IMMUNOGLOBULIN SYNTHESIS AND SECRETION : VI. SYNTHESIS AND INTRACELLULAR TRANSPORT OF IMMUNOGLOBULIN IN NONSECRETORY LYMPHOMA CELLS

Cells from an established line of Burkitt's lymphoma (Daudi) and a mouse myeloma (P₃K) were pulse-labeled in vitro with ³H-leucine, and immunoglobulin was immunologically precipitated from cell lysates and secretions. In contrast to P₃K cells, Daudi cells synthesize a small amount of Ig which is not secreted. Subcellular fractionation experiments indicated that Ig of Daudi cells is synthesized on membrane-bound polyribosomes and enters the cisternae of the microsomes. Ig in the microsomes could be labeled with either ³H-galactose or ³H-fucose suggesting that transport proceeds to the Golgi complex. Additional evidence indicates that Ig molecules are transported to the plasma membrane but are not cleaved from the cell surface. These results together with other studies of Burkitt lymphoma cells suggest that the Daudi line may represent a clone of neoplastic cells derived from normal lymphocytes which synthesize but do not secrete Ig. Similarities between lymphoma cells and antigen-binding cells are discussed.     

COMPLEMENT FIXATION BY A TWO-COMPONENT ANTIBODY SYSTEM: IMMUNOGLOBULIN G AND IMMUNOGLOBULIN M ANTI-GLOBULIN (RHEUMATOID FACTOR) : PARADOXICAL EFFECT RELATED TO IMMUNOGLOBULIN G CONCENTRATION

Complement-mediated lysis of sheep erythrocytes coated with optimal concentrations of rabbit IgG hemolysin was inhibited by euglobulin fractions from the sera of patients with seropositive rheumatoid arthritis. That this was due to direct interaction with the IgG coat on the red cell rather than a nonspecific reaction with complement in the fluid phase was confirmed by controls using cells coated with IgM hemolysin. The inhibitory activity was recovered in purified IgM rheumatoid factor preparations and could be absorbed out with insoluble aggregated human IgG. The inhibitory potency of the rheumatoid factors correlated well with their sheep cell agglutination titers. Inhibition was not the result of physical aggregation of the erythrocytes by rheumatoid factor. Kinetic studies were consistent with the view that rheumatoid factor displaces C1q from its binding to IgG. Paradoxically, at suboptimal sensitizing concentrations of IgG hemolysin, rheumatoid factor enhances the fixation of complement. These results can be interpreted on the basis of the blockage of complement fixation by IgG and its replacement by a relatively weak direct fixation by the IgM rheumatoid factor. Thus, the interaction of RF with IgG generates only a limited ability to fix complement which, when contrasted with the fixation at suboptimal concentrations of IgG hemolysin alone, appears as net enhancement; when this is contrasted with fixation occurring with optimal concentrations of IgG, it appears as net inhibition.     

巢式RT-PCR对肺癌外周血中微转移灶的检测和半定量测定

目的对肺癌外周血中微转移灶的检测和半定量测定。方法以CK-19为标记,采用巢式RT-PCR和半定量方法对肺癌患者外周血中微转移灶的检测,比较治疗前后外周血中癌细胞数的相对变化。结果60例肺癌中有29例CK-19阳性,18例健康人中,仅1例检测出CK-19阳性,7例肺良性疾病患者有2例为CK-19阳性。实验组CK-19阳性率48%(29/60)与对照组12%(3/25)相比有极显著差异(P<0.01)。2例肺癌患者化疗前相对癌细胞数分别为25和16个,经1周期化疗后,相对癌细胞数分别为5和1个。结论以CK-19为标记,采用巢式RT-PCR方法检测肺癌患者外周血中的癌细胞有较高敏感性和特异性,半定量RT-PCR法,能早期反映外周血中癌细胞数的相对变化情况,有助于对临床疗效及早做出评价。     

白血病大鼠化疗后血液中集落刺激因子、造血干细胞和残留白血病细胞动态观察

动态观察了白血病大鼠大剂量化疗后血液中血清集落刺激因子(CSF)、造血干细胞(HSC)和残留白血病细胞的变化。研究结果证明,白血病化疗后血清CSF最早出现,然后HSC增殖并向周围血液迁移,接着周围血细胞开始恢复,血液中白血病细胞出现最晚。随着血液中白血病细胞的增加,正常血细胞随之下降。揭示正常造血细胞与白血病细胞相互竞争而消长的规律,无疑有助于我们了解白血病复发的机理。     

