Preparation of an IgM and IgA Enriched Fraction for Clinical Use

Fraction III obtained during large acale fractionation of human plasma served as starting material for obtaining an immunoglobulin fraction enriched in IgA and IgM. Caprylic acid was used for the precipitation of most proteins other than the immunoglobulins present in fraction III. The proteins remaining in the supernatant are then precipitated vith ethanol at pH 6. Prealbumin is the main contaminant of this immunoglobulin preparation which contains 20–25% IgM and 15–20% IgA. The residual IgG can be removed by adsorption of the other proteins on DEAE-cellulose at pH 5.7 (0.03 M acetate), elution being achieved at pH 5.7 (0.2 M acetate).     

Studies on the proteolytic activity of γ-globulin preparations

1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.     

Regulation of mechanical stability of erythrocytes by serum immunoglobulins

Autologic plasma protects human erythrocytes from hemolysis induced by their suction through glass microfiber filters. The protective effect is related to the protein fraction with molecular mass above 100 kD and reproduced by gamma-globulin. The action of proteins is abolished after heating the erythrocytes above 45 degrees C as well as in the presence of galactose and ribose but not glucose, mannose and lactose. It is suggested that an increase in mechanical stability of erythrocytes is caused by interaction of immunoglobulins with glycoproteid and glycolipid membrane receptors and mediated by the changes of structural state of the cytoskeleton.     

Study of the action of phytohemagglutinin on continuous human cells]

A study was made of the agglutinating and growth-stimulating properties of phitohemaagglutinin (PHA) preparations in experiments on continuous human cells (clone line HeLa k-41 and CaVe). PHA and PHA gamma-globulin fraction possessed poor hemagglutination properties against human erythrocytes of A, B and O groups, but expressed marked cytoagglutination properties against HeLa k-41 and CaVe cells. With high PHA doses (100 and 500 microng/ml) proliferation and mitotic indx of the culture cells was lower and the percentage of dead cells and the agglutinin titre in the preparations was greater (1 : 256). With lower PHA doses (5 and 25 microng/ml) the growth was much stronger and the percentage of dead cells lower. The agglutinin titre in the preparations decreased to 1 : 16-1 : 32. PHA gamma-globulin fraction produced the strongest growth-stimulating action with the least amount of dead cells. However, the agglutinin titre in the preparations remained high (1 : 128). A conclusion was drawn that the action of PHA preparations stimulating and inhibiting the growth of continuous human cells proved to be directly connected with the agglutinin content in the preparations, since gamma-globulin PHA fraction expressed the greatest cytoagglutinating and growth-stimulating action.     

Preparation of immunoglobulins G,A,M (IgGAM) for therapeutic use. Conditions for enrichment in IgA or in IgM]

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...     

In vitro inhibition of the cytopathic action of herpes simplex virus, type 1, with a natural cytokine complex

The antiviral action of a natural cytokine complex (NCC)--the preparation Superlymph and its peptide antimicrobial fraction (AMF)--in the culture of Vero cells infected with type 1 herpes simplex virus (HSV-1), strain VR-3, was studied. The NCC preparation did not alter the morphology of the cells for 6 days and was not toxic for the culture of Vero cells. The NCC and AMF produced a protective antiviral effect, which was manifested by the inhibition of the cytopathic action (CPA) of the virus. In the presence of the preparation, the CPA of HSV-1 was equal to 10(-4.67) ICPD50, while in the control CPA was equal to 10(-5.60). The fraction containing antimicrobial peptides (protegrins) and isolated from NCC, characterized by the method of mass spectrometry, produced the maximum antiviral effect on the cell strain Vero (10(-4.58) ICPD50). Thus Superlymph, an immunomodulator with antiviral activity, could be regarded as an effective preparation for the treatment of HSV infection. The action of such preparation was aimed at the inhibition of the CPA of the virus and the stimulation of the antiviral protective mechanisms of the cell.     

Response and Specificity of Antibodies for Candida albicans

Rabbit antibodies for Candida albicans, reacting in agglutination and fluorescent-antibody reactions, were present in both IgM and IgG protein fractions. The two types of immune globulins were separated from ammonium sulfate-precipitated gamma-globulin either by filtration through a column of Sephadex G-200 or by diethylaminoethyl column chromatography performed by stepwise elution with various concentrations of sodium chloride. In the fluorescent-antibody test, initial separation of the IgG fraction, prior to its conjugation with dye, proved to be essential for the high specificity of this reaction. Investigation of the specificities of the two types of antibodies revealed that the IgG was highly specific, whereas the IgM was not very specific. Each antigen fraction, extracted by various methods, demonstrated its own characteristic antibody response. Only the IgG fraction yielded serotype-specific antibody useful for detection of a serotype of C. albicans in agglutination and fluorescent-antibody tests. The results indicate the importance of IgG for specific serological reactions with the Candida species.     

