Showing papers in "Annals of the New York Academy of Sciences in 1983"

Covalent Structure of Fibrinogen

Abstract: It is well established that the fibrinogen molecule is made up of two identical halves, each containing three different peptide chains. The overall structure may therefore be described as (Aa, Bp, y ) 2. On thrombin-digestion the fibrinopeptides A and B are released and fibrin, with the structure (a, p, yl2, is formed. The complete primary structure of human fibrinogen is known.l-16 The sequence information may be used to study relationships between structure, function and evolution. In genetically determined abnormal fibrinogens the correlation between the structural error and the dysfunction of the molecule may reveal the functional importance of single amino acid residues.

IgA class endomysium antibodies in dermatitis herpetiformis and coeliac disease.

Abstract: Dermatitis herpetiformis (DH) is a vesicular skin disorder with characteristic histologic features (PMNs, microabscesses in dermal papillae adjacent to the vesicles) and disease-specific immunopathological findings (granular or fibrillar IgA deposits most commonly found in dermal papillae) usually associated with gluten-sensitive enter~pathy.~.’.’ Cases with linear IgA deposits in the basement membrane zone (BMZ) have been referred to by various names: “DH with linear IgA deposit^,\"^,' “lgA bullous pemphigoid,”’O or “polymorphic variant of bullous pemphigoid.”’ It is becoming increasingly evident that this disease is a distinct We refer to it here as “linear IgA bullous dermatosis” or LABD. Detailed reviews appear elsew Two types of antibodies have been detected in sera of DH patients, notably anti-reticulin antibodies (ARA) with reported frequencies of 5-1 8% and anti-gliadin antibodies (AGA) in about 40% of the cases. However, these antibodies occur predominantly in coeliac disease and are chiefly of IgG class: ARA have been reported in 38% of adults and in 59% of children; AGA in 40% of adults’ and over 90% of ~hi1dren.I~ Both types of antibodies are occasionally detected in diseases other than with gluten-sensitive enteropathy.” This report sets forth the first findings on IgA class antibody reacting with endomysium IgA-EmA of smooth muscle, particularly of monkey esophagus. We have found these IgA-EmA in a significant proportion of patients with DH, coeliac disease, and other gut diseases but not in cases of LABD. We have also shown that both IgA-EmA and IgG-AGA can be detected on monkey esophagus in the same serum sample by the indirect IF test.

Criteria for selecting electrodes for electrical stimulation: theoretical and practical considerations

Abstract: Smaller, more charge-intensive electrodes are needed for "safe" stimulation of the nervous system. In this paper we review critical concepts and the state of the art in electrodes. Control of charge density and charge balance are essential to avoid tissue electrolysis. Chemical criteria for "safe" stimulation are reviewed ("safe" is equated with "chemically reversible"). An example of a safe, but generally impractical, charge-injection process is double-layer charging. The limit here is the onset of irreversible faradaic processes. More charge can be safely injected with so-called "capacitor" electrodes, such as porous intermixtures of Ta/Ta2O5. BaTiO3 has excellent dielectric properties and may provide a new generation of capacitor electrodes. Faradaic charge injection is usually partially irreversible since some of the products escape into the solution. With Pt, up to 400 muc/cm2 real area can be absorbed by faradaic reactions of surface-adsorbed species, but a small part is lost due to metal dissolution. The surface of "activated" Ir is covered with a multilayer hydrated oxide. Charge injection occurs via rapid valence change within this oxide. Little or no metal dissolution is observed, and gassing limits are not exceeded even under stringent conditions.

Hepatocyte-stimulating factor: a monocyte-derived acute-phase regulatory protein.

Abstract: Peripheral subcutaneous infections undergo a well-defined sequence of clinically noticeable changes known collectively as acute inflammation. For example, following infection, changes in vascular permeability give rise within hours to the rapid biphasic influx of polymorphonuclear leukocytes followed by monocytes into the injured site. Eight to 10 hours after the initial infection, serum levels of specific acute-phase responsive plasma proteins (i.e., a, antitrypsin, fibrinogen, a? macroglobulin, a, acid glycoprotein, C3) increase. While numerous studies reported during the past century have explored the roles of leukocytes in bacterial killing, little is known about hepatocyte stimulation during the acute phase response. The factor(s) responsible for this stimulation, their mechanism(s) of action, site(s) of origin, and the regulation of their own production following infection have, until recently, remained a most difficult and controversial subject for lack of a suitable experimental model system. Studies initiated by Hornburger,' using leukocytes obtained from a turpentine-induced abscess, demonstrated the ability of these leukocytes to secrete soluble factors, which, when injected back into dogs, caused an increase in serum fibrinogen levels. This same protocol, with minor changes, was closely followed by subsequent investigators during the 1960s and '70s. Taken together, these studies all demonstrated that symptoms mimicking those found during the acute inflammatory response (i.e., increased hepatic amino acid flux, increased hepatic protein synthesis, fever, and decreased serum iron and zinc levels) could be passively conferred upon experimental animals by factor( s) secreted from whole leukocyte preparations.2-6 Thus, while these studies provided no definitive data to any of the aforementioned questions regarding hepatocyte stimulation, they succeeded in fostering the notion that leukocytes found at the site of an infection are capable of releasing modulating factors in addition to their bactericidal activity. We have been particularly interested in identifying and characterizing those leukocyte-derived factors responsible for regulating the synthesis of hepatic acute-phase responsive plasma proteins. Initial studies in our laboratory demonstrated the feasibility of using cultured fetal hepatocytes as a bioassay for hepatocyte-stimulating factor (HSF) . In these studies, the rate of fibrinogen secretion (as measured by an enzyme-linked immunosorbent assay, ELISA) was found to be dependent on HSF concentration.7 More recently, we have developed a quantitative HSF bioassay (using cultured adult hepatocytes) that can be used to determine the specific activity (i.e., units/mg protein) of any

