Mouse thymus-independent and thymus-derived lymphoid cells. II. Ultrastructural studies.

The ultrastructural features of B-, T-, and surface Ig(sIg)-bearing cells have been studied on cell suspensions from lymphoid organs of mice at different stages of immunization. The cells were identified by exposure to rabbit antibodies against mouse-specific lymphocyte antigens (MSLA) or brain-associated theta antigen (BAtheta) for T cells, mouse-specific bone marrow-derived lymphocyte antigens (MBLA) for B cells, and mouse Ig for sIg-bearing cells. The rabbit antibodies fixed on the cell surfaces were detected by peroxidase-labeled sheep anti-rabbit Ig antibodies or by a "bridge" technique using southern bean mosaic virus or bacteriophage T4 as the final markers. In some experiments, short-lived lymphoid cells were labeled in vivo with repeated tritiated thymidine and the ultrastructural detection of their surface antigens was combined with radioautography. MBLA+ lymphoid cells showed a whole range of ultrastructural patterns. Most were small and medium-sized lymphocytes with a clear cytoplasm containing mono- and polyribosomes, but they comprised also blasts and large cells with various amounts of endoplasmic reticulum, as well as plasma cells at different stages of maturation. sIg-bearing cells appeared to be identical with MBLA+ cells, except that sIg was less easily detectable on large blasts, and only very rarely observed on plasma cells. MSLA+ and BAtheta+ cells fell into three categories. One of them (T(1) cells) consisted of small to medium-sized lymphocytes with a clear cytoplasm and few organelles, mostly monoribosomes. A second consisted of large cells (T(2) cells) characterized by numerous polyribosomes often in a "rosette"-like pattern, occasional dark, membrane-bound granules, and a developing "filamentous network." The third, very characteristic type, (T(3) cells) was represented by dark small to medium-sized lymphocytes, usually containing large amounts of closely packed ribosomes and showing a striking accumulation of filamentous network, often condensed in large areas devoid of cell organelles. This filamentous network appeared to correspond to the cytochalasin B-sensitive system of microfilaments found in other cells and considered to be one of the contractile elements of the cell. The T(3) lymphocytes showed frequently vesicles suggestive of a strong pinocytic activity, and assumed a variety of shapes, including uropods. Evidence is presented that T(1) lymphocytes represent "virgin" T cells, T(2) "activated," and T(3) "differentiated" lymphocytes.

Mouse thymus-independent and thymus-derived lymphoid cells. I. Immunofluorescent and functional studies.

The simultaneous use on mouse lymphoid suspensions of heterologous antisera directed against thymus-derived (T) cell mouse-specific lymphocyte antigen and brain-associated theta antigen (MSLA and BAtheta) or thymus-independent (B) cell mouse-specific bone marrow-derived lymphocyte antigen (MBLA) surface antigens allowed direct proof of the different specificity of these antisera by double immunofluorescence (IF) staining with selective visualization of fluorochromes. These antisera and antisera against mouse Ig and its different types of chains were then used with technique of either double IF staining or IF combined with radioautography, allowing the following conclusions: (a) Surface Ig (sIg) was found exclusively on B cells and never on T cells, but not all B cells had sIg. Cells containing detectable amounts of Ig were MBLA+, but had less sIg than other B cells or none at all. There was evidence for the existence of a significant number of MBLA+ lymphocytes, neither bearing nor containing detectable Ig. (b) micro-Chains were the most frequent but not the only heavy chains found on spleen cells; however, it could not be decided with the technique used, if a single cell can bear more than one type of heavy chain. No cell containing gamma-chains was found to bear surface micro-chains, although a very few cells containing both micro- and gamma-chains were observed. (c) The antigen-binding cells detected after immunization with bacteriophage T4, bovine serum albumin, Maia squinado hemocyanin, and sheep erythrocytes were analyzed for MSLA, MBLA or sIg using double IF, a combination of IF and radioautography, or inhibition of "rosette" formation. Practically all the antigen-binding cells detected were MSLA-, MBLA+, sIg+. (d) More B cells than T cells were found among short-lived lymphoid cells labeled by repeated in vivo injections of tritiated thymidine, but the results did not support a simplified concept equating T cells to long-lived and B cells to short-lived lymphocytes. (e) Cells dividing rapidly in the lymph nodes draining the sites of immunization with various antigens were predominantly T cells 2 days after immunization and in majority B cells a few days later. (f) Incubation of lymphoid cells at 37 degrees C with rabbit anti-mouse Ig or anti-kappa chains led to complete disappearance of sIg and to decrease of MBLA ("antigenic modulation"). In the same conditions, anti-MBLA gave partial modulation of MBLA and of sIg; MBLA, however, reappeared much faster than sIg. No modulation of T cell surface antigens by the appropriate antisera was observed. Cell treatment with Pronase could remove MBLA, sIg, MSLA, and BAtheta, which reappeared within a few hours. Neuraminidase treatment was without detectable effect on these antigens.

