[Secondary immune response of mice immunized with a protein-cellulose complex]. / Vtorichnyi immunnyi otvet u myshei pri immunizatsii belkovo-tselliuloznym kompleksom.

It has been previously established that an intravenous injection of a protein antigen solution into mice primed with the same antigen in the form of a protein-cellulose complex induces an intensive antibody production (up to 10,000 antibody-forming cells/10(6) splenocytes and up to 3 mg of antibodies/ml of serum). The present study has shown that secondary immune response can be considerably enhanced if large amounts of the antigen are administered intraperitoneally in a protein-cellulose complex during secondary immunization. In these experiments the mean number of antibody-forming cells was 50.000/10(6) splenocytes and the antibody serum level averaged 10 to 12 mg/ml. The effect persisted for a long time: as late as on day 80 the antibody concentration was 2 mg/ml of serum.

[Cultivation of cells on modified porous cellulose beads]. / Kul'tivirovanie kletok na modifitsirovannykh poristykh tselliuloznykh sharikakh.

New microcarriers for the growth of animal cells have been synthesized and studied. The preparations are porous cellulose beads, modified by diamines. Spreading and growth of L cells, MEVO and HETR cells on these beads were observed. As a result of the cultivation the number of animal cells increased 5-10-fold.

Induction of abundant antibody formation with a protein-cellulose complex in mice.

A method is described for immunizing mice with a protein-cellulose complex obtained by covalent coupling of antigenic protein molecules to suspended cellulose particles. Primary immunization of the animals with the complex led to a pronounced immune response persisting for 20-30 days. Subsequent administration of the same antigen in a soluble form resulted in extremely active antibody formation equal to or better than that induced with Freund's complete adjuvant. In BALB/c mice the antibody response was about ten times greater than in the C57BL/6 strain.

[Use of a protein-cellulose complex for inducing intensive antibody formation in mice]. / Ispol'zovanie belkovo-tselliuloznogo kompleksa dlia induktsii u myshei intensivnogo antiteloobrazovaniia.

The protein-cellulose complex prepared by covalent immobilization of single protein molecules on insoluble cellulose particles was used for priming C57BL/6 and BALB/c mice. The serum antibody content and the number of spleen AFC were assayed after animals' boosting with the soluble protein. Such a complex was shown to have marked advantages over the same protein injected both in complete Freund's adjuvant and in a soluble form, in particular. The immune response of BALB/c mice was more than 10-fold higher than that of C57BL/6 animals.

[Effect of Fab-fragments of antibodies against spleen cell surface antigens on the development of antibody formation in cultures of different densities]. / Vliianie Fab-fragmentov antitel protiv poverkhnostnykh antigenov kletok selezenki na razvitie antiteloobrazovaniia v kul'turakh raznoi plotnosti.

The role of surface antigens in the density-dependent inhibition of primary immune response in mouse spleen cell cultures was investigated. For this purpose Fab-fragments of rabbit IgG obtained after immunization with mouse splenocytes were used. Such Fab-fragments alone had no effect on immune response in both optimal and dense cultures. However, successive treatment of cells with Fab-fragments and with ass antibodies against rabbit IgG dramatically augmented the density-dependent inhibition of antibody formation.

[Synthesis of a high-volume immunosorbent with oriented immobilization of Fab'-fragments for antigen separation]. / Sintez vysokoemkogo immunosorbenta s orientirovannoi immobilizatsiei Fab'-fragmentov dlia vydeleniia antigena.

A highly capacious immunosorbent for antigen demonstration has been synthesized. Use has been made of oriented immobilization of antibody Fab'-fragments on porous cellulose balls at the expense of the reaction of disulfide exchange. Up to 100 mg of Fab' was fixed on the sorbent, with the capacity reaching 200 mg antigen (rabbit IgG) per g of dry weight sorbent. The molar ratio Fab'/IgG was equal to 0.8-0.9, i.e. approximated the theoretical limit: one molecule of antigen per one active center antibody.

[Immunosorbent based on porous cellulose sphere]. / Immunosorbent na osnove poristykh tselluloznykh sharikov.

