Electron microscopic observations on antibody-producing lymph node cells.

Lymph node cells of rabbits injected with sheep erythrocytes, identified as antibody-producing by their ability to produce plaques of hemolysis in erythrocyte-containing agar layers, have been examined by electron microscopy, by the use of a procedure devised for subjecting single cells to such examination. The antibody-producing cells thus examined were found to fall into two classes, according to the current terminology: some were in the category of lymphocytes, and others, in the category of plasma cells. Within each class, cells were found to vary in certain characteristics, especially in the degree of development of such organelles as the nucleolus, Golgi apparatus, and the endoplasmic reticulum. In the case of the endoplasmic reticulum especially, it could be seen that a series of these plaque-producing cells, ranked in order of increasing size and development of the endoplasmic reticulum, would extend over a considerable range from those lymphocytes with the least developed organelles to the mature plasma cells with the greatest development of these structures.

Studies on antibody-producing cells. II. Appearance of 3 H-thymidine-labeled rosette-forming cells.

A study of the kinetics of antibody-producing cells has been carried out by the use of rosette formation for detection of individual antibody-producing cells, and labeling with tritiated thymidine, in cells obtained from mouse spleens at intervals after injection of SRBC. Following a primary injection of the antigen, the number of RFC per million cells was found to increase to a peak at 5 days, then, after a decrease, to a second peak at about the 10th day. The curve of tritium labeling of RFC was also biphasic, with peaks on the 3rd and 7th day. The second increase in rosette-forming cells could be shown to involve, especially between the 7th and 9th day, a second increase in lymphoid cell RFC and, among these, 7S antibody-producing cells. When the population examined was restricted to large lymphocytes, two peaks of RFC per million cells and two peaks of labeling were again found. In this case, however, the peaks of RFC and of labeling were reached on the same day in each instance, rather than with the 2 day difference found in the entire spleen cell suspension or the entire lymphoid cell population. Electron microscopic examination of labeled rosette-forming cells showed these to be largely lymphocytes, but to include rather well differentiated plasmablasts as well. No macrophages were found among labeled RFC in the primary response. A substantial number of labeled lymphocytes were found in close contiguity with rosette-forming macrophages. The percentage of labeling in such lymphocytes was as high, on the respective days, as the percentage of labeled cells among the RFC of the entire suspension.

Studies on antibody-producing cells. I. Ultrastructure of 19S and 7S antibody-producing cells.

Antibody-bearing cells of spleen and lymph node of the mouse and rabbit detected by rosette formation with the antigenic red blood cells were collected by micropipet and studied by electron microscopy. More than 300 such cells were examined. In the lymph nodes, rosette-forming cells were all in the lymphocytic and plasmacytic categories. In cells of the mouse spleen, macrophages were also found among the RFC, especially in the later days after immunization. The great majority of the RFC, 70-100%, were of the lymphocytic category. These included small, medium, and large lymphocytes with fine gradations of differentiation, and blast forms with little heterochromatin. The endoplasmic reticulum of these cells occurred in short, very narrow pieces, usually in contact with a mitochondrion. The cells of the plasmacytic category also showed fine gradations from plasmablasts to typical mature plasma cells. Plaque-forming cells of mouse and rabbit were also collected by micropipet. Of 162 such cells, fine gradations were also found throughout the lymphocytic and plasmacytic categories, but in this case the great majority were in the plasmacytic group, and more plasma cells showed amorphous nuclear chromatin. Among antibody-forming cells detected by both reactions, some of the more highly differentiated large lymphocytes contained ER which differed from that in the other large lymphocytes in that the channels were slightly and variably distended, with deposition of some precipitate, and with some tendency to a more nearly parallel orientation of the few channels seen. These were considered transitional cells. Of 10 RFC found in mitosis, all were in the lymphocytic category, in various stages of differentiation, the most advanced of which (in 2 of the 10 cells) was that of the transitional lymphocyte described here. Cells producing plaques facilitated by antisera vs. IgG of the mouse or rabbit (7S) showed the same distribution between cell categories and the same fine gradations as the direct (19S) PFC. Cells producing rosettes which were resistant to lysis in the presence of complement, and were thus presumably producing 7S antibody, showed a distribution similar to that found generally with rosette-forming cells, approximately 80-90% in the lymphocytic category.

Studies on antibody-producing cells. 3. Identification of young plaque-forming cells by thymidine- 3 H labeling.

Mice injected with sheep RBC and then, 4 days later with thymidine-(3)H, were sacrificed on the day of thymidine-(3)H injection or 1 or 2 days later. Hemolytic antibody plaque preparations were made of cells from the draining lymph nodes by a thin-plating procedure permitting collection of isolated PFC for electron microscopic examination and radioautography. Of cells obtained on the day of thymidine-(3)H injections, 65% of the labeled PFC were in the lymphocytic category, in comparison with 13% found previously in the entire population of such cells. The remaining 35% were plasmablasts in early stages of differentiation. Cells obtained 1 day after the thymidine-(3)H injections showed a shift to a majority of labeled cells in the plasmacytic category. Also, the plasmablasts were substantially more differentiated than those of the previous day, and some mature plasma cells were now seen. The labeled PFC obtained on day 2 gave no indication of further differentiation. Cells of rabbit lymph nodes labeled in vitro with thymidine-(3)H showed a range of labeled PFC. The majority were in the plasmacytic category, including some mature plasma cells. The data from the experiments with in vivo labeling suggest a direct differentiation from antibody-synthesizing lymphocytes to plasma cells. Further, the in vivo experiments indicated that differentiation from nascent lymphocyte to plasma cell could be essentially completed within 1 day.