Leukocyte interleukins induce cultured endothelial cells to produce a highly organized, glycosaminoglycan-rich pericellular matrix.

We report here that interleukins have a dramatic effect on extracellular matrix production by cultured endothelial cells. Human umbilical vein endothelial cells incubated with growth media conditioned by lectin-activated human peripheral blood mononuclear leukocytes undergo marked changes in cell shape and elaborate a highly organized extracellular material that is not detectable in untreated cultures. This material has the following characteristics: (a) it is not recognizable by electron microscopy unless the cationic dye, Alcian blue, is added to the fixative; (b) it is visualized as a network of branching and anastomosing fibrils of various thickness that can be resolved into bundles of fine filaments; (c) it is associated with the cell surface, extends between contiguous cells, and coats the culture substrate; (d) it is removed by digestion with glycosaminoglycan-degrading enzymes, such as crude heparinase and chondroitinase ABC. These results demonstrate that soluble factors released by activated peripheral blood mononuclear leukocytes (interleukins) stimulate cultured human umbilical vein endothelial cells to produce a highly structured pericellular matrix containing glycosaminoglycans (probably chondroitin sulfate and/or hyaluronic acid) as a major constituent. We speculate that this phenomenon corresponds to an early step of angiogenesis as observed in vivo as a consequence of interleukin release.

Colon carcinoma immunoscintigraphy by monoclonal anti-CEA antibody labeled with gallium-67-aminooxyacetyldeferroxamine.

Previous experimental results in nude mice showing that radiolabeling the monoclonal antibody anti-CEA 35 with 67Ga-aminooxyacetyldeferroxamine could give better tumor localization than radioiodination prompted us to initiate the present clinical study. The 67Ga-labeled antibody anti-CEA 35 (185 MBq, 0.7-1.7 mg) was injected preoperatively into 14 patients for colorectal carcinoma imaging. The same antibody labeled with 125I (3.7 MBq, 0.25 mg) was injected simultaneously to compare the 67Ga and 125I dose recoveries in surgical specimens. Twelve of 14 primary tumors gave a positive 67Ga scintigraph. The mean %ID/g recovered in all tumors 3-9 days after injection was significantly higher for 67Ga (0.019%) than for 125I (0.005%) (p < 0.001, paired t test). The tumor-to-normal tissue ratios were generally higher for 67Ga, with the exception of liver. We conclude that 67Ga-aminooxyacetyldeferroxamine improved immunoscintigraphy outside the liver, particularly in the pelvic region. We also show that deferroxamine infusion accelerates the excretion of 67Ga in eight patients and propose that this could lead to further improvement of immunoscintigraphy.

Radiolabeled chimeric anti-CEA monoclonal antibody compared with the original mouse monoclonal antibody for surgically treated colorectal carcinoma.

