Proteolysis of blood plasma proteins in the stomach in early ontogeny

Proteolysis of milk substitutes containing fibrinogen, casecid and bovine serum albumin as protein components was studied. Artificial feeding of newborn rats has shown high casecid and fibrinogen utilization. Casecid and fibrinogen contained in milk substitutes delayed protein evacuation from the stomach, while bovine serum albumin intensified protein evacuation. Adaptive changes in enzyme-forming gastric function depend on the protein quality.     

Future prospects for artificial blood

Concern about potential infective agents in donated blood has stimulated the recent development of blood substitutes. Chemically cross-linked hemoglobins are already undergoing clinical trials and might soon be ready for routine use. New generations of modified hemoglobin are being prepared to modulate the effects of nitric oxide and oxygen radicals, and artificial red blood cells are also under development.     

Block formation capacity in fleas infected with a plague agent after their feeding with blood substitutes

By feeding plague infected Xenopsylla cheopis fleas with blood substitutes free of blood formed elements blocked fleas were obtained. These fleas are able to transmit plague agent to white mice and cause their mortality.     

Biochemical aspects of the reaction of hemoglobin and NO: implications for Hb-based blood substitutes

The role of Hemoglobin (Hb) on nitric oxide (NO) biology has received much attention. Until recently, the reaction between erythrocytic Hb and NO was generally considered in the context of mechanisms that safely detoxify NO. However, recent insights suggest that properties associated with the red blood cell limit NO-Hb interactions under physiological conditions, and provide some resolution to the question of how NO functions in the presence of blood. Furthermore, Hb-dependent mechanisms that preserve, not destroy NO bioactivity in vivo have also been proposed. The emerging picture suggests that the interplay between NO and erythrocytic Hb is important in regulating the functions of both these molecules in vivo. However, Hb-dependent scavenging and loss of NO function is significant when this heme protein is present outside the red blood cell. This can occur during hemolysis or administration of Hb-based blood substitutes. Scavenging of NO is a significant problem that limits the use of Hb-based blood substitutes in the clinic, and development of Hb molecules that do not efficiently react with NO remains an important area of investigation. In this article, the reactions between NO and erythrocytic Hb or cell-free Hb are described and the effects on NO and Hb function in vivo and development of blood substitutes discussed.     

Prospects for the use of blood substitutes made in the USSR with the expired date of validity]

As revealed, blood substitutes--aminopeptide, caseine hydrolysate, and hydrolysine with the expired date of validity could be used as the basis of nutrient media for the cultivation of bacteria. Bacterial growth proved to be the optimum in addition to the blood substitutes of a 0.25--0.5% of yeast extract.     

Electrophoretic analysis of PEGylated hemoglobin-based blood substitutes

Polyethylene glycol (PEG)-conjugated hemoglobins, a novel class of blood substitutes, were investigated by a combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) coupled with the microspectrophotometric characterization of single bands and the functional analysis of electrophoretically separated fractions. For these intrinsically heterogeneous products, the molecular mass, the size distribution, and the degree of PEGylation are strictly correlated to their side effects and, therefore, are crucial pieces of information to evaluate their safety and efficacy. The PEGylation pattern was shown to strongly depend on the quaternary conformation of hemoglobin during the reaction, and the degree of conjugation was shown to correlate with the oxygen binding properties of the individual electrophoretically separated fractions. Moreover, small but not negligible fractions of underivatized tetramers, known to be responsible for serious side effects, were detected even in preparations with a high average degree of PEGylation. Overall, this approach might be exploited to characterize other products of protein PEGylation, an increasingly relevant technology for the optimization of the pharmacokinetic properties of protein-based drugs.     

Nonencapsulated hemoglobin-based red blood cell substitutes: an updated view.

The characteristics and properties of the nonencapsulated hemoglobin (Hb)-based materials which have been used as substitutes for red blood cells are reviewed. The chemical and physical criteria such as oxygen dissociation, oxygen carrying capacity, antigenicity, viscosity, and circulatory retention time are described. The critical issue of Hb purity assurance testing is examined. The methods of the analyses which should be used to evaluate the lipid purity, endotoxin levels, and protein profile of Hb solutions are described. Additionally, the various approaches to Hb isolation, purification, and production are examined.     

Rheological aspects of mucin-containing solutions and saliva substitutes.