INDUCED ANTIBODIES THAT REACT IN VITRO WITH SEDIMENTABLE CONSTITUENTS OF NORMAL AND NEOPLASTIC TISSUE CELLS : PRESENCE OF THE ANTIBODIES IN THE BLOOD OF RABBITS CARRYING VARIOUS TRANSPLANTED CANCERS

Antibodies were found in the blood of certain rabbits carrying one or another of four transplanted cancers (Brown-Pearce and V2 carcinomas; RSI and Kato sarcomas) which will fix complement in vitro in mixture with saline extracts of various normal and neoplastic rabbit tissues—including liver, kidney, spleen, and the four tumors mentioned—and chick embryo tissue as well. These antibodies, which have been called induced tissue antibodies, are similar to the natural antibodies previously described (2) in that they react with those constituents of the various tissue cells that prove readily sedimentable in the high speed centrifuge; they differ from the natural antibodies in being absent from the blood of normal rabbits and in withstanding 65° C. for 30 minutes. Certain quantitative differences suggest that the induced tissue antibodies have somewhat various affinities, depending in part upon the type of neoplasm carried by the host. They may perhaps be consequent on antigenic differences between the sedimentable constituents of the tumor cells and those of the new hosts; for they were not found in the blood of rabbits carrying papillomas and cancers composed of the animals'' own cells, and not in that of rabbits in which multiple vaccinia or fibroma virus lesions had recently regressed. The characters of the sedimentable constituents of normal and neoplastic tissue cells, as revealed thus far by chemical, morphological, and serological studies, have recently been discussed (2,8). In this relation, it has seemed essential to recognize the induced antibodies here described, particularly since they may complicate serological studies aimed at disclosing distinctive sedimentable substances in tissue cells. In an associated paper experiments are reported which bear upon the relation between the induced tissue antibodies and an antibody that reacts specifically with a distinctive sedimentable constituent of Brown-Pearce carcinoma cells (7).     

CELLULAR DIFFERENTIATION OF THE IMMUNE SYSTEM OF MICE : VI. STRAIN DIFFERENCES IN CLASS DIFFERENTIATION AND OTHER PROPERTIES OF MARROW CELLS

Marrow cells and 5 x 10⁷ thymocytes of unprimed (C57BL/6 x DBA/2)F₁, (C57BL/10 x WB)F₁ and (C3H x C57BL)F₁ donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. Upon injection of sheep erythrocytes, splenic plaque-forming cells (PFC) secreting IgM (direct PFC or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By grading the numbers of marrow cells, inocula were found that contained few immunocompetent cells reaching the recipient spleens, interacting with thymocytes or other accessory cells (or both), and generating PFC. The frequency of responses in BDF₁ mice conformed to Poisson statistics, indicating that immunocompetent marrow cells participated in a single-hit interaction limiting PFC responses. The marrow cells assayed were not restricted for the antibody class (IgM versus IgG) to be secreted by mature PFC. Unrestricted marrow cells could have been either the precursors of PFC or accessory cells. Different results were obtained in BWF₁ and C3BF₁ mice. The frequency of responses in relation to the number of marrow cells grafted did not follow Poisson statistics, and the limiting cells were restricted for antibody class. Presumably, immunocompetent cells of these strains were more heterogeneous than those of BDF₁ mice and participated in a multiplicity of cell-to-cell interactions. The strain differences reflected inherent properties of marrow cells and not influences of the environment in which PFC were produced. The results confirmed for bone marrow the heterogeneity of immunocompetent cells reported by others for spleen, and suggested that genetic factors such as "immune response" genes regulate cellular differentiation also for functions other than those related to antibody specificity.     

STUDIES UPON THE EFFECT OF LIGHT ON BLOOD AND TISSUE CELLS : I. THE ACTION OF LIGHT ON WHITE BLOOD CELLS IN VITRO.