UNLABELLED: Biodistribution and tumor uptake of a chimeric human-mouse monoclonal antibody (MAb) and the original mouse MAb have been comparatively studied. METHODS: Eighteen patients with suspected colorectal cancer scheduled for surgery underwent immunoscintigraphy with 123I-labeled chimeric anti-CEA MAb. Iodine-125 and 131I trace-labeled chimeric and original mouse MAb were simultaneously injected for biodistribution studies. RESULTS: Similar serum kinetics and a low immunogenicity were observed for both antibodies. Mean binding capacity to CEA measured in PBS after radiolabeling was identical for both MAbs and it was slightly decreased when measured in serum 1-4 hr after injection. Radiochromatograms of patients sera showed immune complex formation related to the amount of circulating CEA. Postoperative ex vivo radioactivity counting in tissue samples revealed similar antibody distributions with notably similar antibody uptakes in tumors. High tumor uptakes (between 0.02 to 0.06% injected dose per g) were observed in 3 of 13 patients operated for primary or metastatic colorectal cancer. CONCLUSION: In this dual-label technique, the radioiodinated anti-CEA IgG4 chimeric MAb and the original mouse IgG1 MAb were shown to have very similar behavior in colorectal cancer patients.

[76Br]Bromodeoxyuridine, a potential tracer for the measurement of cell proliferation by positron emission tomography, in vitro and in vivo studies in mice.

5-Bromo-2'-deoxyuridine (BrUdR) labeled with 77Br and 76Br was compared with 5-iodo-2'-deoxyuridine (IUdR) labeled with 125I or 131I, first in vitro then in in vivo experiments in mice. The results showed a significantly higher incorporation of BrUdR into DNA than IUdR, which can be explained by the greater similarity (size and surface hydrophilicity of the molecules) of BrUdR to thymidine. Both tracers are dehalogenated quickly in vivo but not in vitro. Free bromide is excreted more slowly than iodide, resulting in a higher background activity level after the application of [76Br]BrUdR and compensates for the favorable DNA incorporation. 76Br has more favorable properties than 124I for imaging purposes with positron emission tomography (PET) because of a very convenient half-life (16 h vs. 4.15 days) and about double the positron yield per decay. However, the more favorable physical properties are balanced by the slower excretion and thus the estimated radiation dose is higher in the case of 76Br than 124I. Thus, both tracers, [124I]IUdR and [76Br]BrUdR are potentially suitable but not optimal to measure cell proliferation in vivo. The difference between the two tracers is small and the extrapolation from mice to human difficult, and thus it cannot be concluded if one of the tracers would be better than the other for imaging of cancer patients.

Elimination of free radionuclide by a chelating agent improves tumor-to-nontumor ratios following radioimmunotargeting with antibody labeled with 67Ga.

To circumvent radionuclide accumulation in nontarget tissues when employing metallic radionuclides for radioimmunoscintigraphy or radioimmunotherapy, we have investigated the effect of the chelating agent deferroxamine (DFO) on the biodistribution of 67Ga following its administration attached to intact monoclonal antibody MAb35 and its F(ab')2 fragment. Following administration of 67Ga-labeled MAb35, DFO accelerated whole-body elimination of 67Ga and reduced its accumulation in several normal tissues, including liver, spleen and kidney. No reduction in tumor accumulation of 67Ga was observed. Following administration of 67Ga-labeled F(ab')2 fragment, kidney accumulation was higher than with the intact antibody (29% and 4% ID/g, respectively) and blood levels lower (0.69% and 5% ID/g, respectively). Again, no alteration in tumor accumulation of 67Ga was seen following DFO, although liver, kidney and blood levels were reduced and whole-body elimination accelerated.

[76Br]Bromodeoxyuridine PET in tumor-bearing animals.

5-bromodeoxyuridine (BUdR) provides in vitro measures of tumor cell proliferation. We used positron emission tomography to study tissue and plasma kinetics of [76Br]BUdR in tumor-bearing animals. In order to account for the slow washout of the major plasma metabolite, [76Br]bromide, a mathematical correction for the distribution volume of [76Br]bromide was applied. However, following correction specific tumor tracer retention was low or even zero and did not correlate with independent measures of proliferation. The kinetic characteristics of [76Br]BUdR make this tracer unsuitable for proliferation imaging.

