Plasma protective effect on red blood cells exposed to mechanical stress

Hemodilution with plasma expanders is a widely applied practice during extracorporeal circulation and hemodialysis. Despite the immediate beneficial effects of hemodilution, such as reduction of blood viscosity and red blood cell (RBC) aggregation, elevation of blood flow in the microcirculation, etc., the dilution of plasma may cause some unfavorable effects on RBCs, amplifying the mechanical damage caused by circulatory assist devices. The authors investigated the effect of partial and total replacement of plasma on susceptibility of human and bovine RBCs to mechanical stress in vitro. Hemolysis was measured after the exposure of RBCs suspended in different media to similar mechanical stress. Experiments were performed at room temperature with control of osmolality and viscosity of the suspension media. The lowest hemolysis was obtained for RBCs suspended in serum, plasma, and albumin solutions. Hemolysis in PBS and Dextran suspensions was more than three times higher than that in plasma (p < 0.001). The protective effect depended upon protein concentration. Human RBCs were found to be significantly more sensitive to mechanical stress than bovine RBCs in all investigated suspension media (p < 0.005). Human RBCs from men suspended in plasma were found to be significantly (p < 0.05) more fragile than RBCs from women. The presence of even small amounts of plasma (such as 25%) in the suspension media significantly (p < 0.001) decreased hemolysis. However, a 30% replacement of plasma with PBS or Dextran solutions caused a statistically significant (p < 0.001) increase in mechanical hemolysis. This suggests that a decrease in the concentration of plasma proteins due to hemodilution may elevate blood damage during extracorporeal circulation and hemodialysis.     

Bacterial cross-contamination of meat during liquid nitrogen immersion freezing

Prerigor beef carcass surface tissue (BCT) was used to simulate lamb carcasses on a processing line with a 15-min liquid nitrogen (LN) immersion freezing step, and the potential for the dissemination of bacteria during freezing was examined. Streptomycin-resistant strains of Listeria innocua and Escherichia coli O157:H7 spiked into a fecal slurry were inoculated onto BCT pieces that were introduced into the freezing process to represent contaminated carcasses. Following this introduction, subsequently frozen uninoculated BCT, LN, and LN containers were examined for the inoculated organisms. In the first study, BCT samples were inoculated with ca. 7 log CFU/cm2 of both L. innocua and E. coli O157:H7, spray washed with water and frozen, distributed among uninoculated BCT, in LN for 15 min. In two separate trials, L. innocua was recovered by enrichment from all uninoculated BCT and LN samples. E. coli O157:H7 was also recovered from uninoculated BCT and LN, but this cross-contamination was more sporadic. Both species were recovered from the LN container following freezing. Attempts to enumerate cross-contaminating bacteria in the second trial indicated that contaminating levels were low (< 1.0 CFU/cm2 BCT). In a second study, a 2.0% lactic acid spray wash was used to reduce further the numbers of L. innocua introduced into the freezing system and resulted in fewer positive samples, although this organism was still recovered from many uninoculated BCT samples. When either bacterium was inoculated at lower initial levels (1.35 to 1.77 log CFU/cm2) and BCT was water or 2.0% lactic acid spray washed prior to freezing, neither L. innocua nor E. coli O157:H7 was recoverable by enrichment from uninoculated BCT, LN, or from the freezing container. Results demonstrate that bacterial cross-contamination of meat during LN immersion freezing can occur but indicate that the use of good sanitation practices and product with low microbial numbers can limit this occurrence.     

The effect of trehalose on human lung fibroblasts stored in euro-collins and low potassium dextran solutions

BACKGROUND: High asthma morbidity has been reported in certain demographic groups in geographical areas of low socioeconomic status (SES). AIM AND METHODS: We tested for an ecological association between the gender of people being frequently hospitalised for asthma and the SES of the area in which they lived, using a cross-sectional study. RESULTS: Women represented 75% of the readmission population at The Queen Elizabeth Hospital (TQEH--low SES hospital) and 55% at Modbury Hospital (moderate-high SES hospital). Women at TQEH were significantly more likely to have one readmission within 12 months and over 30 times more likely to have two or more readmissions than women at Modbury Hospital. CONCLUSION: The ecological association observed in this study needs to be confirmed elsewhere in Australia.     