The stability of gas bubbles in liquid‐gas solutions*

Abstract: A study has been made of the conditions under which a single gas bubble is in equilibrium within a closed volume of liquid that is maintained at constant pressure and temperature. It is predicted that, depending upon the amount of gas present in the closed volume, there may be no equilibrium state, one equilibrium state, or two equilibrium states for the bubble. For the case where one equilibrium state is predicted, it is also shown that a bubble in such a state is stable against growth yet unstable against dissolution. Where two equilibrium states are possible, it is predicted that the state corresponding to the smaller bubble size is in unstable equilibrium and the state

The aqueous pore in the red cell membrane: band 3 as a channel for anions, cations, nonelectrolytes, and water.

Abstract: This article develops arguments for the existence of an aqueous pore in the red cell membrane as the principal route for passive flux of ions, water, and small nonelectrolytes and proposes a molecular model for the pore. In principle, such an aqueous pore would provide easy passage into and out of the cell for all solutes small enough to enter the channel. The red cell membrane, however, regulates the fluxes of cations and anions closely and discriminates carefully among other small solutes. These constraints have been incorporated into the model, which visualizes the channel and its associated regulatory system as governing passive transport of ions of either sign, as well as water and small nonelectrolytes into and out of the cell. The model, which was formulated to consolidate a number of observations already in the literature, has caused us to look for new interrelations between inhibitors specific to cation, anion, and nonelectrolyte transport. The results of these experiments, presented below, demonstrate that interrelations d o exist and provide evidence that supports the view that a common aqueous channel provides primary access to the red cell cytoplasm.

Screening of toxic compounds in mammalian cell cultures

Abstract: The cytotoxic concentrations of about 100 randomly selected drugs and chemicals, tested on HeLa cells in the MIT-24 system and/or in primary cultures of fetal chicken cells, were compared with the lethal doses and/or concentrations of the agents in the mouse and in man. Most agents (80%) had a similar toxicity in vitro and in vivo, suggesting a lethal interference in man with basal functions common to all specialized human tissues as well as cultured cells, i.e., basal cytotoxicity. This high frequency of basally cytotoxic agents opens possibilities for screening chemicals for toxicity and for studying cytotoxic mechanisms with a standard battery of a few appropriate cell tests. This battery may be used in three ways: (1) to study cytotoxic mechanisms of all chemicals, and apply the resulting knowledge to understanding toxicity in man of basally cytotoxic agents; (2) to supplement conventional animal tests in acute toxicity test programs; (3) to screen chemicals and extracts for their potential basal cytotoxicity. To validate these ideas and to select suitable tests for the battery, results from cytotoxicity tests on a wide variety of chemicals in several in vitro systems must be compared with one another and with the toxicity of the agents in animals and man.

Histopathology of profound sensorineural deafness.

Abstract: In all the cases of profound deafness that we examined, the sensory epithelium along the basilar membrane had severely degenerated. However, ganglion cell counts and peripheral fiber estimates demonstrated a high degree of variability when analyzed with respect to the state of the organ of Corti or the etiology of the original cochlear disorder. We conclude that a complex interplay of factors determines the number of cells remaining in the spiral ganglion at a given time. It appears impossible at present to predict which diseases or toxic states produce primarily a sensory loss while leaving a uniform and adequate number of cochlear neurons functionally intact. We would like to emphasize that although the ganglion cell counts were consistently reduced in all our cases of profound sensorineural deafness, it has always been a surprise to us to find that the actual numbers of surviving cells are considerable. Of 15 patients, all of whom were totally deaf, only 3 had ganglion cell counts less than 10,000, whereas the majority had counts well above 15,000. Observations of this kind are possible only if accurate ganglion cell counts are carried out. Estimating the number of spiral ganglion cells by only looking at microscopic sections usually results in estimates of greater cell loss than is actually present.

Dynamics of Aerosol Formation by Chemical Reaction

Abstract: Aerosols form by chemical reactions in process and combustion gases, stack plumes, and the open atmosphere. Aerosol formation proceeds as follows. ( I ) Aerosol precursor molecules react i n the gas, producing condensable molecules. (2 ) The condensable molecules deposit on stable particles and/or on subcritical molecular clusters. (3) The subcritical clusters grow into the stable size range and/or are scavenged by the stable aerosol. (4) Stable particles coagulate, while condensation continues to take place on their surface. The critical cluster diameter, d r , corresponds to the equilibrium particle diameter calculated from the Kelvin relationship,

Initiation of the alternative complement pathway due to spontaneous hydrolysis of the thioester of C3.