In this study rheological properties of aqueous solutions of mucin, albumin and mucin-albumin have been investigated in search for saliva substitutes. They were compared with commercially available saliva substitutes on the one hand and natural human saliva on the other hand. For the latter a few measurements on saliva are reported here in addition to previously reported measurements done in our laboratory. Proteins absorb at the interface and saliva proteins do so strongly and rapidly. Therefore rheological measurements were carried out on the interface and on the bulk underneath the layer. In both cases the flow curve and the complex viscosity was determined. The results show that specific mucin-albumin solutions were rheologically similar to human whole saliva with respect to both bulk liquid and surface properties. The rheological properties of commercial saliva substitutes were essentially different from those of human saliva. It is concluded that mucin-albumin solutions have good perspectives as saliva substitutes.     

Tissue engineering applications and stem cell approaches to the skin, nerves and blood vessels

Tissue engineering applies the principles of life sciences and engineering to the development of biological substitutes to restore, maintain or improve tissue function. Developments in this multidisciplinary field have yielded advances in the reconstruction of skin, peripheral nerves and blood vessels. In this review we highlight the fundamental principles and challenges of engineering products which can mimic both the structure and function of these tissues in their healthy state. We discuss the recent progress in the field with its implications for revolutionising healthcare in the future.     

Towards the Development of Hemerythrin-Based Blood Substitutes

Hemerythrin is proposed as an alternative to hemoglobin-based blood substitutes. In contrast to hemoglobin, hemerythrin exhibits negligible reactivity towards oxidative and nitrosative stress agents (peroxide, nitric oxide, nitrite). Protocols for attachment of polyethylene glycol and glutaraldehyde cross-linking of Hr are described. These derivatizations appear to have favorable effects on O₂ affinity and autoxidation rates for use in blood substitutes. Based on lessons learned from hemoglobin-based blood substitutes, these derivatizations should also help limit extravasation and antigenicity of a hemerythrin-based blood substitute.     

Microvascular experimental evidence on the relative significance of restoring oxygen carrying capacity vs. blood viscosity in shock resuscitation

The development of volume replacement fluids for resuscitation in hemorrhagic shock comprises oxygen carrying and non carrying fluids. Non oxygen carrying fluids or plasma expanders are used up to the transfusion trigger, and upon reaching this landmark either blood, and possibly in the near future oxygen carrying blood substitutes, are used. An experimental program in hemorrhagic shock using the hamster chamber window model allowed to compare the relative performance of most fluids proposed for shock resuscitation. This model allows investigating simultaneously the microcirculation and systemic reactions, in the awake condition, in a tissue isolated from the environment. Results from this program show that in general plasma expanders such as Ringer's lactate and dextran 70 kDa do not sufficiently restore blood viscosity upon reaching the transfusion trigger, causing microvascular collapse. This is in part restored by a blood transfusion, independently of the oxygen carrying capacity of red blood cells. These results lead to the proposal that effective blood substitutes must be designed to prevent microvascular collapse, manifested in the decrease of functional capillary density. Achievement of this goal, in combination with the increase of oxygen affinity, significantly postpones the need for a blood transfusion, and lowers the total requirement of restoration of intrinsic oxygen carrying capacity.     

Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin

The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolerance induction and outlines in detail current available strategies and alternatives that may allow achieving full-thickness skin replacement and repair.     

Characterization of Hair Follicle Development in Engineered Skin Substitutes

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.     

How safe is blood,really?

Blood is safer than it has ever been, however the progression of transfusion from dangerous intervention to reliable supportive care been non-linear. Disparities resulting from geography, economy, and social class persist. Some risks are known, others are unknown but predictable, and still others may be totally unpredictable. Among the known risks are infectious and immunologic events that can be calculated per unit of blood transfused. These risks vary by component. Among the unknown risks are the potential for emerging pathogens transmitted by blood and for processing or storage lesions to result in short or long-term toxicity. National registries provide some reassurance that transfusion may not affect mortality significantly beyond the first few weeks after administration. Nevertheless, transmission of novel pathogens, repeated allogeneic stimulation, and infusion of cytokines or chemokines may have unrecognized consequences. Blood safety can be effected dramatically with small investment in developing countries. In the developed world, technologies such as pathogen inactivation, antigen camouflage, component substitutes, or cell expansion promise relatively small advances in safety at substantial cost. No strategy guarantees zero-risk.     

Multistep production of bioengineered skin substitutes: Sequential modulation of culture conditions

Many studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboidal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.     