Single photon emission computed tomography study of human cancer: utilization of 57Co-labelled bleomycin.

The sensitivity and specificity of single photon emission tomography with 57Co-labelled bleomycin (57Co-BLM) for the detection of cancer was determined from a prospective study involving a large group of patients selected to investigate roentgenographic abnormalities. Eighty-four of the 104 patients studied had malignant disease, of whom 76 had a positive scintigram. Eighteen of the 20 patients with benign disorders had a negative scintigram. The sensitivity and specificity was therefore 90.5 and 90% respectively. For the subset of patients who underwent investigation below the diaphragm, the sensitivity was 85.7%, while for investigation above the diaphragm, it reached 95.2% (this excluded reconstructions on the bladder level, because it produced large artifacts). This study leads to the conclusion that SPECT can be used specifically to investigate unidentified X-ray abnormality and diagnose malignancy using 57Co-BLM. In addition, we propose further investigation to evaluate the usefulness of this method in staging cancer.

[Positron emission tomography (PET) in the preoperative evaluation of cervical lymph node metastasis of ORL cancer]. / La tomographie par émission de positrons (PET) dans l'évaluation préopératoire des métastases ganglionnaires cervicales des cancers ORL.

The distribution of specific radiolabeled biological compounds in tumor tissues can be imaged by positron emission tomography (PET). The substance used is fluorodeoxyglucose, labeled with the positron emitter fluorine-18. This substance is partly trapped in tumor cells with increased glucose metabolism. This noninvasive imaging technique allows to assess quantitatively and in three dimensions the extent of metastatic disease in ENT cancer. The case presented illustrates the important value we foresee for this new imaging modality in the presurgical staging of cervical metastatic disease of ENT tumors. Sensitivity and specificity of the PET-FDG imaging technique for the loco-regional staging of ENT cancer are, according to preliminary results of an ongoing, prospective clinical study, very high.

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.

The humoral immune response of mouse bone marrow lymphocytes in vitro.

Mouse bone marrow (BM) small lymphocytes are shown to contain competent precursors for a primary haemolytic plaque forming cell (PFC) response to heterologous red blood cells and TNP in an in vitro culture system. Their response is dependent on T co-operative factors, which can be provided by irradiated spleen cells activated by concanavalin A or the supernatant of an allogeneic culture, added at the beginning or after 24 h of culture. The frequency of PFC precursors for the response to SRBC is found to be equal or higher in BM than spleen cultures. However, BM lymphocyte cultures stimulated by E. coli lipopolysaccharide show an increase of DNA synthesis but contain only few polyclonal PFC, in contrast to spleen.

Cell-mediated immunity to antigens associated with murine sarcoma virus-induced tumors: augmentation of cytolytic T lymphocyte activity by successive specific and nonspecific stimulation in vitro.

It has been shown previously that lymphoid cells from mice which have rejected a tumor induced by the Moloney sarcoma virus-leukemia virus (MSV-MLV) complex develop high levels of specific H-2-restricted cytolytic T lymphocyte (CTL) activity after in vitro stimulation with syngeneic, irradiated MLV-induced lymphoma cells in mixed leukocyte-tumor cell cultures (MLTC). Attempts to further increase lytic activity by restimulating long-term MLTC cells with syngeneic, irradiated lymphoma cells have met but with limited success. This report shows that, in contrast to the lack of increased activity observed after specific stimulation with lymphoma cells, nonspecific stimulation of long-term MLTC cells either with supernatants from secondary mixed leukocyte cultures (2 degrees MLC SN) or with supernatants from concanavalin A-stimulated spleen cells leads, on a per cell basis, to a further 5 to 10-fold increase in CTL activity. The stimulatory activity of 2 degrees MLC SN is due to a factor(s) of apparent mol. wt. of 25,000 to 40,000. The activity of the CTL populations formed under these conditions is at least 100-fold higher against syngeneic as compared to allogeneic MLV-induced or unrelated tumor cells.