Abstract: Initiation of the alternative pathway of complement involves the random attachment of C3b on all biological particles in contact with plasma. This process requires continuous proteolytic generation of metastable C3b. The responsible protease arises as a result of conformational changes in C3 that occur upon thioester hydrolysis. The product, C3(H2O), is functionally C3b-like and forms a C3 convertase with Factors B and D. The resulting biomolecular enzyme, C3(H2O),Bb, is a serine protease that cleaves C3 generating metastable C3b. The rate of spontaneous generation of C3(H2O) under physiological conditions is between 0.2 and 0.4%/hr. C3(H2O),Bb produces only three to five metastable C3b molecules per enzyme before being inactivated by regulatory proteins. The thioester bond in metastable C3b has been estimated to be 10(10) times more reactive than that in native C3 and mediates attachment of C3b to biological particles. Once bound, C3b is subject to two competing processes: (1) inactivation and (2) a chain reaction-like amplification process that can deposit large numbers of C3b molecules on the particles within a very short period of time after the initial C3b binds. On activators of the alternative pathway, inactivation of C3b is restricted and amplification of C3b results in activation of the cytolytic pathway of complement.

The secretory immune system of lactating human mammary glands compared with other exocrine organs

Abstract: It is well known that human colostrum and milk are much richer in IgA than other exocrine secretions. Thus, colostrum contains about 300 times more IgA than stimulated parotid secretion and about 60 times more than normal unstimulated whole saliva.' The idea has therefore emerged that lactating mammary glands either harbor a remarkably active secretory immune system or, alternatively, by some unknown mechanism, drain selectively dimeric IgA from serum.' The former possibility appears paradoxical because the mammary glands, like the major salivary glands, are sheltered from a continuous exposure to antigens and mitogens. in contrast to the secretory immune systems of respiratory and gastrointestinal mucosae. Elucidation of the mentioned possibilities, however, has not been feasible because no previous study has characterized quantitatively the secretory immune system of lactating mammary glands with reference to data available for other secretory tissues. This lack of basic information concerns all mammalian species; although some immunohistochemical investigations have been carried out on lactating mammary glands from sheep,3 pig: mice.? rats.' and cows,' the results have not been compared on a quantitative basis with the situation in other exocrine glands. The only immunohistochemical study of human lactating glands that I am aware of, likewise provided no quantitative information." The purpose of the present investigation was twofold. First, I wanted to characterize in situ the cellular aspects of the secretory immune system of human lactating mammary glands and to relate the findings to information about other glandular tissues subjected to comparable studies in our laboratory. The number of lactating glands available for investigation was unfortunately small, because of the difficulties involved in obtaining such fresh material; but the studied specimens are collectively thought to afford a representative map of the activated mammary gland immune system. Second, by applying available information about the secretion of IgA by parotid and lactating mammary glands, I wanted to compare the secretory capacity of these organs in relation to tissue wet weight and complements of immunoglobulin (1g)-producing cells.

Adaptation of the DNA-repair and micronucleus tests to human cell suspensions and exfoliated cells.

Abstract: In vitro assays for genotoxicity have been applied to over 11,000 chemicals, many of which have been found to have the capacity to induce mutations, chromosome aberrations, sister chromatid exchanges, mitotic recombination, gene conversion, nondisjunction, DNA fragmentation, DNA repair, and a host of other nuclear anomalies. The results of these observations have profoundly changed our attitude toward environmental carcinogenesis. Only two decades ago, chemicals with carcinogenic and mutagenic properties were considered to be rare. The induction of a DNA alteration and mutation in somatic cells was thought to be an extremely rare event. Today, one has the impression that we are living in a brew of carcinogenic and genotoxic compounds and that hundreds, if not thousands, of mutagens and clastogens enter man daily through the regular diet. Previously, the development of cancer was thought to occur in individuals accidentally exposed to an excessive dose of one carcinogen. Now one is inclined to accept the idea that, since relatively large amounts of carcinogens enter man and carcinogen-DNA adducts are formed in many organs, cancer will develop primarily in individuals who ingest too small an amount of anticarcinogenic agents or who have a defective defense mechanism. If this assumption is correct, then in vitro tests, which lack the complete metabolic activation and inactivation systems of a whole organism, may predict a potential but not the actual carcinogenic hazard to man. The design, development, and validation of short-term in vivo tests that are applicable to man are needed to obtain a higher and more reliable predictive value.

Occurrence and nature of bacterial IgA proteases.