Studies on antibody-producing cells. IV. Ultrastructue of plaque-forming cells of rabbit lymph.

Efferent lymph of the popliteal lymph nodes of rabbits was collected 4 days after a single footpad injection of SRBC. Thin-layer agar plating was done to isolate plaque-forming cells of the lymph for electron microscope examination, and the numbers of plaque-forming cells (PFC) in cells from the lymph and lymph nodes were determined. Of 71 PFC of lymph isolated and examined, 93% were lymphocytes, most of them with signs of substantial levels of physiologic activity. The cytoplasm showed an abundance of free ribosomes and many finger-like projections. The endoplasmic reticulum (ER) was barely detectable in most of the active lymphocytic PFC, and in some, a few short narrow channels of ER could be seen. Approximately one-fifth of the lymphocytic PFC presented an appearance of senescence, with signs of degeneration: rounded cells, with amorphous nuclear chromatin, and very few microvilli. The remaining 7% of the PFC of the lymph showed an unusual combination of features: small round cells with a narrow ring of cytoplasm which, however, contained well-organized channels of ER. Such cells had been found only among PFC of peripheral blood of the rabbit. The number of PFC per million cells was higher in the lymph than in the suspensions of lymph node cells. In both the contralateral lymph node and its efferent lymph, the number of PFC was less than 1% that of the injected side.

Electron microscopic observations on antibody-producing cells in lymph and blood.

Antibody-producing cells have been identified among cells obtained from efferent lymphatic vessels, the thoracic duct, and peripheral blood. These cells, which produced plaques of hemolysis and which were quite rare (20 to 50 per million), due in most instances to 19S antibody, were located and studied by electron microscopy. Of the antibody-producing cells found in these three sites there were several features common to all: small size (5 to 8 micro), generally spherical shape, approximately central position of the nucleus, and retention in the nucleus of the condensations of chromatin characteristic of the lymphocyte. The differences among the cells of these sources were largely in the relative amount and state of organization of the organelles of the cytoplasm. In cells of the efferent lymphatic vessel and the thoracic duct, the endoplasmic reticulum showed a range from relative scarcity to considerable numbers of well organized channels. Between these extremes were cells with a considerable amount of endoplasmic reticulum, the channels disorganized and sectioned at various angles. The cytoplasmic matrix of all of these contained a profusion of polyribosomes. Antibody-producing cells obtained from peripheral blood showed, around the roughly spherical nucleus, a ring of cytoplasm which was narrow, but wholly organized into parallel lamellae of endoplasmic reticulum, with many polyribosomes between these, and a large Golgi body. Some similarities and some differences of these cells, in comparison with antibody-producing cells obtained from lymph nodes, have been indicated.

Human cystinosis: intracellular deposition of cystine.

Membrane-limited inclusions were found in the cytoplasm of cells of the rectal mucosa, leukocytes, and cultured fibroblasts from two humans with cystinosis. Most of the inclusions contained amorphous material, presumably cystine. In cells of the rectal mucosa the material appeared frequently crystallized. This was rarely seen in leukocytes, and never in cultured fibroblasts. The fact that acid phosphatase could be demonstrated consistently in the organelles responsible for sequestration of cystine indicates that they are lysosomes.

Neuronal properties of neuroectodermal tumors in vitro.

Cell lines of medulloblastoma, retinoblastoma, and neuroblastoma, three childhood tumors derived from neuroectoderm, have been compared with respect to their neuronal properties. Neuroblastoma, a neural crest derivative, has been shown to express specific neuronal enzymes and the action potential sodium ionophore. Cell lines of medulloblastoma and retinoblastoma also express neuronal specific enzymes and therefore are considered to be of neuroblastic origin.

Glycopeptides from the surgace of human neuroblastoma cells.

Glycopeptides suggesting a complex oligosaccharide composition are present on the surface of cells from human neuroblastoma tumors and several cell lines derived from the tumors. The glycopeptides, labeled with radioactive L-fucose, were removed from the cell surface with trypsin, digested with Pronase, and examined by chromatography on Sephadex G-50. Human skin fibroblasts, brain cells, and a fibroblast line derived from neuroblastoma tumor tissue show less complex glycopeptides. Although some differences exist between the cell lines and the primary tumor cells, the similarities between these human tumors and animal tumors examined previously are striking.

Establishment and characterization of human neuroblastoma cell lines.