The synthesis of an immunosorbent in the form of porous beads is described. The beads were prepared by emulsification of cuprammonium glucose solution in organic solvents, followed by precipitation of beads with a mixture of acetone and sulfuric acid. The product was oxidized with NaIO4 and conjugated with protein antigen or antibodies to obtain an immunosorbent fit for column chromatography. The capacity of the immunosorbent thus obtained was found to be 300-1000 mg of antibodies or 50-70 mg of antigen per 1 g of the sorbent.

[Antigen-induced increase in the number of cells producing non-specific immunoglobulins]. / Narastanie chisla kletok, produtsiruiushchikh nespetsificheskie immunoglobuliny, pri deistvii antigena.

The appearance of antibody- and nonspecific immunoglobulin-forming cells (AFC and nIFC) in spleen cell suspensions from normal mice and from those immunized with sheep red blood cells (SRBC) was studied. The cell suspensions were cultivated in the modified Mishell-Dutton system. The induction of the immune response in vitro resulted in a sharp increase of nIFC formation. Their amount reached 51 218 per 10(6) spleen cells, i. e exceeded the number fo AFC 25-172-fold. Much more was the increase in the absolute number of nIFC during cultivation in the presence of antigen of spleen cells from animals immunized with SRBC 3--4 days before experimentation. In this case, the maximum number of nIFC reached 165 000 per 10(6) spleen cells, i. e. 33% of the total number of B-cells. The kinetics of AFC and nIFC formation and regulatory mechanisms of the populations varied under different conditions. The nature and potential mechanisms of nIFC formation under the influence of the antigen are discussed.

[Synthesis of a high-capacity immunosorbent based on a cellulose suspension]. / Sintez vysokoemkogo immunosorbenta na osnove suspenzii tselliulozy.

A method is suggested for preparation of an immunosorbent on the basis of cellulose suspension. Antigen was coupled to periodate-oxidized cellulose via aldehyde groups. Optimal conditions (time of oxidation, amount of an oxidant, quantity of protein antigen added) for immunosorbent synthesis were determined. The amount of antibodies bound to the immunosorbent was approximately equal to the weight of the immunosorbent (up to 950 mg of antibodies per 1 g sorbent). Thus each molecule of antigen coupled to cellulose bound 5-8 molecules of antibodies.

A sharp antigen-induced increase in the number of cells secreting non-specific immunoglobulins in vitro.

The cultivation of normal mouse spleen cells in a modified Mishell-Dutton system for 1-4 days in the presence of a water-soluble antigen of sheep red blood cells results in a sharp increase in the number of cells secreting non-specific immunoglobulins (nIFC). This increase is much more visible if spleen cells from mice primed with the same antigen 3-4 days before cultivation, are used. The rise in NIFC becomes apparent on day 1 and runs up to maximum on day 3. At this time a peak of 165 X 10(3) nIFC per 10(6) cells is attained, i.e. the nIFC quantity reaches approximately 33% of total B-cells. Kinetics of the antibody-forming cells and nIFC appearance under varying conditions is different. Clearcut differences are also revealed between the mechanisms of regulation of both these populations. The initial population of cells destined to form non-specific immunoglobulin is estimated to be 363/10(6) cells during a primary immune response in vitro; if splenocyte donors are primed with a homologous antigen, this population become approximately 800-1,900/10(6) cells.

[Effect of mitogens on suppression of antibody formation and proliferation in dense cultures]. / Vliianie mitogenov na podavlenie antiteloobrazovaniia i proliferatsii v plotnykh kul'turakh.

A study was made of the effect of mitogens on general proliferation and primary immune response to sheep red blood cells in density-inhibited cultures of mouse spleen cells. The mitogens applied included fetal calf serum and both B cell- and T cell-specific mitogens (dextran sulfate, LPS and ConA). Experiments with 3H-thymidine incorporation demonstrated that the proliferation was equally enhanced by any mitogen in both optimal and density-inhibited cultures. The mitogens did not remove the density inhibition of antibody formation.

[Changes in membrane permeability following a rapid increase in the density of a lymphoid cell suspension]. / Izmenenie pronitsaemosti membran pri bystrom uvelichenii plotnosti suspenzii limfoidnykh kletok.

Incorporation of uridine into the cells was studied under conditions of rapid increase in density of the cell suspension either freshly-prepared from spleen of mice strain C57BI/6 or subjected to precultivation. Absorption of uridine by the cells after an increase in their density was rapidly decreased within several minutes. The phenomenon was not due to presence of dead cells in the culture. An inhibitor of protein synthesis puromycin partially removed the suppression of uridine absorption in dense cultures.