Abstract: Secretory immunoglobulin A (sIgA) is the principal mediator of specific immunity on human mucosal surfaces. Although the exact mechanisms are partly unclear, it is generally accepted that sIgA antibodies maintain the integrity of the mucous membranes by reducing their colonization by microorganisms, by neutralizing toxins and viruses, and by preventing the penetration of microbial and other antigens and allergens through the surfaces.' IgA in serum appears to play an important role in the regulation of inflammatory responses particularly in submucosal tissues.' Recent studies in rodents have suggested, furthermore, that serum IgA is involved in the disposal of antigens from the circulation.' For sIgA to successfully protect mucosal surfaces it must resist proteolytic attack from digestive and microbial enzymes that occur in these environments. Changes in the structure of IgA induced by bacteria was first demonstrated in vitro by Miiller in 1971.3 He reported that strains of Neisseria meningitidis and N. gonorrhoeae possess proteolytic activity that invariably induce selective changes in the electrophoretic mobility of human IgA. Although the exact nature of these changes were not identified, Milller suggested that bacterium-induced alterations of IgA is a possible factor in the pathogenesis of infections caused by N. meningitidis and N. gonorrhoeae. Strains of a number of other bacterial species, including Pseudomonas aeruginosa, Bacteroides melaninogenicus, and Proteus vulgaris, were shown to have a strong proteolytic activity capable of causing progressive degradation of IgA. In addition, several human-pathogenic organisms induced changes in the electrophoretic mobility of IgA and various other human serum glycoproteins by attacking their carbohydrate moieties. The existence of a bacterial proteolytic enzyme with the capacity to induce specific cleavage of IgA in the hinge region, yielding intact Fab, and Fc, fragments was first reported by Metha and coworkers in 1973.4 The enzyme was demonstrated in human feces of six normal subjects examined. The intestinal bacterium responsible for this cleavage has never been identified. Subsequent studies by Plaut, Genco, and Tomasi revealed that a similar enzymatic activity is present in human saliva, and that an organism indigenous to the oral cavity, Streptococcus sanguis, produces an enzyme with this characteristic activity.'

The breakup of small drops and bubbles in shear flows

Abstract: The problem of determining the deformation and burst of a single drop freely suspended in another fluid undergoing shear is of fundamental importance in a variety of physical processes of practical significance; for example, the rheology of emulsions and the dispersion of one fluid phase into another. It was studied both theoretically and experimentally by G. I . Taylor,’.’ who, as he has with many of the other topics in fluid mechanics, obtained quantitative results that were not only the first on the subject but which remain among the most important and fundamental in this field. The systems considered experimentally by Taylor’ are depicted in FIGURES l a and I b. An initially spherical liquid drop of radius a and viscosity Ap was placed in a fluid, with which it was immiscible, of equal density and of viscosity p. A steady shear of strength G was then applied and the drop was found to deform into a steady shape if G was maintained below a critical value G,, but the drop broke when G exceeded G,. The two shear flows set up by Taylor’ were: ( I ) the “hyperbolic” flow, u, = Gx, ug = Gy3 with u , and u , being the corresponding velocity components along the x and y directions, respectively, which is a pure straining motion without vorticity, and (2) the simple shear flow, u, = Gy3 ug = 0, which, as is well known, consists of a pure straining motion, with its principal axis of extension along the diagonal in the xy plane, plus a solid body rotation about the origin. In the absence of inertial erects, which were indeed negligible in Taylor’s experiments,’ the independent parameters, in addition to the type of shear being impressed, are: C , the strength of the shear flow; a, the radius of the initially spherical drop, p, the viscosity of the ambient fluid; A, the viscosity ratio; and y. the interfacial tension. Hence, the deformation, D = ( L B ) / ( L + B ) , where L and B are the half-length and the half-breadth of the drop, respectively, becomes a function of only two dimensionless groups, i.e., the capillary number ( k ’ = Gwa/y) and the viscosity ratio, A. Taylor’ found that, for fixed A, D was linear in Gpa/y for small values of the capillary number k ’ , but that, beyond a certain range, the slope of the D versus k ’ curve increased rapidly in many cases until a point was reached where a steady drop shape could no longer be maintained and the drop burst. There were conditions, however-most notably with high viscosity drops (A >> 1) in a simple shear flow-for which a limiting deformation was attained and drop breakup did not occur. Examples of these two types of deformation curves are sketched in FIGURE 2. From a practical point of view, the quantity of primary interest is the critical shear

Electron Microscopy of Fibrinogen, its Plasmic Fragments and Small Polymers

Abstract: When we began our project on the electron microscopy of fibrinogen four years ago, the most widely accepted structural model was the trinodular rod proposed by Hall and Slayter in 1959.l They imaged individual fibrinogen molecules that had been sprayed on mica in a volatile buffer, dried in vacuum, and shadowed with heavy metal. There was some controversy over these results because other labs had difficulty reproducing the specimens. Nevertheless, most of the data from biochemistry and physical chemistry fit the trinodular model quite nicely. A more serious controversy arose from laboratories attempting to visualize the molecules by negative staining, which is generally recognized as a higher resolution technique than shadowing. Large globular particles were the predominant species found in these specimens.2. Although strikingly different in shape from the trinodular rod, models based on these images of spherical particles could be made to accommodate most of the hydrodynamic and solution scattering data. We wanted to find which structure was correct and to demonstrate the same structure by these different specimen preparation techniques. Our first goal was to obtain improved shadowed specimens that showed large fields of clean, unaggregated molecules. We achieved this goal with a crucial innovation in the shadowing technique: inclusion of glycerol, at a concentration of 20% to SO%, in the buffer in which the protein was sprayed onto the mica.4 The glycerol seems to affect the spreading of the drops on the mica and the deposition of the protein molecules as the drop recedes during vacuum drying. Glycerol is not always required, in particular for fibrinogen, which has yielded good specimens from a wide range of solution conditions. In our experience, however, the use of glycerol results in a marked increase in both the frequency and the quality of good specimen areas. In the absence of glycerol the protein is often deposited in aggregates or the molecular structure is distorted by drying; in the presence of glycerol most of the droplets have clean areas of unaggregated molecules. Typical fields of unidirectionaland rotary-shadowed specimens of fibrinogen dried from glycerol are shown in FIGURES 1A and 1B. It may be useful to recount briefly the history of the glycerol innovation. When we were beginning our project we were particularly impressed by images of shadowed myosin presented by Elliott and Offer.s Their specimen preparation involved freeze-drying a sample that had been sprayed on mica;

The structure and evolution of vertebrate fibrinogen.