In vitro effect of dextran-benzene-tetra-carboxylate hemoglobin on human blood rheological properties

While conducting pharmacological investigations into oxygen carriers, it is important to study the in vitro and in vivo rheological behavior of blood cells in the presence of such preparations. With regard to the original nature of human hemoglobin bound to benzene tetracarboxylate substituted dextran (Dex-BTC-Hb), it seemed necessary to study its rheological effect in a simulated in vitro hemorrhagic shock compensated by a blood substitute. The viscosity of substitutes was determined as well as several rheological parameters after 0, 3 and 6 hours incubation periods of red blood cells with substitutes: viscosity of blood-substitute mixtures at different levels of plasma substitution erythrocyte aggregation of blood-substitute mixtures by determining the velocity of rouleau formation and the cohesion of rouleau network. This work yielded several observations: The viscosity of Dex-BTC-Hb was slightly higher than those of solutions of native Hb, Dex-BTC T10, Dextran 40 (Plasmacair, modified fluid gelatin (Plasmion and hydroxyethyl starch 200 (Elohes). The substitution of a blood volume with Dex-BTC-Hb, corresponding to a compensated 45% hemorrhagic shock, slightly increased the viscosity of hemodiluted blood as compared to other substitutes. In the presence of Dex-BTC-Hb, the aggregation of erythrocytes appears to be increased as compared to standard solutions. Yet, the effect was close to that of Plasmion or Elohes.     

An in vitro model of human red blood cell production from hematopoietic progenitor cells

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.     

The effect of calf lymph and bovine red blood cells on in vitro cultivation of Theileria parva-infected lymphoid cells

Calf lymph introduced to cultures of Theileria parva-infected lymphoid cells caused a stimulation of both cells and parasites resulting in the formation of rapidly dividing cells and the developmental stages of the parasite. The presence of piroplasms was confirmed when washed bovine red blood corpuscles were added to the culture     

Lithium-magnesium relationship in red blood cells during lithium prophylaxis

The authors investigated lithium and magnesium levels in serum and RBC in 30 patients with affective disorders during lithium prophylaxis. No differences in magnesium levels were found in relation to diagnosis (unipolar, bipolar), sex and age. Mean RBC lithium index was higher in females than in males, particularly in bipolar patients. Magnesium levels in erythrocytes showed a significant negative correlation with RBC lithium and RBC lithium index. Serum magnesium levels did not correlate of lithium concentrations. It was concluded that RBC magnesium may play a role in lithium penetration of RBC and this was discussed in relation to clinical and pathogenetic factors.     

Frequency of nucleated red blood cells in maternal blood during the different gestational ages

PURPOSE: To study the course and visual outcome of Vogt-Koyanagi-Harada (VKH) syndrome in children. METHODS: The medical records of 97 consecutive patients with VKH syndrome were reviewed. Patients had been seen at King Khaled Eye Specialist Hospital in Riyadh, Saudi Arabia. The records were divided into two groups: those in whom the onset of the disease occurred at the age of 14 years or younger (pediatric group) and those in whom the disease manifested after the age of 14 years (adult group). RESULTS: Eight (61%) of the 13 children required cataract surgery compared to 14(17%) of 84 adult patients. Despite medical and surgical therapy, eight (61%) of the pediatric group had a final visual acuity of 20/200 or worse compared with 22 (26%) of the 84 adult patients (p=0.0103). CONCLUSION: The course of early-onset VKH (before the age of 14 years) appears to be aggressive. The ocular complications are more severe in children than in adults, leading to rapid deterioration in vision. Despite therapy, the final visual outcome of VKH in children is poor.     

Temperature transitions of protein properties in human red blood cells

Human red blood cells (RBC) undergo a sudden change from blocking to passing through 1.3 +/- 0.2-micrometer micropipettes at a transition temperature (Tc) of 36.4 degrees C. For resealed RBC ghosts this transition occurs at 28.3 degrees C (Tg). These findings are attributed to an elastomeric transition of hemoglobin from being gel-like to a fluid and to an elastomeric transition of membrane proteins such as spectrin. Spectrin shows a uniform distribution along the aspirated RBC tongue above Tg in contrast to the linear gradient below Tg.     