1. An extreme and rapid degeneration which occurred in tissue cultures of leucocytes from the blood of cats, guinea pigs, and rabbits, is described in detail. 2. This degeneration was found to appear in the culture when the cells were planted in any of the culture media tried, some of which were autogenous heparin plasma, autogenous plasma, autogenous serum, Tyrode solution, and mixtures of these with embryo juice. 3. The specific cellular changes which occurred are described for the different leucocytes. In general, there was first a latent period during which no change could be observed in the cell. Following this there was a period of stimulation during which the motion of the cell was greatly accelerated. This second stage has been observed in all cells except the lymphocyte, in which it may possibly occur to a slight degree. Finally there was the terminal stage, the stage of degeneration, in which the cell rounded up, lost its motility, and either became badly swollen or else underwent a more or less complete coagulation. 4. The factor causing this degeneration was found to be exposure of the culture to light, as, for example, during microscopic examination. 5. By a reduction of the infrared part of the spectrum, it was indicated that the effect was not due to a heat coagulation of the cells. 6. This degeneration was also found to occur in the complete absence of ultra-violet wave-lengths. 7. Further, it was shown that this degeneration was caused by light which lay within each of the three wave-length zones (1) 430µµ to 550µµ; infra-red; (2) 475µµ to 630µµ; 690µµ to infra-red; (3) 600µµ to infra-red. 8. No indication was given as to whether all regions of these zones were active in causing the degeneration, or whether the active rays are limited to certain wave-length bands lying within these zones. 9. This degeneration of the leucocytes under the action of light was also found to occur upon irradiation of hanging drops of whole blood. This is interpreted as showing conclusively that the degeneration was not dependent upon the additional factors of centrifugation, continued lowering of temperature, or the presence of abnormal saline solution. 10. It was noted, however, that the leucocytes in hanging drop cultures required a markedly longer time for their degeneration under the action of light than did the leucocytes in cultures prepared from the buffy coat and inoculated in serum. This is considered as possibly due, either to injury to the cell during centrifugation and subsequent handling, or to some action of the red blood cells present in large amounts in the hanging drops of whole blood. 11. In these hanging drop cultures of whole blood degeneration of the leucocytes was also found to occur when the light reaching the culture was first freed from the larger part of its infra-red and from all of its ultra-violet. 12. It was also shown that the same degeneration was produced by wave-lengths of light lying within each of the three wave-length zones defined in Section 6 of this summary.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : XVI. THE RESPONSE OF IMMUNE RABBIT CELLS TO PHYTOHEMAGGLUTININ, ANTIGEN, AND GOAT ANTI-RABBIT IMMUNOGLOBULIN ANTISERUM

There exists in the rabbit a population of lymphocytes carrying immunoglobulin-like receptors on their surface. These receptors interact with antigen and with anti-immunoglobulin antibodies and appear to mediate the recognition process leading to the humoral immune response. There exists in the rabbit a second population of lymphocytes capable of reacting with phytohemagglutinin. This population of lymphocytes is different from the one capable of reacting with soluble protein antigens or anti-immunoglobulin antiserum and is probably involved in the mediation of cellular immunity.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : XII. THE DIFFERING RESPONSES OF NORMAL RABBIT LYMPHOID CELLS TO PHYTOHEMAGGLUTININ, GOAT ANTI-RABBIT IMMUNOGLOBULIN ANTISERUM AND ALLOGENEIC AND XENOGENEIC LYMPHOCYTES

Cells of the different lymphoid organs in the normal adult rabbit were investigated for their capacity to respond in vitro to a number of stimuli, such as phytohemagglutinin (PHA), anti-rabbit immunoglobulin antiserum (GARIG) and allogeneic and xenogeneic lymphoid cells, and for their capacity to adsorb radioactively-labeled anti-immunoglobulin antiserum. The bone marrow cells responded minimally to PHA, GARIG, and the allogeneic and xenogeneic stimuli. The thymus cells were unable to respond to stimulation with GARIG although they responded to the other stimuli. The cells of the other lymphoid organs tested responded to all the mitogenic agents, to varying degrees. On the basis of the results presented and the findings of other investigators, it is concluded that: 1. The response of the cells to GARIG indicates a potential capacity to mediate humoral immunity and requires the presence of immunoglobulin or immunoglobulin-like recognition sites on the cell surface. 2. The response of the cells to PHA and allogeneic and xenogeneic cells indicates a potential capacity to mediate cellular immunity and does not necessitate the presence of immunoglobulin-recognition sites on the cell surface. 3. The thymus in the normal adult rabbit consists of cells capable of mediating cellular immunity only. 4. The other lymphoid organs appear to possess cells capable of mediating humoral and cellular immunity.     

IMMUNOLOGICAL FUNCTIONS OF HUMAN T-LYMPHOID CELL LINE (MOLT) : I. RELEASE OF IMMUNOSUPPRESSIVE FACTORS FROM THE MIXTURE OF MOLT-4 CELLS AND SHEEP RED BLOOD CELLS

A short term incubation of the mixture of established human T-lymphoid cells (MOLT) and sheep red blood cells (SRBC) resulted in the release of factors which nonspecifically suppressed the response of mouse spleen cells against heterologous erythrocytes in vitro. Neither human B-cell line (RPMI 1788), nor the supernate of MOLT cell suspension in the absence of SRBC had such suppressive effects. The supernate of the mixture of MOLT cells with chicken red blood cells (CRBC) did not suppress either anti-CRBC or anti-SRBC responses of mouse spleen cells. Since CRBC did not form rosettes with MOLT cells, it is suspected that the origin of the production of these factors might be MOLT cells forming SRBC rosettes. Some of these factors are dialysable.     