Abstract: The past three decades have given rise to extraordinary advances in our understanding of the fibrinogen-fibrin system. Thus, at the beginning of the 1950s it was discovered that a small amount of peptide material was proteolytically released from fibrinogen by thrombin, and that as a result the parent molecules spontaneously assembled into a fibrin In the intervening thirty years the general shape of the fibrinogen molecule has been determined,6-s its complete covalent structure established,g-16 the mode of its polymerization described,l6, l7 the details of its covalent stabilization revealed,l8, and very much more discovered, some of which is reviewed elsewhere,2o. 21 and many other aspects of which will be reviewed or reported at this meeting. The polymerization event itself is well enough understood that peptide antipolymerants have been designed on the basis of that understanding, synthesized, and shown to be effective.22+ 23 It has been a privilege merely to witness the astonishing progress during this period, much less to participate in it. This is not to say that no mysteries about the fibrinogen-fibrin system remain. There are still many points that are poorly understood, including, for example, details of the lateral packing in fibrin fibers and the geometry of covalently cross-linked a chain multimers. Moreover, the exact contacts in the initial polymerization event are still unknown, although they may be tantalizingly close to 25 We still suffer from the lack of a detailed x-ray structure, and the mechanics of the intracellular bioassembly of the molecule have yet to be unveiled. The mystery I want to focus on in this article, however, is one that I have been stuck on for the last 25 years: how did the vertebrate fibrinogen molecule evolve? The problem is that the fibrinogen molecule seems just to “appear”fully developed-with the dawning of vertebrate animals.2E This is to say that fibrinogen of the type found in vertebrates has so far not been found in an invertebrate or a protochordate. Certain invertebrates are known to have coagulable proteins,279 28 but these molecules have no obvious common ancestry with the vertebrate type, nor are they activated by thrombin. As far as we know, all vertebrates have fibrinogen molecules composed of three pairs of nonidentical chains (a&y2), and all are converted to fibrin by thrombin-releasing fibrinopeptides from the amino-terminal segments of the a and /3 chains (TABLE 1). The selective advantage of a fibrinogen-fibrin system for vertebrates, what with their valuable pressurized and iron-laden blood, is obvious. What is not obvious is where the ingredients for the fibrinogenfibrin system ever came from. Certainly a protein as complex as fibrinogen is not invented instantaneously as the result of a random assemblage of amino acids. In this article we will first consider the structure of the human fibrinogen molecule, making some inferences about fibrinogen evolution on the basis of

Energetics of membrane deformation and adhesion in cell and vesicle aggregation.

Abstract: Membrane-membrane adhesion processes are prominent aspects of cellular and subcellular life, whether as precursors to fusion events in phagocytic, endocytotic, and secretory processes or as the primary mechanism in blood cell aggregation and agglutination. The general impression is that the factors that influence aggregate formation simply alter the chemical affinity between opposing membrane surfaces and that affinity is the sole determinant of aggregation. However, other physical factors play equally important roles in aggregation-disaggregation processes. Most obvious are the external forces that act on cell or vesicle bodies produced by fluid shear stresses in circulation and convection. Less obvious, but intrinsically significant, is the mechnical deformability of the cell or vesicle because it directly regulates the adhesion process. Simply stated: \"rigid bodies have limited ability to adhere (unless they fit together perfectly), whereas \"flaccid or easily deformed bodies can form large contact areas with little work. This article will present first a brief outline of the determinants of cell and vesicle deformability and how these factors influence aggregation in the absence of fluid shear stresses. Next, the specific energetics of deformation and adhesion of membrane-bound capsules (like red blood cells and lipid bilayer vesicles) will be discussed; this will be followed by examples of red cell and lipid vesicle experiments which have been used to evaluate the levels of surface affinity associated with aggregation in dextran solutions and plasma. Finally, a short discussion of the factors involved in disaggregation of adherent cells will be given, with estimates of the fluid shear stress required to disaggregate cells.

Analysis of the effector functions of different populations of mucosal lymphocytes

Abstract: Lymphocytes separated from the epithelial layer of mouse small intestine, IEL, were tested for their NK cytotoxicity against Yac-1 targets. There was little NK activity in a 4 hour assay, but high activity in an 18 hour assay, and the NK activity of IEL did not parallel that in the spleen in any of the mouse strains tested. Furthermore, IEL exerted a suppressor activity on mouse spleen NK activity. Specific T-cell cytotoxicity appeared in IEL in mice immunized with an intraperitoneal injection of P-815 tumor cells. By contrast with IEL, LPL had little NK or NK suppressor activity, but higher levels of specific T-cell cytotoxicity in tumor-immunized mice than intraepithelial lymphocytes. A high proportion of IEL had granules that stained with Giemsa and Astra blue. Furthermore many IEL carried Lyt-2+ phenotype and no other T-cell surface antigen. Intraepithelial lymphocytes appeared, therefore, to have staining and phenotype characteristics of both granular NK cells and suppressor cells. It was clear that the intestinal mucosa contained populations of immune effector cells that were heterogeneous in nature and function.