Evidence for the presence of aquaporin-3 in human red blood cells

A facilitated diffusion for glycerol is present in human erythrocytes. Glycerol transporters identified to date belong to the Major Intrinsic Protein (MIP) family of integral membrane proteins, and one of them, aquaporin-3 (AQP3), has been characterized in mammals. Using an antibody raised against a peptide corresponding to the rat AQP3 carboxyl terminus, we examined the presence of AQP3 in normal and Colton-null (aquaporin-1 (AQP1)-deficient) human erythrocytes. Three immunoreactive bands were detected on immunoblots of both normal and Colton-null red cells, very similar to the bands revealed in rat kidney, a material in which AQP3 has been extensively studied. By immunofluorescence, anti-AQP3 antibodies stained the plasma membranes of both normal and Colton-null erythrocytes. Glycerol transport was measured on intact erythrocytes by stopped-flow light scattering and on one-step pink ghosts by a rapid filtration technique. Glycerol permeability values, similar in both cell types, suggest that AQP1 does not represent the major path for glycerol movement across red blood cell membranes. Furthermore, pharmacological studies showed that Colton-null red cells remain sensitive to water and glycerol flux inhibitors, supporting the idea that another proteinaceous path, probably AQP3, mediates most of the glycerol movements across red cell membranes and represents part of the residual water transport activity found in AQP1-deficient red cells.     

In vitro and in vivo protective effect of honokiol on rat liver from peroxidative injury

Honokiol, a compound extracted from the Chinese medicinal herb Magnolia officinalis, has a strong antioxidant effect on the inhibition of lipid peroxidation in rat heart mitochondria. To investigate the protective effect of honokiol on hepatocytes from peroxidative injury, oxygen consumption and malondialdehyde formation for in vitro iron-induced lipid peroxidation were assayed, and the mitochondrial respiratory function for in vivo ischemia-reperfusion injury were evaluated in rat liver, respectively. The inhibitory effect of honokiol on oxygen consumption and malondialdehyde formation during iron-induced lipid peroxidation in liver mitochondria showed obvious dose-dependent responses with a concentration of 50% inhibition being 2.3 x 10(-7) M and 4.96 x 10(-7) M, respectively, that is, 550 times and 680 times more potent than alpha-tocopherol, respectively. When rat livers were introduced with ischemia 60 min followed by reperfusion for 60 min, and then pretreated with honokiol (10 micrograms/kg BW), the mitochondrial respiratory control ratio (the quotient of the respiration rate of State 3 to that of State 4) and ADP/O ratio from the honokiol-treated livers were significantly higher than those of non-treated livers during reperfusion. The dose-dependent protective effect of honokiol on ischemia-reperfusion injury was 10 microgram-100 micrograms/Kg body weight. We conclude that honokiol is a strong antioxidant and shed insight into clinical implications for protection of hepatocytes from ischemia-reperfusion injury.     

In vitro culture of human peritoneal fluid cells

The author has studied the behaviour of cells from human ascitic fluid in long-term culture (5-6 months). Three cellular types are described with different morphological features, namely the cellular shape, the fashion in which the cell spreads on the glass, the nucleocytoplasmic ratio, the chromatin appearance and the abundance of mitochondria. The three cellular types can phagocytose, but each one in a different way. The first one phagocytoses exclusively erythrocytes 'by contact' without emission of pseudopods; the second one phagocytoses degenerating nucleated cells in the same way as the first; the third type phagocytoses degenerating nucleated cells by emission of long pseudopods. The origin of these three cellular types is discussed; it is felt that they are transformed mesothelial cells. According to this study, it cannot be excluded, especially for the second and the third type, that they are histiocytes coming from serous membranes. The life in vitro of the three cellular types is depending upon the composition of nourishing medium. Cells can divide by mitosis only during the first 10 - 15 days of culture (mitotic index 0.1-3.0(0/00). Nuclear amitosis, nucleolus expulsion into cytoplasm and cytoplasmatic DNA synthesis can be observed in healthy cells.     

In vitro effect of garlic powder extract on lipid content in normal and atherosclerotic human aortic cells

In the present study, the mechanism of the in vitro effect of garlic powder extract (GPE) on lipid content of cultured human aortic cells was investigated. The addition of GPE abolished atherogenic blood serum-induced accumulation of free cholesterol, triglycerides, and cholesteryl esters in smooth muscle cells derived from uninvolved (normal) intima. In cells isolated from atherosclerotic plaque, GPE lowered these lipids. GPE inhibited lipid synthesis both in normal and atherosclerotic cells. It inhibited acyl-CoA:cholesterol acyltransferase activity that participates in the cholesteryl ester formation and stimulated cholesteryl ester hydrolase that degrades cholesteryl esters. This may explain the lipid reduction caused by GPE in atherosclerotic cells. GPE inhibited the uptake of modified low density lipoprotein and degradation of lipoprotein-derived cholesteryl esters, thus considerably reducing the intracellular accumulation of cholesteryl esters. This suggests the mechanism responsible for the prevention of lipid accumulation in aortic cells caused by atherogenic blood serum.     