THE OCCURRENCE DURING ACUTE INFECTIONS OF A PROTEIN NOT NORMALLY PRESENT IN THE BLOOD : III. IMMUNOLOGICAL PROPERTIES OF THE C-REACTIVE PROTEIN AND ITS DIFFERENTIATION FROM NORMAL BLOOD PROTEINS

The C-reactive protein present in the albumin fraction of the serum of patients during certain acute bacterial infections is highly antigenic upon injection into rabbits. The antiserum thus prepared reacts specifically with this protein and does not react with the proteins of normal human serum. Immunological specificity has been demonstrated by both precipitin and complement-fixation tests. Antiserum prepared in rabbits to the C-reactive protein from human sources also reacts specifically with the similar protein in the serum of monkeys acutely ill with experimental pneumococcus infection. By means of immunological reactions it is possible to detect amounts of reactive protein which are too small to yield a visible precipitate in tests with the C polysaccharide. Certain of the properties are discussed which distinguish the C-reactive protein from the proteins of normal human serum.     

SEROTONIN (5HT) IN MIGRAINE: LEVELS IN WHOLE BLOOD IN AND BETWEEN ATTACKS

SYNOPSIS
Fluorimetric estimations of total 5HT in blood of 11 migrainous patients during an untreated migraine attack were made. Total blood 5HT dropped during a migraine attack to about 68% of its former level returning to preheadache values by the time the pain ceased. Mean values for total blood serotonin (ng/ml) were 65.1 at the beginning of a migraine attack, 57.9 at the peak, 61.0 at the end, and 91.2 in the headache free period (p<0.05). No correlation between the intensity of the headache and the concentration of 5HT during a migraine attack was found (p>0.5). The influence of some drugs upon fluorescence in blood of migraineurs was tested.     

STUDIES UPON THE EFFECT OF LIGHT ON BLOOD AND TISSUE CELLS : II. THE ACTION OF LIGHT ON ERYTHROCYTES IN VITRO.

1. It was found that when hanging drops of whole blood, drawn from a rabbit, were subjected to irradiation from certain light sources, a striking degeneration of the white and of the red cells occurred. In this degeneration of the red cells there was (a) a preliminary period of 15–30 minutes during which no effect was noted; (b) following this, there was a period during which the cells swelled and became first almost biplanar, then biconvex, and finally spherical. After a spherical shape had been assumed, the liberation of blood pigment from the cells began. This process progressed with varied rapidity, sometimes being very slow, and being incomplete; sometimes being of almost explosive rapidity. This liberation was not accomplished in either case through a gross rupture of the cells, but through the agency of some submicroscopic change produced either in the cell contents, the cell wall, or both. Following this liberation of pigment, in some cultures the red cells showed signs of coagulation, as did the white cells. In other cultures, however, the red cells were reduced to achromatic shadows. 2. When whole blood was diluted with unbuffered Locke solution and then irradiated in hanging drop preparations, the erythrocytes swelled, as in whole blood, but then, instead of an almost instantaneous hemolysis of every cell present, or a slow liberation of hemoglobin from each individual cell, all the red cells hemolyzed, one or two at a time, the hemolyzed cells being left as achromatic shadows. 3. This hemolysis of the red cells, as in the case of the degeneration of the white cells, occurred upon irradiation of the culture with white light, or with light lying in each of the three spectral zones of the visual spectrum, defined by Wratten filters Nos. 45, 58 and 29 respectively, as follows: (a) 430µµ–550µµ; infra-red; (b) 475µµ–630µµ; 690µµ-infra-red; (c) 600µµ-infra-red. 4. Within the range of intensities of light employed, there was little or no difference in the rate at which light of these three regions acted on the red cells. Further, this rate was the same as was the rate when the red cells were irradiated with no colored filter interposed in the optical path, or when a 5 per cent total transmission neutral filter was interposed. 5. It was shown that this degeneration of the red cells under the action of light was not dependent on the presence or absence of serum, with the possible exception of that trace which might have been adherent to the red cells even after repeated washing. 6. The white cells in the culture generally showed no changes until after traces of red cell pigment could be seen free in the surrounding medium. 7. On the other hand, no neutrophils were observed moving around for longer than a few minutes after the liberation of red cell pigment had occurred. 8. The liberation of substances from the red blood cells, as a result of irradiation of such preparations, may play some major rôle in the degeneration of irradiated white cells.     