Three new tissue culture cell lines, CHP-100, CHP-126, and CHP-134, have been established from explant cultures of human neuroblastoma. The cell lines have been characterized with respect to morphology, chromosomes constitution, growth, neural enzyme content, and their ability to grow in nude mice. The cells grow as dense masses comprised of fibroblast-or neuroblast-like cells with small processes. The cell lines differ in their neural enzyme acitivity. The chromosomal content of the 3 cell lines is near diploid, and all are capable of forming tumors in nude mice. The morphological findings indicate that the cells in culture resemble those found in the tumor, and the enzyme activities are consistent with those of nervous tissue. This the morphological, biochemical, and tumorigenic properties confirm that the 3 cell lines are neoplastic cells of neural origin.

Early events in herpes simplex virus infection: a radioautographic study.

The early events in herpes simplex virus infection were studied by means of radio-autography. The virus was rapidly taken up by the host cells and uncoated. Viral deoxyribonucleic acid (DNA) reached the nuclear sites of replication in 15 to 30 min after infection. The viral DNA occasionally associated with chromosomes or condensed chromatin but was more frequently found to be randomly distributed. Viral progeny appeared 3 hr after infection. These particles did not show any particular spatial relationship to the parental DNA. The morphological latent period lasted 2.5 hr.

Morphological aspects of the uptake of simian virus 40 by permissive cells.

After exposure of permissive cells to simian virus 40 (SV40), single particles were engulfed by the cell membrane and transported to the nucleus. The cell membrane closed tightly around the particles, increasing their diameter from 40 to 55 nm. The cell membrane was lost during interaction with the nuclear membranes, and particles of the original size were found in the nucleus 1 hr after infection. Uncoating of these nuclear particles occurred rapidly, and none could be found 4 hr after infection. Viral progeny appeared 24 hr after infection.

Morphogenesis of the nucleoprotein of vesicular stomatitis virus.

Accumulation of the nucleoprotein of vesicular stomatitis virus (VSV) in the cytoplasm of BHK-21 cells and in two of four human cell lines was demonstrated. Appearance and progression of the nucleoprotein inclusions paralleled development of virus-specific immunofluorescence and production of virus progeny. The inclusions appeared early as discrete foci of filamentous material which eventually increased in size to form large masses which replaced normal cytoplasmic constituents. The filamentous strands were found in close proximity to budding virions. The inclusion material was extracted from infected cells and purified in cesium chloride gradients. The isolated filaments resembled the ribonucleoprotein isolated from purified virions. They incorporated (3)H-uridine, exhibited virus-specific complement-fixing activity, had a buoyant density of 1.32 g/cm(3), and appeared as single wavy strands the width of which varied from 2.5 to 8.5 nm, depending on the angle of viewing.

Structure and development of rabies virus in tissue culture.

Structure and development of two fixed rabies virus strains in baby hamster kidney cells (BHK/21) were investigated by electron microscopy. The morphological development was correlated with fluorescent-antibody staining and infectivity titration. The uptake of virus was enhanced by addition of diethylaminoethyl dextran, and structural changes became apparent in the cytoplasm 8 to 9 hr after infection, when fluorescent-antibody staining was first discernible. These changes consisted of matrices containing fibers replacing normal cytoplasmic structures. Virus particles appeared at the edges of these matrices and inside them at 24 to 48 hr. This corresponded to significant rises in intracellular infectious virus. Formation of virus particles by budding from cell membranes was seen at 72 hr. Further incubation of the infected cells resulted in synthesis of bizarre structural elements. The complete virus particle was bullet-shaped with an average size of 180 by 75 mmu. It consisted of an inner core of filamentous material surrounded by two membranes of different densities. The surface showed a honeycomb arrangement with surface protrusions 60 to 70 A long having a knoblike structure at their distal end. These surface protrusions were absent at the flat end of the virus particle.

Comparison of the lipids of intracellular and extracellular rabies viruses.

The lipid composition of both intracellular and extracellular forms of the ERA strain of rabies virus grown in BHK/21 cells was determined. The lipids from purified preparations of both intracellular and extracellular virus yielded 57 and 58% neutral lipid, respectively. The phospholipids of the intracellular and extracellular virus constituted 43 and 42%, respectively. Triglyceride and cholesterol appear to be the major neutral lipids, whereas sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine comprise the major bulk of phospholipid in both virus types. The molar ratio of cholesterol to phospholipid was 0.87 (intracellular) and 0.92 (extracellular). On the basis of the data presented, it is reasonable to assume that the lipids of both intracellular and extracellular rabies virus are similar.

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.

New assay procedure for separation of mycoplasmas from virus pools and tissue culture systems.

Presence of mycoplasma organisms in tissue culture systems and virus pools was detected by titration of the contaminated material on agarose-suspended BHK21/13S cells. The use of this method permitted isolation of mycoplasmas which could not be detected by standard assay methods. Mycoplasma colonies at concentrations ranging from 10(4) to 10(6) colony-forming units/ml in agarose-BHK21/13S media could be distinguished from virus plaques, and the two populations of microorganisms could be easily disassociated either by electron microscopy or by biological methods. All isolated mycoplasmas were identified in growth inhibition tests as belonging to the GDL group. The growth inhibition test on agarose-BHK21/13S cell suspension plates could also be applied directly to those strains which could not be isolated by standard assay procedures.