Physico-chemical and hygienic property modifications of stainless steel surfaces induced by conditioning with food and detergent.

The effect of repeated conditioning procedures (25 runs), consisting of soiling (milk and meat products) and cleaning steps, on the hygienic status, physico-chemical properties and surface chemical composition of stainless steel (SS) surfaces, was investigated. Five SSs differing in grade and finish were used. Both soiling and surface cleaning/conditioning procedures resulted in a similar increase in the surface contamination with carbon, while the changes in the basic component of the surface free energy depended on the conditioning procedure. The passive film was also affected, the Fe/Cr ratio in particular. The hygienic status was also changed, especially with milk as shown by monitoring the number of residual adhering Bacillus cereus spores after contaminating the surface with spores followed by cleaning. The results show that in food environments, the presence and the nature of conditioning molecules play a major role in the hygienic status of SS surfaces.

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.

[Novel criteria for assessing red blood cell viability in blood banks]. / Nouveaux critères d'évaluation de la viabilité des hématies destinées à la transfusion.

UNLABELLED: In light of recent results on the mechanism of programmed cell death of human red blood cells (RBC), the aim of the present study was to solve the enigma of the rapid clearance of transfused RBCs. MATERIALS AND METHODS: We describe new criteria of RBC viability founded on the use of flow cytometry. They were applied, in association with the classical ones: ATP and hemolysis measurements, to RBCs stored in SAGM medium for 42 days. RESULTS AND CONCLUSIONS: Application of an original method of flow cytometric quantitation of in vitro erythrophagocytosis showed that an important proportion of stored RBCs were phagocytized although the following classical signals for phagocytosis were absent, i.e.: desialylation, phosphatidylserine exposure in the outer leaflet of the RBC membrane, loss of CD47 receptor, an antiphagocytosis signal. In addition, ATP was still present and hemolysis was very low. This enigma was solved by the use of scanning electron microscopy, which showed the disappearance of discocytes and the presence of an important proportion of spheroechinocytes, which are the phagocytable forms of RBCs. The mechanism of this dramatic morphological transformation remains to be elucidated.

Heat treatment of whole milk by the direct joule effect--experimental and numerical approaches to fouling mechanisms.

The development of alternative technologies such as the direct Joule effect to pasteurize and sterilize food products is of great scientific and industrial interest. Our objective was 1) to gain insight into the ability to ensure ultra-high-temperature treatment of milk and 2) to investigate the links among thermal, hydraulic, and electrical phenomena in relation to fouling in a direct Joule effect heater. The ohmic heater [OH; E perpendicular to v (where E is the electrical field and v is the velocity); P (power) = 15 kW] was composed of 5 flat rectangular cells [e (space between the plate and electrode) = 15 mm, w (wall) = 76 mm, and L (length of the plate in plate heat exchanger or electrode) = 246 mm]--3 active cells to ensure heating and 2 (at the extremities) for electrical insulation and the recovery of leakage currents. In the first step, the thermal performance of the OH was investigated vs. the flow regimen [50 < Re (Reynolds number) < 5,000], supplied power (0 < P < 15 kW), and electrical conductivity of fluids (0.1 < sigma(20 degrees C) < 2 S/m) under clean conditions with model fluids. This protocol enabled a global thermal approach (thermal and electrical balance, modeling of the temperature profile of a fluid) and local analysis of the wall temperature of the electrode. An empirical correlation was established to estimate the temperature gradient, T(w)-T(b) (where T(w) is the wall temperature and T(b) is the product temperature) under clean conditions (without fouling) and was used to define operating conditions for pure-volume and direct-resistance heating. In the second step, the ability of OH to ensure the ultra-high-temperature treatment of whole milk was investigated and compared with a plate heat exchanger. Special care was taken to investigate the heat transfer phenomena occurring over a range of temperatures from 105 to 138 degrees C. This temperature range corresponds to the part of the process made critical by protein and mineral fouling. The objectives were 1) to demonstrate the ability of an OH to ensure heat treatment of milk, 2) to study the thermal and hydraulic performance with an increasing power and temperature difference between the inlet and outlet of the OH, 3) to define and validate a criterion to follow heat dissipation efficiency, and 4) to compare the fouling propensity with the different configurations. A heat dissipation coefficient, Rh(CO), was defined and validated to monitor the fouling propensity through global electrical and thermal parameters. Finally, a numerical simulation was developed to analyze heat profiles (wall, deposit, bulk). Because of an increasing Joule effect in the static deposit, the simulation showed how wall overheating would definitively cause fouling to spiral out of control.

Apoptosis in a unicellular eukaryote (Trypanosoma cruzi): implications for the evolutionary origin and role of programmed cell death in the control of cell proliferation, differentiation and survival.