STUDIES UPON THE EFFECT OF LIGHT ON BLOOD AND TISSUE CELLS : III. THE ACTION OF LIGHT ON FIBROBLASTS IN VITRO.

1. In the presence of autogenous red blood cells, fibroblasts, grown in vitro from the heart and intestine of embryo chicks of from 6–12 days incubation, underwent a rapid degeneration when exposed to light of the visual spectrum. 2. In this degeneration, the cells showed a marked increase in refractive index, and a massive formation of colorless vacuoles of low refractive index. These vacuoles gave no reaction with osmic acid or Sudan III, but took up neutral red. Later in the degeneration the cells showed marked signs of rounding up and of coagulation. In some cases this degeneration process was modified in such a manner that, instead of the formation of vacuoles, the whole cytoplasm became much less viscid and the cells swelled greatly and became spherical. 3. The similarity of this degeneration to that shown by the erythrocytes, and more particularly, to that shown by the polymorphonuclear neutrophils, under the action of light, is pointed out. 4. Degeneration of the fibroblasts occurred when the cells were irradiated by light of any one of the following wave-length zones: (a) 430µµ–550µµ; infra-red; (b) 475µµ–630µµ; 690µµ-infra-red; (c) 60µµ-infra-red. 5. This degeneration was much slower for the cells irradiated through the blue or red filters of less than 5 per cent, and less than 6.6 per cent, respective total transmissions. When irradiated through the green filter of 23 per cent total transmission it was much more rapid, and even more rapid than for cultures irradiated with white light of 100 per cent total transmission. These data were obtained on a short series of cultures and must be considered only as suggestive. 6. In the apparent absence of red cells, the fibroblasts underwent only a slight degree of degeneration after irradiation lasting from 13–24½ hours. It is possible that even this minor degeneration may depend upon the presence of traces of disintegration products of red cells in the culture. Much more advanced degenerative processes were obtained, when many red cells were present, after only 3 hours irradiation.     

STUDIES ON THE NATURE OF THE AGENT TRANSMITTING LEUCOSIS OF FOWLS : I. ITS CONCENTRATION IN BLOOD CELLS AND PLASMA AND RELATION TO THE INCUBATION PERIOD

The concentration of the transmitting agent of leucosis in fowls, as determined by titration, is approximately the same in the suspensions of blood cells and in cell-free plasma; the smallest amount of plasma producing leucosis was 0.000001 cc. and of cell suspension 0.00001 cc. This observation excludes the possibility that transmission of leucosis by plasma is due to the presence of a small number of leucemic cells in the plasma. The success of inoculations with plasma (20 to 28 per cent of fowls) is, within wide limits, independent of the amount injected (10^–1 to 10^–6 cc.). The percentage of successful inoculations with varying quantities of plasma is lower than with corresponding amounts of suspensions of cells (33 to 71 per cent). When plasma containing the transmitting agent is injected in decreasing amounts the incubation period of the leucosis is conspicuously lengthened. With decreasing amounts of a suspension of leucemic cells the incubation period is not so frequently nor so greatly prolonged.     

IMMUNE LYSIS OF NORMAL HUMAN AND PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) RED BLOOD CELLS : III. THE MEMBRANE DEFECTS CAUSED BY COMPLEMENT LYSIS

1. The defects produced on the membrane of the human red blood cell by the action of complement and antibody have been studied by the use of the electron microscope. These are round to slightly ovoid holes and are surrounded by an irregular ring, about 20 A thick. The mean diameter of the holes is about 103 A if human complement is used (regardless of the antibody used for sensitization) and about 88 A if guinea pig complement is used. 2. The holes in normal and PNH red cells appear to be identical, under the same conditions. The membrane defects produced by lysis of PNH cells with acidified normal serum (the Ham's test) are identical to those produced by complement lysis with specific antibody, indicating that complement is undoubtedly the cause of such lysis. 3. Evidence is presented that when human complement acts on human red cells sensitized with anti-I antibody, each complete activation of complement leads to the production of a cluster of holes. This contrasts to the action of guinea pig complement, on sheep cells, each activation of which leads to a single hole. 4. The maximum number of anti-I antibody molecules which can attach to a human red cell (i.e. the minimum number of antigen sites) is about 500,000 for both normal and PNH cells. 5. The number of holes produced during lysis of the PNH cell is the same as that of the normal cell. When all cells are lysed by am excess of C', a mean of about 90,000 holes are present on each membrane. When complement is limited, a larger proportion of PNH cells are lysed due to their peculiar sensitivity to C' but the number of holes on each lysed cell is the same as for normal cells lysed by the same concentration of C'.