Localization of immunological complexes fixing beta1C (C3) in germinal centers of lymph nodes.

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the beta(1C) component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit beta(1C), IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, beta(1C), and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for beta(1C), IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the beta(1C) component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.

Marrow cytometry and prognosis in myeloma.

We have previously shown that flow cytometric analysis of acridine orange-stained bone marrow cells is useful for the objective enumeration and characterization of plasma cells from patients with myeloma, frequently exhibiting an abnormal DNA and an elevated RNA content. In this report on 77 previously untreated patients, we have investigated the biologic and prognostic implications of these quantitative tumor cell parameters. The degree of marrow involvement by tumor, both by microscopic and cytometric analysis, correlated with the clinically derived tumor mass stage. Examination of the product of relative tumor cell RNA content and marrow tumor infiltrate (as a measure of metabolic capacity for immunoglobulin production) in relationship to the myeloma protein concentration in the serum revealed differences in the efficiency of immunoglobulin production and/or catabolism. There was an inverse relationship between the degree of marrow tumor involvement and RNA index, suggesting a more aggressive behavior of myeloma in patients with a low tumor cell RNA content. Prognostically, high tumor cell RNA content identified patients with a high likelihood of response to both initial treatment (32 patients, P = 0.004) and salvage therapy (29 patients, P = 0.01). Favorable factors for survival were low clinical tumor mass stage (P = 0.07) and low marrow tumor infiltrate as determined morphologically (P = 0.04) and cytometrically (P = 0.004). Thus, the direct examination of marrow cellular DNA and RNA content permitted assessment of tumor burden and was useful in the prediction of response and survival.

Altered expression of growth-regulated protooncogenes in human malignant plasma cells.

The expression of three growth-regulated protooncogenes, c-myc, c-myb, and p53, and the S-phase-specific histone H3 gene, was compared in bone marrow cells from multiple myeloma patients and normal controls by measuring the amount of specific RNA by Northern blot analysis. Four samples contained at least 72% of myeloma cells, one sample 43%, and one 11%. Expression of the protooncogenes was similar in normal and myeloma bone marrow cells, whereas that of histone H3 gene was significantly reduced (between 10 and 15 times) in samples containing at least 43% of malignant plasma cells and not detectable in those containing more than 72% of neoplastic cells. Protooncogene levels of expression were compared to those of the H3 gene to distinguish the increased expression of a growth-regulated gene due to a true deregulation from overexpression reflecting solely an increase in the fraction of cycling cells. The ratios of expression of protooncogenes to histone H3 were markedly increased in multiple myeloma cells; the highest ratios were found in the patients with the highest number of malignant plasma cells. These results suggest that the expression of three growth-regulated oncogenes (c-myc, c-myb, p53) is altered in myelomatous plasma cells.

The localisation of intracellular immunoglobulin and alpha-1-antitrypsin by immunoelectron staining of post-osmicated, resin-embedded tissue.

An indirect immunoperoxidase method is described to demonstrate intracellular immunoglobulins and alpha-1-antitrypsin in semithin and ultrathin sections from human tissue. The tissue was primarily fixed in glutaraldehyde, post-osmicated and resin-embedded.

Biclonal and hypodiploid multiple myeloma.

Plasma cell DNA and RNA content was measured by flow cytometry of acridine orange-stained bone marrow cells from 72 untreated patients with multiple myeloma. Biclonal or hypodiploid DNA stemlines were identified in 10 patients, nine of whom had a low RNA content and did not have response to chemotherapy. Biclonal tumors often showed atypical myeloma protein changes with chemotherapy, suggesting that one clone was reduced whereas the other remained unchanged. These findings suggest a genetic basis for the resistance of low RNA tumors to chemotherapy.

Immunohistochemistry in bone marrow diagnosis. Value of a panel of monoclonal antibodies on routinely processed bone marrow biopsies.