The origin of programmed cell death (PCD) has been linked to the emergence of multicellular organisms. Trypanosoma cruzi, a member of one of the earliest diverging eukaryotes, is a protozoan unicellular parasite that undergoes three major differentiation changes and requires two different hosts. We report that the in vitro differentiation of the proliferating epimastigote stage into the G0/G1 arrested trypomastigote stage is associated with massive epimastigote death that shows the cytoplasmic and nuclear morphological features and DNA fragmentation pattern of apoptosis, the most frequent phenotype of PCD in multicellular organisms. Apoptosis could be accelerated or prevented by modifying culture conditions or cell density, indicating that extracellular signals influenced the epimastigote decision between life and death. Epimastigotes responded to complement-mediated immunological agression by undergoing apoptosis, while undergoing necrosis in response to nonphysiological saponin-mediated damage. PCD may participate into the optimal adaptation of T. cruzi to its different hosts, and the avoidance of a local competition between a G0/G1 arrested stage and its proliferating progenitor. The existence of a regulated cell death programme inducing an apoptotic phenotype in a unicellular eukaryote provides a paradigm for a widespread role for PCD in the control of cell survival, which extends beyond the evolutionary constraints that may be specific to multicellular organisms and raises the question of the origin and nature of the genes involved. Another implication is that PCD induction could represent a target for therapeutic strategies against unicellular pathogens.

Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria.

Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.

Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis.

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.

Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review.

Human red blood cells (RBCs) have a life-span of 120 days in circulation, after which they are phagocytized by resident macrophages. Extensive studies have been undertaken by many investigators in order to elucidate the cellular and molecular mechanisms of the erythrophagocytosis. The critical questions addressed by physiologists, clinicians and biochemists are: 'which of the many traumatic blemishes that appear on the erythrocyte surface as it winds its way through the circulation is the primary signal for clearance of the effete RBC from the circulation?', or 'What is the critical signal that it, and it alone, will activate the resident macrophage to adhere to and engulf it?'. Numerous, and often conflicting, hypotheses have been proposed. Each investigator focusing on but one of the many modifications that afflict the cell surface of the ageing erythrocyte, viz changes in either or both the carbohydrate or peptidic moieties of glycoproteins; abolishment of the pre-existing asymmetry in the lipid bilayer with the exposure of phosphatidylserine residues; or alterations in spectrin, to mention but a few. Many of these investigators also have invoked an intermediary role for auto-immune antibodies that recognise the change(s) on the erythrocyte surface and thereby serve as opsonins as a prelude to the erythrophagocytosis. The objective of the present review is to evaluate the data in support of the various hypotheses, and to submit some of our own recent observations involving the use of flow cytometric procedures that: i) provide evidence that the cell surface sialic acid serves as a determinant of the life-span; ii) characterise the senescent erythrocyte population that is specifically captured and phagocytized by macrophages (utilising the rapid and sensitive procedure we developed for quantification of in vitro erythrophagocytosis); and finally iii) provide evidence for the existence of an alternative pathway that is independent of immunoglobulins.

Fouling of Heat Transfer Surfaces Related to beta-Lactoglobulin Denaturation During Heat Processing of Milk.

Denaturation of beta-lactoglobulin during heating of milk in a plate heat exchanger has been investigated as an important factor in fouling the heat transfer surface. Using, on one hand, data on chemical composition of deposits obtained from biochemical analysis technics and, on the other hand, kinetic data of beta-Iactoglobulin denaturation, the distribution profile of deposits along the surface and the experimental fouling curves can be adequately predicted.

Experimental device and methods for studying milk deposit formation on heat exchange surfaces.

An experimental mini-heat-exchanger was developed in our laboratory.It can be inserted between the heating and holding sections of a pilot milk pasteurizer without modifying its temperature gradient. Representative soil samples from both heating and holding sections were obtained. Changes in soil composition according to running time were studied over a range of 1 min to 4 hours. Previously cleaned 0.4 mm thick stainless steel plates (304L) had to be cut into pieces before analysis. Different kinds of results were obtained. Scanning electron microscopy showed that the deposit was composed of two different types of soil. A dense deposit could be observed near the metal surface after only 1 min of treatment; the thickness of this deposit increased to 15 mum during the process. Over this layer, a spongy deposit only slightly bound to the former, could be observed Lightly fouled plates were analyzed by "in situ techniques" (X.P.S.)Thick deposits were embedded and sectioned for transmission electron microscopy and histochemical analyses.A microanalytical method was applied to deposits after removal and resulted in detection ranges exceeding 10 mg/cm(2).

Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium.

The effect of a high hydrostatic pressure treatment on the Gram-positive Listeria monocytogenes strain Scott A and the Gram-negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions. Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5.6) and sodium phosphate (pH 7.0) suspension buffers. Increasing pressure treatments resulted in an exponential decrease of cell counts. Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments. Progressive morphological changes were evident with the pressure increase. Cell lysis only appeared with the highest pressure treatments. Cell volume was not affected by pressure treatment. A progressive decrease of deltapH (pHin - pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase. A parallel lowering of membrane potentials was measured.

Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells.

Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10(7) cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 microliter mg of protein-1 for the culturable form and 10.96 microliter mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages.

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.