Development of immunological capacity under germfree and conventional conditions

Abstract: The development of immunocompetency in mammals occurs during fetal life.'-3 The immunological capacity of the newborn organism, however, is not fully mature; the insufficiently adapted organism is delivered into an environment full of microorganisms. At the beginning of life the defence capacity depends to a great extent on a supply of maternal antibodies from placenta, colostrum, and milk. It is the intestinal lymphoid tissues that are exposed to the load of microflora and food antigens. The significance of microbial antigens for a clonal expansion of lymphocytes of certain idiotypes and isotypes was recently critically e ~ a l u a t e d . ~ . ~ The lymphatic tissue of the intestine reacts to antigenic stimuli by a rapid development that is particularly apparent during the first days after birth.e.7 Signs of activation of an organized lymphatic tissue (Peyer's patches, appendix) can be followed on both morphological and functional l eve l~ .~~ ' ' Later this activation is reflected in a presence of the IgMand IgA-producing cells in the intestinal mucosa.\" Cells originating in Peyer's patches and primed by intestinal antigens are supposed to migrate through the mesenteric lymph nodes, the thoracic duct and blood into the gut and other mucosal surfaces, forming the so-called common mucosal system.''-'e A similar migratory pathway as for IgA-producing cells was described for T-cells originating in the The nature of factors involved in the homing of these cells to distant mucosal surfaces and exocrine glands is still u n k n ~ w n . ' ~ ~ ' ~ In our previous studies we compared the immunological capacities of germfree (GF) piglets and rabbits with those of their conventionally raised (CONV) partners and showed that the antigenic stimulation fundamentally affects development of the immunological capa~ i ty .~ . '~ . ' ~ Nevertheless, for some other species, for example mice, the development of the immunological capacity in the GF and CONV individuals was reported to be comparable.'' We therefore inquired into the development of another available species-rat-and studied factors that affect development and recirculation of lymphocytes. Because the six-layered placenta of the pig considerably limits transfer of maternal antibodies, the GF piglets seem to be a suitable model for the study of problems concerning the development of immunological capacity, especially in the GALT. In addition, a higher weight of lymphatic organ makes easier some quantitative assays. Our experimental model was therefore used for the study of

Large‐scale Production of Mammalian Cells and Their Products: Engineering Principles and Barriers to Scale‐up

Abstract: Mammalian cell products have great medical and clinical importance, but to date, production methods employed to manufacture these products on a large scale are not as cost efficient as they could be. The implementation of process control would greatly improve the productivity of these products. Recently developed methods to produce cells on a large scale, such as microcarriers, artificial capillaries, tubular spiral film, and microencapsulation must be optimized, and the problem of oxygen transfer limitation must be solved. The accumulation of potentially toxic waste products can inhibit growth and reduce productivity. This effect can be reduced by either adjusting the environmental parameters of a fed-batch culture, so that the cell's metabolism is shifted away from producing these compounds, or by continually perfusing medium through the culture. If these technical barriers can be overcome, the cost of producing products derived from mammalian cells can be greatly reduced.

Tuftsin, A Natural Activator of Phagocyte Cells: An Overview

Abstract: The recognition that a small oligopeptide was responsible for the full stimulation effect of specific cytophilic gamma-globulin on blood neutrophils arose from a study of the kinetics of phagocytosis. These were unusual in that the stimulation was short lived and that preincubation of the phagocyte with the gamma-globulin rendered the latter inactive. The oligopeptide was isolated, its structure determined (Thr-Lys-Pro-Arg) and synthesized. The discovery of human mutants with tuftsin deficiency exhibiting signs and symptoms of frequent severe infection further emphasized the specific biological function of the tetrapeptide. The mutant peptide was isolated, sequenced (Thr-Glu-Pro-Arg), and synthesized. Further studies showed that tuftsin requires two enzymes for its liberation from the parent carrier gamma-globulin. One enzyme is in the spleen that cleaves distal to the arginine end, and the other, on the outer side of the plasma membrane, cleaves proximal to the threonine residue. The tetrapeptide tuftsin stimulates all functions of phagocytic cells: phagocytosis, pinocytosis, motility, immunogenic activity including processing of the antigen and augmentation of the number of antibody-forming cells, bactericidal activity, and, above all, tumoricidal activity. The latter has been shown by several laboratories.