To see if immunohistochemistry can be used on routinely processed bone marrow biopsies for diagnostic purposes, 73 biopsy specimens, fixed in sublimate-formaldehyde, decalcified in an acetic acid-formaldehyde mixture, and embedded in paraffin, were studied with a panel of antibodies. The specimens included "normal," lymphomatous, and myeloproliferative disorders as well as some biopsies with metastatic carcinoma. The results show that the different cell lines and their localization in the bone marrow can be easily identified and quantitative and qualitative changes can be assessed. Megakaryopoiesis is identified with Factor VIII-related antigen and Ulex europaeus agglutinin (UEA); myelopoiesis stains with MT-1, elastase, Leu M-1, LN-2, LN-3, HECA 452, and 115D8; and in myeloproliferative conditions, myeloblasts and promyelocytes stained with leukocyte common antigen (LCA). Erythroid cells stained with UEA, glycophorin A, and LN-1. Lymphocytes were marked with LCA, MB-2, and LN-2. Plasma cells were stained best with immunoglobulin light chain antisera; only occasional reactivity with LCA and 115D8 was observed. Carcinomas all reacted with MB-2; occasional reactivity with 115D8, HECA 452, LN-1, LN-2, MT-1, and Ber H-2 was seen. A small panel of selected antibodies, such as UEA, Leu M-1, and LCA, and the immunoglobulin light chain antisera can be very helpful in bone marrow diagnosis and would cover most indications.

Immunoglobulin-producing cells in CSF and blood from patients with multiple sclerosis and other inflammatory neurological diseases enumerated by protein-A plaque assay.

Using the Protein-A plaque assay, numbers of IgG + IgA + IgM producing cells determined in patients with multiple sclerosis (MS) were 0.1-5% in CSF and 0.1-0.7% in peripheral blood; interestingly, 7 of 11 MS patients had IgM producing cells in CSF. In patients with aseptic meningitis (AM), the corresponding values were 0.04-7.5% in CSF and 0.4-2.4% in peripheral blood. There were more Ig producing cells in peripheral blood from patients with AM and MS than in healthy subjects. A correlation between numbers of IgG producing cells in CSF and the concentrations of intrathecally produced IgG (CSF IgG index) was registered in patients with AM; the same was the true for IgA. The Protein-A plaque method, adopted for 20 X 10(3) lymphocytes, makes possible enumeration of Ig-producing cells in CSF and discrimination among cells secreting different Ig classes, thereby being a powerful tool for studying immune reactions in the CNS-CSF compartment.

Immunofluorescence labeling indices in myeloma and related monoclonal gammopathies.

We measured plasma cell labeling indices (LI) in 52 patients with monoclonal gammopathies. Cytoplasmic reactivity with polyspecific or kappa- and lambda-specific light chain anti-Ig reagents identified monoclonal plasma cells, plasmablasts, and lymphocytoid plasma cells. Among newly diagnosed untreated patients, a high immunofluorescence LI distinguished those with multiple myeloma (MM) from those with stable monoclonal gammopathies (P less than 0.002). Among treated patients with MM, those in the plateau phase of the disease had low LI, whereas patients in the relapse phase or early in treatment had high LI. The immunofluorescence LI correctly classified three more patients with newly diagnosed MM than did the tritiated thymidine LI technique. LI specific for the neoplastic plasma cells resulted in excellent discrimination of patients with active disease. Because results are easily and rapidly obtained, this technique is useful clinically.

Plasma cell iron--clinical and morphologic features.

In order to identify the major clinical features and laboratory findings in patients with plasma cell iron, the authors reviewed the medical records and marrow aspirates of 53 consecutive patients with plasma cell iron hospitalized at Nashville Veterans Administration Hospital over a seven-year period. Plasma cell iron was associated most commonly with alcoholism and occurred in marrows with increased, normal, and decreased iron stores. In patients with decreased marrow iron, plasma cells were the major site of stainable iron. Plasma cell iron was found in patients without other morphologic changes of alcoholism such as megaloblastosis, erythroid vacuolization, and ringed sideroblasts. Plasma cell iron could be demonstrated in biopsy and autopsy material from extra-marrow sites. Ultrastructural studies showed iron always was located in membrane bound lysosomal vesicles of plasma cells.

Eosinophilia and plasmacytosis of the bone marrow in Hodgkin's disease.

Eosinophilia and plasmacytosis of the bone marrow were found in a group of patients with newly diagnosed Hodgkin's disease. An attempt was made to correlate these cytologic findings with other modalities used in the staging of patients with Hodgkin's disease and believed to have prognostic importance, such as age, sex, histologic type of Hodgkin's disease, presence or absence of bone marrow involvement, and pathologic stage. Statistical analyses of these finding s indicated that eosinophilia and plasmacytosis occur frequently but to date appear to be nonspecific findings. Whether the occurrence of eosinophilia and plasmacytosis is related to an immune response in unknown.