[Immunologic specificity of local inhibitory intercellular interactions]. / Immunologicheskaia spetsifichnost' lokal'nykh tormoziashchikh mezhkletochnykh vzaimodeistvii.

The authors previously reported local intercellular interactions suppressing the increase of the antibody-forming cell (AFC) count in the suspension of the spleen cells of nonimmunized mice and sharply elevating with increase in density of the cultivated suspensions. This work showed that preliminary immunization of mice with an antigen eliminated or sharply reduced the accretion inhibition of the AFC cells against the given antigen, but failed to influence the accretion inhibition of cells producing antibodies against another antigen, and the proliferation inhibition of the dividing bulk cells in the culture.

[Role of distant lymphoid cell interactions for the development of in vitro antibody formation]. / Rol' distantnykh vzaimodeistvii limfoidnykh kletok dlia razvitiia antiteloobrazovaniia in vitro.

In order to clarify the nature of the recently described intercellular local interactions which were shown to inhibit the antibody-forming cells (AFC) proliferation in vitro, a possibility of realization of this effect at a distance was studied. It was shown that the population with a great number of cells could inhibit in vitro at a distance an increase of the AFC in the population of cells separated from the former by a millipore filter, impermeable for the cells. The results were the same with the use of polymethylmetacrylate film (5 to 10 micrometer in thickness) impermeable for proteins with a molecular weight of 150000 daltons (125I-IgG-antibodies) and some ions (51CrO4), but permeable for other substances with low molecular weight.

[Relationship between antibody formation in cultures of lymphoid cells and incubation temperature]. / Zavisimost' antiteloobrazovaniia v kul'turakh limfoidnykh kletok ot temperatury inkubatsii.

An increase of the plaque-forming cells (PFC) and of the 3H-thymidine incorporation in the spleen cell cultures of immunized and nonimmunized C57BL/6 mice were studied in increase of the incubation temperature from 2 to 37 degrees C. An exponential rise of the PFC cells with the elevation of temperature and the presence of the "breaking" temperature, above which the rate of increase of the PFC cells displayed a sharp elevation, was demonstrated. The curves of intensification of the 3H-thymidine incorporation with the temperature elevation failed to follow the pattern of the PFC growth curve in a number of cases. Cultivation of immune cells at low temperatures led to the accumulation in the medium of some factors simulating the AFC formation.

The rapid onset of inhibition of antibody-forming cell proliferation upon an increase in density of cellular suspensions cultured in vitro.

It was shown previously that the development of primary immune response in vitro in the Mishell-Dutton system was strongly inhibited by increased cell densities (Gurvich et al., Immunology 1975. 28: 271). In the present work, the rate of onset of this inhibition was studied in C57BL/6 mouse spleen cell suspensions during the phase of exponential increase of antibody-forming cells (AFC) after 3 days' cultivation in vitro with the antigen. It has been shown that the increase in AFC number is very soon inhibited following an increase in the cellular density, the inhibition already being evident within 2 or 4 h. Parallel reduction in incorporation of radioactive precursors in DNA and especially RNA is observed, whereas inhibition of protein synthesis was delayed.

[Mechanism of antibody formation suppression in high density suspension cultures]. / Mekhanizm podavleniia antiteloobrazovaniia v suspenzionnykh kul'turakh s bol'shoi plotnost'iu

There was a sharp decrease in the number of antibody producing cells formed in induction of primary immune reaction in vitro with increase of surface density (number of cells per 1 cm2 of the bottom of the incubation vessel); the cause of this phenomenon was studied. It was shown that depression of antibody formation in dense cultures was associated not with disturbance of the induction of the clone of antibody-forming cells, but with the inhibition of the subsequent proliferation of the cells of this clone. Disturbance of proliferation was reversible: a reduction of the density of the cultures 1 or 3 days after the beginning of the incubation led to the restoration of the growth of the number of antibody-producing cells. The depressive effect could not be transferred by humoral way from the cultures with a high density to the cultures with a low density; it could not be eliminated by daily replacement of the incubation medium in dense cultures for a fresh nutrient medium.