On subdifferentiability spaces

Abstract: Fifteen years ago, Edgar Asplund introduced the classes of strong and weak differentiability spaces’ (later called Asplund and weak Asplund spaces, or A-spaces and WA-spaces), which were Banach spaces characterized by differentiability properties of continuous convex functions defined on them. This was the principal analogy that led us to define subdifferentiability and weak subdifferentiability spaces (S-spaces and WS-spaces) it terms of subdifferentiability properties of lower semicontinuous (Isc) nonconvex functions. The initial impulse came, however, from nonsrnooth analysis, where a subclass of weak subdifferentiability spaces appeared quite naturally.’ Elements of the subclass were called weak trustworthy spaces (WT-spaces), by which we meant that such spaces could be trusted, in the sense that a reasonably good calculus of Dini (weak) c-subdifferentials did exist for Isc functions defined thereon. (Exact definitions will be given in the next section.) By analogy, we define here a class of trustworthy spaces (T-spaces) in terms of Frkhet t-subdifferentials. To avoid confusion, we have to say that we also use the words “subdifferentiability spaces” as a collective name for all spaces characterized by their subdifferentiability properties. The purpose of the paper may, therefore, be described as studying subdifferentiability spaces and their connection with some significant classes of spaces (Asplund spaces, spaces with differentiable norms, efc . ) . We refer to References 2 and 3 for results demonstrating the role of subdifferentiability spaces in nonsmooth analysis. All spaces are assumed to be Banach and all functions to be extended-real-valued (if nothing more specific is said). For a functionf, we set

Relation of cochlear implant function to histopathology in monkeys.

Abstract: Although a number of patients are currently benefiting from cochlear implants, it is widely believed that considerable improvement can be made in this method of providing hearing to the deaf. Many investigators are currently gathering data with this goal in mind. We believe that one of the most fruitful approaches is to examine the relations between implant function, measured psychophysically, and the condition of the implanted cochlea, measured histologically. This approach yields data that are applicable at several levels: (1 ) Observing the relation of implant function to various levels and types of pathology will aid in establishing criteria for selection and treatment of implant candidates. (2 ) These observations yield data relevant to physiological models of implant function. If we can model what neural elements are being excited by electrical stimulation, and how various percepts are encoded, we will be in a much better position to specify optimal electrode placements and parameters for electrical stimulation. ( 3 ) Studies of hearing in the absence of hair cells and basilar membrane mechanics, and in many cases in the absence of a large percentage of the normal complement of eighth nerve fibers, provide unique opportunities to analyze central neural mechanisms. A better understanding of central auditory processing would be of great benefit to those who are trying to devise paradigms for translating acoustic stimuli into electrical signals that can be delivered by singleor multichannel cochlear implants. Realistically, achievement of these goals will require data from both implanted human patients and laboratory animals. The human data ultimately demonstrate the relevance of the animal data, and in addition, some percepts are more readily tested in humans than in animals. On the other hand, the procurement of human histological data is naturally very slow, and the human psychophysical data are often inadequate owing to a variety of complicating factors. For example, human data are usually collected from patients who have been deaf for a number of years and whose ability to perform the required psychophysical experiments is not known, whereas animal data can often be obtained from a deafened, implanted ear and a normal hearing

Mechanisms of toxic injury.

Abstract: 2. The rate of production of initiating factor(s) and its rate of detoxication. 3. The accessibility of the initiating factor to the crucial “target” cellular molecules and the extent and persistence of the reaction between them. 4. The biological role of these “target” molecules (which may vary with cell type, physiological state of the cell, etc.). 5. The ability of cellular defense mechanisms to repair, replace, or compensate for these altered “target” molecules. 6. The nature and extent of release of products from the injured cell that may cause cytotoxic or stimulatory effects on other cells of the same or different cell types. initiating factor.

Fibrinopeptide B in fibrin assembly and metabolism: physiologic significance in delayed release of the peptide.

Abstract: The delayed release of peptide B that accelerates towards the end of fibrin formation unmasks accessory (b-) epitopes for monomer interaction. Ultracentrifuge and chromatographic analysis of the composition and dissociation of soluble complexes formed by monomers in fibrinogen solution indicate that the b-epitope augments aggregation by acting cooperatively with the a-epitope to reinforce rather than cross-bridge oligomer assembly. Monomer/fibrinogen association by coordinated interactions through both epitopes is strengthened by an additional order of magnitude over associations (10(7) and 1.6 X 10(6) M-1) through the a- and b-epitopes individually, without affecting oligomer thickness. It is suggested that the delayed release of B has purpose in allowing early complexes to dissociate for (1) rapid equilibration across interstitial fluids, and for (2) rapid uptake by phagocytic cells which depend on access to the a-epitope for monomer absorption. In late stages of coagulation, stabilization of oligomer assembly imparted by the b-epitope blocks both equilibration of fibrin concentrations and phagocytic clearance of the fibrin to localize deposition.