Preparation of unaltered hemoglobin from human placentas for possible use in blood substitutes

Hemoglobin extracted from human placentas could be used as the basis of blood substitutes provided it could be prepared on a large scale with appropriate oxygen-binding properties. Unfortunately, the industrial conditions under which it is extracted, produce hemoglobin with high oxygen affinity and which is no longer influenced by the classical effectors. These characteristics were shown to be caused by a degradation of the alpha-chain brought about by an arginine carboxypeptidase present in the placental tissues and leading to the disappearance of the C-terminal arginine residue. This carboxypeptidase which is released from the tissues during the process of crushing the frozen placentas, degrades the protein during the chromatographic purification procedure. The addition of an inhibitor of this carboxypeptidase (for example, arginine) as soon as the placentas are thawed and during the chromatographic process, makes it possible to obtain placental hemoglobin with oxygen-binding properties quite similar to those of HbA prepared from peripheral venous blood.     

Biochemical and rheodynamic properties of red blood cells crosslinked with glutaraldehyde.

New data on the properties of red blood cells (RBC) cross-linked with glutaraldehyde are presented (see also Biochem. Biophys. Res. Commun., 1988, 156, 970-977). Equilibrium and kinetic data show that by carrying out the fixation procedure in the absence of oxygen but in the presence of allosteric effectors (e.g., stabilizing the low-affinity (T) quaternary state of hemoglobin), it is possible to maintain, at least in part, the biochemical functions of the crosslinked hemoglobin inside the cell. Moreover, we show that the oxygen affinity of fixed red blood cells (RBC) is still modulated, even though to a smaller degree, by the allosteric effector bezafibrate (BZF), which is able to cross the fixed RBC membrane. Membrane filtration experiments indicate that the higher rigidity of fixed RBC alters significantly their rheodynamic properties and show that in order to exploit "engineered" RBC as "blood substitutes," more flexible cross-linking reagents may offer significant advantages.     

Western honey bee (Apis mellifera L.) attraction to commercial pollen substitutes and wildflower pollen in vitro

Many beekeepers feed their western honey bee (Apis mellifera) colonies artificial pollen substitutes to provide colonies with adequate nutrition during times of limited pollen quantity or quality. We provided caged worker bees commercially available pollen substitutes (AP23, MegaBee, UltraBee) and wildflower pollen in a choice-test to determine their relative attraction to/preference for the diets. We measured diet consumption by honey bees and observed honey bee behaviour to evaluate bee preferences for certain diets. The bees interacted with and consumed more wildflower pollen than they did any of the commercially available pollen substitutes. Our data suggest that bees have a strong preference for wildflower pollen over commercially available pollen substitutes.     

Oral mucous membrane flora in patients using saliva substitutes

Salivary substitutes are sometimes valuable for elderly people and radiotherapy patients, and may be used indefinitely. It is possible that this change in the ecology may effect the oral flora. Objective: To analyse the presence of micro‐organisms on oral mucous membranes during use of saliva substitutes. Design: Cross‐over single‐blind study. Setting: Clinic for Maxillofacial Surgery, Malmö University Hospital and Department of Oral Microbiology, Malmö University. Subjects: 19 patients with low salivary secretion who had been radiated for cancer in the head and neck region. Intervention Two saliva substitutes: linseed extract and a carboxymethyl cellulose preparation (Salinum and MAS‐84) were used for 3 week periods. Measurements: Microbial samples taken, processed and analysed. Results: No differences were observed when comparing baseline values with the results after the saliva substitutes and no significant differences between the use of different agents. Conclusion: The study suggests that use of linseed extract and carboxymethyl cellulose preparation during periods of weeks does not influence flora commonly related to caries, periodontitis or infections in the oral mucous membranes.     

修饰血红蛋白作为红细胞代用品的研究进展和挑战

血源紧缺和病毒污染问题推动了血液代用品的研究,以血红蛋白为代表的红细胞代用品成为研究的重点。为克服血红蛋白直接使用的毒副作用,各种修饰技术得到了迅速发展,其中包括双阿司匹林交联、戊二醛交联、棉子糖交联、聚乙二醇偶联、脂质体包埋、生物可降解高分子包埋等。其中一些技术已经形成规模化制备工艺,产品进入临床试验,有的已在个别国家上市。鉴于这项研究意义重大,我国有关研究已经起步并正在迅速发展,各国同行的研究有重要的参考价值,因此有必要对近年来血红蛋白作为红细胞的代用品研究状况进行分析,明确面临的挑战,这将有利于发展更全面和有效的研究方案,以期取得突破性进展。