Response and specificity of antibodies for Shigella flexneri: demonstration of type-specific factors in immunoglobulin G fraction of antiserum

Considerable amounts of immunoglobulin G (IgG) antibody appeared in hyperimmune rabbit serum at a late period during a course of immunization with several injections of Shigella flexneri O antigens. High yields of IgG antibody possessing homogenous specificity could be fractionated from crude gamma-globulin solution on a diethylaminoethyl-Sephadex column with 0.02 m phosphate buffer (pH 6.6) containing 0.1 m NaCl. Specificities of IgG antibodies for six serotypes of S. flexneri were demonstrated to be high as compared with those of whole sera and their IgM antibodies. Type-specific factors for antigens I to VI were shown in each IgG fraction according to serotype employed. Further, in most sera, subtype-specific factors could be detected in the IgG fraction. These results suggest that it would be desirable to use IgG antibodies for the typing of S. flexneri.     

Non-histone chromatin proteins of mouse spleen cells during the primary immune response to sheep red blood cells and aggregated human gamma-globulin.

1. The protein/DNA ratio in chromatin of spleen cells increased during immunization; the ratio was the highest at the time of the maximum antibody synthesis, then decreased to the control values. 2. In the spleen cells of mice immunized with sheep red blood cells or aggregated human gamma-globulin, several characteristic fractions of non-histone chromatin proteins were preferentially synthesized: two antigen-specific fractions were observed in the phase of IgM and IgG synthesis, respectively, and the third, unspecific fraction was detected when the antibody synthesis ceased.     

Rat liver mitochondrial aspartate aminotransferase mRNA: isolation of mRNA by polyacrylamide gel electrophoresis

In order to obtain an enriched fraction of mRNA for mAAT we applied the following method: preparation of cytoplasmic polysomes; isolation of total RNA; isolation of mRNA by chromatography on oligo(dT)-cellulose column; polyacrylamide gel electrophoresis; extraction of mRNA from the gel. The procedure seems to allow isolation of mRNA enriched fraction for mAAT. An enriched fraction of mRNAs can be useful for the preparation of corresponding cDNAs.     

Antiglobulin factor in sera from patients with liver disease.

(1) An antiglobulin factor, non-neutralizable by human gamma-globulin, was demonstrated in sera of two patients with liver disease. (2) By absorption and elution techniques, two fractions were differentiated in a serum: one is reactive to rabbit antibody and the other seems cross-reactive to rabbit and human antibodies. (3) In double immunodiffusion test, the antiglobulin factor formed a precipitation band with heat-aggregated human IgG as did rheumatoid arthritis serum. (4) While the antiglobulin activity to rabbit antibody was demonstrated in both the IgM and IgG fractions, the reactivity to human antibody was localized in the IgM fraction. (5) From its selective reactivity to individual anti-D sera (sensitizers), at least a part of the specificity of the antiglobulin factor must be related to anti-Gm (1), and consequently can be regarded as auto-reactive.     

Chromatographic purification of gamma-globulin from human serum on ferric oxide-agarose columns

A fast chromatographic procedure for the purification of the gamma-globulin fraction of human serum on ferric oxide-containing agarose has been developed. The presence of ferric oxide (to which gamma-globulin is absorbed) increases the rigidity of the beads. They can therefore be used at high flow rates without troublesome crosslinking. The separation has been performed on a 14-ml column and on a 1.5-ml disposable column. The yield is 87 +/- 6% and the gamma-globulin fraction is homogeneous in agarose gel electrophoresis.     

Activation of Fc receptor-bearing lymphocytes by immune complexes. I. Stimulation of lymphokine production by nonadherent human peripheral blood lymphocytes

Activation of human peripheral blood lymphocytes by incubation with particulate immune complexes or aggregated human gamma-globulin was studied by measuring the release of leukocyte migration inhibitory factor (LIF) activity. LIF-active supernatants were consistently produced when nonadherent lymphocytes containing less than 1% surface immunoglobulin-bearing cells and less than 0.2% nonspecific esterase-positive monocytes were incubated in the presence of RBC sensitized with rabbit or human antibodies or with pooled heat-aggregated human gamma-globulin. This immune complex-induced lymphokine production (ICLP) was dependent on the presence of cells bearing receptors for the Fc portion of IgG (Fc gamma). ICLP could not be demonstrated with lymphocyte preparations enriched for B cells even though the latter showed vigorous LIF production in the presence of complement-sensitized erythrocytes. ICLP was dependent on the concentration of lymphocytes and of stimulant as well as on the duration of coincubation, and it required active metabolic processes and RNA and protein synthesis but not DNA synthesis. Ca++ but not Mg++ was obligatory. ICLP by non-B Fc gamma receptor-bearing lymphocytes may play a role in antibody-dependent protective inflammation and immunologic injury phenomena, which is similar to that of lymphokine release by antigen-activated T cells in delayed hypersensitivity responses.     