THE PRIMARY STRUCTURE OF α2‐MACROGLOBULIN AND LOCALIZATION OF A FACTOR XIIIa CROSS‐LINKING SITEa

Abstract: The tetrameric plasma glycoprotein a-macroglobulin (~$4)' forms complexes with proteinases from all four classes, E.C.3.4.21-24,'-' that are rapidly removed from the circulation.' The four identical M, 180,000 subunits contain approximately 9% carbohydrate' and are presumably pairwise disulfide bridged? Two such dimers are assembled to form the tetrameric human %M, M, 720,000.'.6.7 Following activation by specific limited proteolysis in the bait region*-'' a,M undergoes a conformational change resulting in the formation of a binding site@.) for proteinases? Furthermore, this change in conformation leads to the activation of a presumed internal /3-cysteinyly-glutamyl thiolester in each subunit of qM. ' ' -14B~th covalent and noncovalent %Mproteinase complexes are formed,''. 15* l6 in which the activity of the bound proteinase is preserved.\", I' The complement components C3 and C4 also contain an activatable internal thiolester in their a'-chains.''< ,'The thiolesters of q M , C3, and C4 are located in strongly homologous sequences.\", During the present investigation of the primary structure of a,M several reports describing the sequences of relatively short stretches of a,M have appeared.'-''* \"-\" In this communication we report the complete sequence of the M, 180,000 subunits of a,M that contain 1450 amino acid residues.

Factors influencing fibrin gel structure studied by flow measurement

Abstract: In order to form a gel, fibrinogen has to be activated. Physiologically, this event is governed by thrombin, which releases two peptides, fibrinopeptide A (FPA) and fibrinpeptide B (FPB), from fibrinogen in the course of its transformation to fibrin. It is believed that release of FPA leads to formation of linear polymers. The polymer molecules appear to constitute elongated structures, having a width double that of fibrinogen. They are formed by end-to-end and side-to-side association of fibrinogen units in a half-staggered fashion. These polymer molecules were first observed in thrombin-fibrinogen systems in which clotting was inhibited by addition of hexamethylene glycol or similar agents (FIGURE 1) . However, polymers do also form in clottable systems and they are present before the time of g e l a t i ~ n . ~ ~ The polymer molecules are very likely the result of interaction between polymerization sites in activated bifunctional fibrinogen molecule^.^^ * The polymers formed when FPA only is released by the snake venom enzyme Batroxobin is different from those which are formed by release of both FPA and FPB by t h r ~ m b i n . ~ The polymers eventually cross-link f to form the infinite network structure which is the gel according to the definition by Flory.lo Ferry and Morrison l1 were the first to perform systematic studies on fibrin gel structures. They distinguished between two types of gels: i.e., the coarse and the fine types. In the fine type, more or less individual polymer strands were suggested to cross-link to form the gel. In the coarse gel, on the other hand, it was assumed that the polymers were laterally aggregated and cross-links occurred between these bundles of polymers. The hypothetical structure of the fine and coarse type of gel is shown in FIGURE 2. Most information on fibrin gel structure has been obtained from studies of the optical and mechanical properties of the gels. We will present here studies we have performed using liquid and particle permeation of fibrin gels under a variety of experimental conditions. This approach was also used by Carr et d.l2 to study thickness of fibrin strands in fibrin gels. We have studied the flow properties of gels produced by Batroxobin and by thrombin. The former gels are called Fibrin I gels, and the latter Fibrin I1 gels.' The simple flow-device we have used is shown in FIGURE 3. The gels were prepared in special cups (FIGURE 3A) as previously described.13

Fibrin polymerization sites in fibrinogen and fibrin fragments

Abstract: The inhibition of blood clotting by degradation products of fibrinogen reflects an interference with fibrin network formation. The original discovery that digestion of fibrinogen by plasmin resulted in the formation of fragments with anticoagulant activity 1. did not recognize the importance of inhibition of fibrin polymerization and was interpreted as an interference with thrombin action. During the past 25 years considerable progress has been achieved in the understanding of fibrin clot formation. The use of fibrinogen and fibrin fragments significantly contributed to the advancement of our knowledge, especially since many biologic functions of fibrinogen were recovered in various fragments. Thrombin releases two molecules of fibrinopeptide A (FPA) and two molecules of fibrinopeptide B (FPB) from the Aa and Bg polypeptide chains of the fibrinogen molecule, respectively. The cleavage of four arginylglycine peptide bonds converts fibrinogen into fibrin monomer, which polymerizes spontaneously forming a fibrin clot. This paper addresses the structure and localization of active contact sites with complementary affinities which are thought to specifically organize fibrin monomers and drive the polymerization reaction. Batroxobin, a protease from Bothrops atrox venom, cleaves FPA from fibrinogen. This event is sufficient to initiate clotting and formation of fibrin I, which lacks FPA but still contains FPB, and provides evidence that the loss of FPA alone is sufficient for fibrin gelation. Thrombin releases FPA at a much faster rate than FPB and the ultimate product, fibrin I1 lacks both fibrinopeptides.

Membrane interaction and deformation.

Abstract: Membrane interaction and membrane deformation should be considered two aspects of the same phenomenon. Because membrane interaction depends on structural features such as curvature, chemical composition, and surface organization, one must expect that these features will change as membranes are brought together. Our observations on model phospholipid membranes have allowed us to measure and to distinguish electrostatic (coulombic or double-layer) forces, electrodynamic (van der Waals, dispersion) interactions, and hydration forces due to the solvation of polar groups that are stabilizing the membrane surface. As a consequence of these interactions, approaching membranes may flatten against each other, may change the packing density of their constituent molecules, may rearrange the composition of components in regions close to the apposing membrane, or may even show conformational changes in the arrangement of the lipid hydrocarbon chains. Abundant examples of such deformation accompanying membrane interaction are evident from x-ray diffraction and electron microscopic observations.