A histochemical study of the distribution of dextran 500 in human corneas during organ culture

Complications arising from limb-lengthening procedures such as muscle contracture, axial malalignment of the bone and traction injuries to the nerves and vessels, are often severe. Often complications arise from the build-up of forces in the biological tissues which are resisting lengthening. Little is known about the origin and magnitude of these forces, although three studies have identified the regenerate (new bone tissue) as the dominant resisting tissue. This study describes the development of a method to examine these forces. It employs load measurement devices in the structural columns of Ilizarov fixators which measure the compressive load on the frame exerted by the biological tissues. The distribution of this load between the columns of the frame, in conjunction with a transverse radiograph of the limb at the regenerate site, is used to examine the origin of the resisting force. Accuracy was determined by a laboratory simulation which found the predicted position of the force to be within 5 mm of the actual position in all four cases tested. Mean error in the total measured force was 2 N (SD, 1 N). A pilot study on a patient undergoing a 60 mm femoral lengthening revealed a peak force of 717 N originating in the Vastus Lateralis or the illiotibial tract. Negligible contribution to resistance was provided by the regenerate, contrary to that found with other studies.     

The effect of lead on intracellular water content of human red blood cells. Part 1. An experiment in vitro (author's transl)

We observed in vitro experiment that lead increased osmotic resistance of normal human erythrocytes and decreased mean corpuscular volume and intracellular potassium. Though the mechanism of the increased osmotic resistance of erythrocytes caused by lead has not yet been completely clarified, the following hypothesis could be accepted. Lead causes leakage of water from erythrocytes, which means that more water can enter the cell before hemolysis occurs. But there has been no report of direct measurement of the intracellular water content of erythrocytes treated with lead. This paper tried to clarify the relationship between intracellular water and osmotic resistance of lead treated erythrocytes in vitro experiment. The results were: 0.05 mumol/ml of lead increases osmotic resistance, trapped water content and plasma water content of normal human blood and decreases intracellular water content after incubation for 2 hours at 37 degrees C.     

Frequencies of fetal nucleated red blood cells in maternal blood during different stages of gestation

High molecular weight forms of tyrosinase have been found to be expressed during spontaneous remelanization of the amelanotic B-16 melanoma cells in culture as well as in melanotic tumors formed from amelanotic melanoma cells grown in C57BL/6J mice. Overnight extraction of the crude melanosomal fractions from such tumors and cultured melanoma cells reveal the presence of an additional DOPA-MBTH positive band well below the stacking gel. This band has been found to be alpha-PEP7 (antibody specific for tyrosinase) positive and alpha-PEP1 (antibody specific for TRP-1) negative on Western blot analysis. Heat treatment at 60 degrees C for 60 min results in the loss of this band and considerable loss of activity of the melanosomal extract. Trypsin treatment of these melanosomal extracts resulted in a minor change in the mobility of the high molecular weight band. SDS-PAGE under reduced conditions followed by Western blotting revealed that the high molecular weight band was lost and not detected by alpha-PEP7 or alpha-PEP1. These findings indicate that high molecular weight, heat sensitive and trypsin resistant forms of tyrosinase are transiently expressed in B-16 melanoma cells and tumors that are initiating remelanization following phenotypic drift towards the amelanotic state.     

In vitro study of pesticide hematotoxicity in human and rat progenitors

Some pesticides are hematotoxic and cause aplastic anemia, agranulocytosis, neutropenia, and thrombopenia. In order to evaluate hematopoietic progenitor cultures in the exploration of pesticide hematotoxicity, human and rat colony-forming unit-granulocyte and macrophage (CFU-GM) were cultured in the presence of different concentrations of pesticides, known to be either hematotoxic or innocuous for blood cells. The results were compared to the control culture of the same sample. Four insecticides (lindane, azinphos, mevinphos, parathion methyl), three herbicides, (2,4,5, T, bromacil, MCPA), and two fungicides (fosethyl-aluminum, DNOC) were tested and exhibited the following data: 1) Pesticides, known to be hematotoxic, inhibited the development of progenitors. Different phenomena were observed and suggested different mechanisms: cell destruction, block in mitosis, decrease or delay in mitosis. 2) Difference in sensitivity to molecules between human and rat progenitors was observed. Human progenitors were more sensitive to pesticides, except for 2,4,5 T.     

In vitro generation of human dendritic cells and cell therapy

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.