Distribution and concentration of cyclosporin in human blood.

In patients receiving cyclosporin to minimise graft versus host disease after allogeneic bone marrow transplantation, whole blood cyclosporin concentration was roughly twice the serum concentration when blood was separated at 37 degrees C. In turn, blood separation at 37 degrees C resulted in a doubling of serum cyclosporin concentration compared with separation at room temperature. In vitro studies showed that the latter phenomenon was due to a temperature dependent partitioning of cyclosporin between plasma and red cells, such that increased cyclosporin was taken up from the serum into red cells at room temperature. Increasing delay in separation of patient blood (at either temperature) resulted in a gradually increasing cyclosporin serum concentration. Further in vitro studies showed that a distribution equilibrium between blood components was reached within 30 min incubation. Red cell uptake of cyclosporin was saturable at an incubation concentration of greater than 4 microgram/ml, while plasma and mononuclear cells showed a linear uptake to 7 micrograms/ml. The cellular cyclosporin content of a mononuclear cell was roughly 1000 times greater than that of an erythrocyte. For clinical monitoring we recommend the measurement of cyclosporin concentration either in whole blood or in serum separated at 37 degrees C without delay after venepuncture.

Ultrastructural study of human myeloma cells in relation to its function.

The ultrastructure of neoplastic plasma cells from a patient with prolonged multiple myeloma was studied in relation to its function, that is, the secretion of immunoglobulin light chain. Peroxidase-labelled antibodies, each monospecific to its immunoglobulin component chain, were used to localize intracellular immunoglobulin within myeloma cells under the electron microscope. By this method, only the kappa type light chain was detected within myeloma cells in bone marrow tissue of this patient, indicating that the occurrence of free kappa type light chains in serum and urine was due to the cessation of heavy chain synthesis within the myeloma cells. The kappa chain was demonstrated as conspicuous electron-dense precipitates in ergastoplasm, its cisternal space, external layer of nuclear membrane, and ribosomes associated with ergastoplasm and nuclear membrane. No immunoglobulin was demonstrated in an atypical Golgi complex, an organelle which is ordinarily engaged in protein synthesis. Numerous crystalline structures and similar inclusion bodies found in myeloma cells appeared to have arisen from the Golgi area, but they did not ever react with the peroxidase label. Discharge of the kappa chain from the cell seems to be carried out through cell fragmentation, possibly caused by progressive distension of the ergastoplasmic cavity.

Cytology of myeloma cells.

A cytological, cytochemical, and cytometric study of plasma cells from 195 cases of multiple myeloma showed that, contrary to earlier reports, flaming cells, thesaurocytes, and intranuclear inclusions are not confined to IgA-secreting cases but are common also in IgG and Bence Jones varieties of myeloma. IgA-secreting cells are not larger, nor do they have a lower nuclear cytoplasmic ratio than other myeloma cells. On average, for a given mass of tumour, Bence-Jones, IgG, and IgA varieties of myeloma produce amounts of paraprotein in the ratio 1 to 1-6 to 2-7.

The plasma cells in inflammatory disease of the colon: a quantitative study.

Immunoglobulin-containing cells have been counted in the mucosa of colectomy specimens from patients with ulcerative colitis and Crohn's disease. An increase in IgA cells occurs in both when compared with normal. In Crohn's colitis the increase is significantly higher than in ulcerative colitis. The importance of these findings in relation to pathogenesis is discussed.

Extracellular paraprotein globules in a patient with monoclonal gammopathy.

A patient with a plasma cell disorder and IgA lambda paraprotein had the unusual finding of numerous, large, opaque, extracellular globules in her bone marrow and liver. While plasma cell intracellular inclusions are well documented, identification of such material in extracellular sites is not. These globules were of various sizes, stained light to dark blue with Wright's stain, were gray in hematoxylin-eosin preparations, and were fluorescent when stained with conjugated antibodies to lambda, but not to kappa, light chains. They strongly resembled the inclusions (Russell bodies) seen in plasma cells and are believed to be massive accumulations of immunoglobulin. The clinical significance of this morphological finding is unknown, but physicians interpreting bone marrow specimens should be aware of this unusual feature.

Enteral and parenteral responses in mice after primary infection with Trichinella spiralis (Nematoda).

