of density-gradient separated grafts. These toxicities may relate to the volumes of cryoprotectant and cell lysis products infused, which were less for the more highly purified density-gradient separated grafts.

Clinical toxicity of cryopreserved bone marrow graft infusion

We prospectively evaluated infusion-related toxicities in 82 recipients of autologous bone marrow grafts. The grafts were cryopreserved in 10% dimethylsulfoxide and stored in liquid nitrogen. All grafts were concentrated and buffy-coat cells were collected. Forty-seven grafts were treated ex vivo with 4-hydroperoxycyclophosphamide (4-HC) at 100 micrograms/mL; 26 grafts were further processed using density-gradient separation and treated with 4-HC at 60 micrograms/mL. Nine buffy-coat concentrates were frozen without drug treatment. Before infusion, patients were medicated with mannitol, hydrocortisone, and diphenhydramine. Grafts were rapidly thawed and immediately infused without further manipulation. During the infusions, 33 (70%) recipients of treated buffy-coat, 5 (56%) recipients of untreated buffy-coat, and 6 (23%) recipients of density-gradient separated grafts experienced varying symptoms including nausea, abdominal cramping, and flushing. Forced vital capacities for 83% of the recipients of treated buffy-coat concentrates decreased after the graft infusion; six of these patients complained of dyspnea and one patient experienced an acute episode of respiratory decompensation. Decreased heart rates were observed in 98% of the recipients of treated buffy-coat cells with asymptomatic bradycardia occurring in 45%. Forty-five patients (96%) in this group experienced transient hypertension, with 18 (38%) requiring additional medications within 6 hours after the infusion for control of blood pressure. Similar cardiovascular changes were observed in the recipients of untreated buffy-coat concentrates. One recipient of an untreated buffy-coat concentrate had 2 degrees heart block after the graft infusion. Twenty-three (88%) recipients of density-gradient separated grafts had decreased heart rates and 21 (81%) had increased blood pressure. However, the degrees of change were less than those experienced by the recipients of treated buffy-coat cells (P less than .01). Forced vital capacities were not affected by the infusion of the density-gradient separated grafts. No renal failure or obvious hemolytic episodes occurred for any patient group. Minor to moderate toxicities were associated with cryopreserved graft infusions. Recipients of buffy-coat separated grafts, both treated and untreated, experienced more complications than the recipients of density-gradient separated grafts. These toxicities may relate to the volumes of cryoprotectant and cell lysis products infused, which were less for the more highly purified density-gradient separated grafts.

Hypomagnesaemia: an underdiagnosed interaction between gentamicin and cytotoxic chemotherapy for acute non-lymphoblastic leukaemia.

Hypomagnesaemia occurred in 6 of 11 leukaemic patients who received gentamicin. This problem has been reported relatively infrequently when gentamicin is used alone. Our hypomagnesaemic patients received concurrent courses of gentamicin with cytotoxic therapy significantly more often but it is not clear whether this represents a true drug interaction or an interaction between gentamicin and the products of cell lysis produced by cytotoxic treatment. The incidence of this problem has been underestimated previously because hypocalcaemia was used as an indicator. Renal wasting of magnesium is well documented as the mechanism by which the hypomagnesaemia is sustained but further investigation is required to see whether other factors are involved in its initiation.

14C-labeling of the compounds excreted by phytoplankton for employment as a realistic tracer in secondary productivity measurements

Preparations of the dissolved organic compounds released by photosynthesizing marine phytoplankton have been obtained with(14)carbon activities as high as 1.5 × 10(5) dpm/ml. The radioisotope content of the preparations resides wholly in dissolved organic compounds of low molecular weight (MW<3500), which are uncontaminated by residual(14)C-labeled inorganic carbon. The labeled compunds arise through photosynthetic fixation and do not appear to be products of cell lysis during the incubation or to originate from cell damage during the filtration step employed.

Electron microscopy of “lysis from within” of Escherichia coli by coliphage T2

The fine structure of cells of Escherichia coli B infected with coliphage T2 and the appearance of the products of cell lysis following replication of this phage were examined by electron microscopy. Cells sampled 10 minutes after infection show an extensive alteration of their cytoplasm as a result of phage infection. Granules that are presumed to be ribosomes have been resolved with great clarity in such cells. Hexagonal, electron dense bodies approximately 500 Å in diameter—presumably the condensed phage DNA—are distributed randomly throughout the protoplast of cells sampled 23 minutes after infection. The membrane that surrounds the viral DNA in intact phages was not resolved although a zone greater than 100 Å separated the cellular ribosomes from the condensed phage DNA. The products of lysis are primarily vesicles bounded by one unit membrane. Some of vesicles consist of concentric rings that are composed of triple-layered membranes of a thickness of approximately 75 Å. Granules were not attached to the membranes bounding the vesicles.