DNA synthesis in rat liver following administration of antihepatocytotoxic serum]

Experiments were conducted on adult female rats. The autoradiographic method was applied to the study of thymidine-3H incorporation into the parenchymatous and reticulo-endothelial cells of the liver under conditions of using low doses (0.06 microgram of protein per 100 of body weight) of antihepatocytotoxic serum (AHCS), gamma-globulin isolated from it (gammaAHCS) and gamma-globulin fraction of normal rabbit serum (gammaNRS) to intact animals and rats with carbon tetrachloride affection of the liver. The labelled nuclei index of both the parenchyma and the reticuloendothelial cells increased in case of gammaAHCS administration, and, to a lesser extent, of AHCS to intact animals. gammaAHCS used against the background of CCl4 administration intensified the reparative regeneration. The action of gammaAHCS has phasic character--the period of the labeled nuclei elevation was followed by their reduction, replaced by new intensification of the proliferative processes.     

Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation

A method is described for the subcellular fractionation of brain to obtain a preparation highly enriched in synaptic plasma membranes. The enriched fraction is recovered from the interface of a two-step sucrose density gradient on which a hypotonically lysed crude mitochondrial fraction from brain has been separated by simultaneous sedimentation and flotation centrifugation. Enzyme marker activities associated with the neuronal plasma membrane are enriched in the synaptic plasma membrane-containing fraction while less than 10% of enzyme markers associated with the major probable contaminants, myelin and mitochondria, are found in the same fraction. Morphological examination of the enriched fraction suggests that about 80% of the profiles are recognisably synaptic in origin. Compared to previously described methods for obtaining synaptic plasma-enriched fractions of equivalent purity, the procedure reported here is simpler, shorter, and of greater capacity.     

Immunoglobulin FAB-fragments: specific activity and reactogenicity. III. Comparative study of the antigenic properties of anti-encephalitic gamma-globulin and FAB-fragments isolated from it]

Parenteral administration of antiencephalytic gamma-globulin and of the Fab-fragments, extracted from it, to rabbits stimulated the formation of specific antibodies. The maximal blood serum specific antibody level in rabbits reimmunized with Fab-fragments was much lower than that following the reimmunization of rabbits with gamma-globulin. Antibodies specific to gamma-globulin and Fab-fragments were concentrated in the gamma-globulin fraction of the immune sera and could be referred to the immunoglobulins of G class.     

Electrodecantation of serum proteins.

The sedimentation of albumin under the action of the electric and gravitational fields was determined as a function of time in discontinuous experiments in a rectangular cell, using serum with the albumin fraction stained blue. It was shown that even under the influence of strong electric fields, the upper boundary of the albumin layer fell no further than the mid-point of the cell. In continuous single-stage separation of gamma-globulin from other serum proteins, only about half the gamma-globulins can be obtained from the solution because it remains homogeneously distributed throughout the solution and is only free from albumin and other proteins in the upper half of the cell. In experiments with continuously operated triangular cells, the process was optimized to give gamma-globulin of 97.5% purity in a yield of 80%, at serum flow-through rates of up to 0.5 l/h in a block composed of 40 cells.     

Fowl Cholera Immunization in Turkeys: I. Efficacy of Various Cell Fractions of Pasteurella multocida as Vaccines1

Cell fractions of Pastuerella multocida (P-1059) were tested as vaccines against fowl cholera in turkeys. These fractions were culture filtrate, cell wall, and cytoplasm. A second culture filtrate preparation made from cells grown on blood-agar rather than the standard medium was also tested along with a "combination" preparation made by recombination of the cell fractions. Each preparation was tested in three vehicles: saline, alum (0.5%), and Freund Incomplete Adjuvant (50%). The turkeys vaccinated with these preparations were challenged by exposure to an experimental epornitic of fowl cholera. The combination fraction appeared to be the most promising vaccine when compared to the protective action of the commercial bacterin included in the test as a positive control.     

Cerebrospinal fluid gamma-globulins in subacute sclerosing leucoencephalitis

Immunoelectrophoresis, gel filtration-electrophoresis and immuno-gel filtration of cerebral fluid samples obtained from five patients with subacute sclerosing leucoencephalitis and known to contain an abnormal gamma-globulin fraction (gammac) produced strong evidence that the gammac-globulin is made up of several components of small molecular size. This fraction is obviously not homogeneous, since some of the cases show more than one precipitate on immuno-gel filtration with immune serum against y-globulin. The gammac appears to be closely related to the Fab-fragment of human gamma-globulin.     

Experimental analysis of the complement-binding activity of gamma-globulin]

It was experimentally demonstrated that the anticomplimentary characteristics of gamma-globulin perparations was associated with disturbances of the colloid condition of the serum system and the absence of any stabilizing action of albumin and other serum proteins. It is also expressed as a result of a high complement-binding activity of protein aggregates. The anticomplementary characteristics of the nonaggregated part of protein in commercial preparations of gamma-globulin could be depressed by the addition of albumin or fresh serum; as to the anticomplementary characteristics of protein aggregates -- it remains unchanged. An intermolecular electrostatic interaction exists between albumin and gamma-globulin; it prevents sorption of the complement on Fc-fragment of IgG, whose destruction leads to the manifestation of the gamma-globulin complement-binding activity.