Enteral and enteral-parenteral infections were produced with T. spiralis in albino, Swiss Webster, outbred mice. Primary enteral infections abbreviated with thiabendazole stimulated inflammatory changes in Peyer's patches and the lamina propria of the small intestine of mice. These changes were accompanied by increased IgA in the intestinal luminal wash. Primary enteral-parenteral infections similarly stimulated the gut, and, in addition, the spleen. Splenic stimulation resulted in production of IgG1, and IgG2 antibodies specific for T. spiralis L3.

Inflammatory cell subpopulations in the middle ear mucosa of ears with effusion.

Middle ear mucosal biopsies were taken from 11 patients with middle ear effusion. In 8 cases the specimens were sufficiently large to allow detailed studies of the submucosal cellular components. It appears that in noncomplicated serous middle ear effusion, due to mechanical obstruction of the Eustachian tube, the submucosa is not infiltrated by inflammatory cells. In all types of mucoid effusion of variable duration, various lymphocyte classes, i.e. T- and B-lymphocytes and plasma cells, were present, suggesting a normal cellular immune response. The lack of granulocytes seems to indicate that there is no submucosal infection.

[The nature of crystals in plasma cells present in a case of apparently benign biclonal gammapathy]. / Sulla natura dei cristalli endoplasmacellulari presenti in un caso di gammapatia biclonale apparentemente benigna.

A 77 years old white man presents, in his blood serum, a moderate gammapathy with 2 monoclonal peaks, IgAk and IgGlambda, and in his urine a little quantity of BJk, without any clinical or radiological manifestation. The sternal bone marrow showed 3-5% plasmacells, mostly containing many needlelike inclusions in the cytoplasm. These inclusions, red-violet at the May-Grünwald-Giemsa, were PAS and Sudan negative; positive at the Danielli reaction (for proteins). The alpha-naphtil-acetate-esterase and the acid phosphatase were present in one outer layer of the inclusions; ATP-ase was absent. At the electron microscopy, the inclusions were localized outside the rough endoplasmic reticulum; they exhibited a crystalline structure and were surrounded by an envelope which reminded the lysosomes. Basing on the morphological pattern, as well as on the presence of some lysosomal enzymes and on the lack of staining of crystals with fluorescinated anti-Ig sera, the hypothesis is stressed of an abnormal lysosomal hyperactivity, possibly leading to crystallization of the enzymatic proteins in the lysosomes.

[Study of macrophages infiltrating rat cardiac allografts. 1.--Description of the technique of heterotopic cardiac transplantation in the rat and morphological analysis of the cells infiltrating the graft (author's transl)]. / Etude des macrophages dans les allogreffes cardiaques expérimentales. I.--Description de la technique de greffe de coeur hétérotopique de rat et analyse morphologique des cellules infiltrant le greffon.

The technique of heterotopic cardiac transplantation in the rat is described in detail. A simple, nontraumatic method of obtaining cellular prints from the heart is proposed for the study of cells infiltrating the allograft. Analysis of the morphological aspect of the cellular infiltrate present at the time of rejection in a series of allografts revealed a very high proportion of cells belonging to the monocyte-macrophage series, thus suggesting an important role for these cells in the rejection process.

[Study of the total nucleic acid content of lymphoid tissue plasma cells for assessing the immunodepressive action of various antirheumatic agents]. / Izuchenie summarnogo soderzhaniia nukleinovykh kislot plazmaticheskikh kletok limfoidnoi tkani dlia otsenki immunodepressivnogo deistviia razlichnykh protivorevmaticheskikh sredstv.

The influence of antirheumatic drugs, acetylsalicylic acid, diacetoxibenzoic acid, imurane and D-penicillamine, on the status of immunocompetent cells in experimental infectious-allergic carditis was studied morphologically in 70 rabbits. The immunosuppressive effect of all 4 drugs was established which was manifested by a decrease in the number of plasma cells in the lymphoid tissue and a decrease in the content of nucleic acids in their cytoplasm. D-penicillamine was the exception as after its use the content of nucleic acids in the cell cytoplasm was found to be increased which was considered to be due to clasmatosis of plasma cells, marginal karyolysis, damage of the nuclear membrane and release of nucleic acids from the nucleus into the cytoplasm. Acetylsalicylic acid and diacetooxybenzoic acid decrease RNA content in the cytoplasm of plasma cells less than imurane and do not cause cell degeneration with contamination of the extracellular environment with products of cells degeneration.