Immunoscintigraphy with biosynthetically-labeled 75Se-antibodies--I. Biodistribution of 75Se-monoclonal antibodies and of free radionuclide.

Biosynthetically-radiolabeled MoAbs provide a tool to test whether structural modifications of the MoAbs influence the results of conventional immunoscintigraphy. When biosynthetically-labeled 75Se-MoAbs from the Mel-14 hybridoma were injected into mice with melanoma xenografts, the high tumor recovery supported the hypothesis of a structural advantage. The increased excretion of 75Se obtained by supplementing the diet of the mice with cold selenium did not reduce the tumor recovery, demonstrating an accumulation of the free radionuclide in normal tissue.

Immunoscintigraphy with biosynthetically-labeled 75Se-antibodies--II. Comparison with antibodies labeled by radioiodination.

The biodistribution of 75Se and 125I were compared in nude mice bearing melanomas and injected simultaneously with biosynthetically-labeled 75Se-MoAbs and radioiodinated 125I-MoAbs from the same anti-melanoma hybridoma. The 75Se dose recovery in the tumor (day 10) was twice that for 125I, but the tumor to normal tissue ratios were identical. It is concluded that biosynthetically-labeled MoAbs survive better in vivo, while the tumor to normal tissue ratios are governed by the excretion rate of labeled MoAb catabolytic products.

[Diagnosis of internal abscesses by scintigraphy: results of a method using purified granulocytes]. / Diagnostic des abcès internes par scintigraphie: résultats d'une méthode utilisant des granulocytes purifiés.

In the 111-indium-leukocyte scan method described by M. L. THAKUR et al. in 1977 for the diagnosis of abdominal abscesses the whole leukocyte fraction from a blood sample is labeled and injected into patients. We propose a modification in which purified granulocytes are used. The results of 43 granulocyte scans show that this modification does not diminish the sensitivity of the method. Furthermore, it allows the detection of abscesses in a shorter time (2-4 hours after injection) and with 111-indium doses (200 muCi) at least 3 times lower than in the original method. Increased specificity can also be expected.

Precorneal residence time in humans of sodium hyaluronate as measured by gamma scintigraphy.

The aim of the present study was to quantify in man the distribution and clearance of two aqueous sodium hyaluronate (SH) solutions of 0.125% and 0.250% after the administration of 25 microliters onto the cornea. Isotonic phosphate buffer (PB) was used as a reference instillation. No systemic or local medication was given to the seven 18- to 30-year-old, healthy male volunteers. A detailed evaluation of the anterior segment of the eye, as well as a Schirmer test and a break-up time measurement, yielded results within the normal range. The clearance of 0.125% and 0.250% SH solutions radiolabelled with sodium pertechnetate Tc-99m was measured by gamma scintigraphy and compared with that of a PB solution tagged with the same radiolabel. There was no statistically significant difference between the quantities of 0.125% SH and PB solutions remaining in the precorneal space at 20 min (paired t-test, P = 0.78, n = 7). However, in comparing the 0.250% SH with the PB solution, we observed a statistically significant difference (P = 0.01, n = 7) in the amount remaining in the precorneal space after the same interval. Actually, 53% of the radiolabelled 0.250% SH solution remained on the cornea as compared with 30% for the 0.125% SH solution and 18.3% for the PB solution. These results suggest that an SH solution of 0.250% might have a prolonged residence time on the precorneal surface, and that SH could therefore be used as an additive in various drug-release systems for the eye.

The area of attachment of cytotoxic T lymphocytes to their target cells shows high motility and polarization of actin, but not myosin.

Conjugates of cytotoxic T lymphocytes attached to their target cells were studied by double immunofluorescence on fixed smears to detect simultaneously the localization of actin and myosin within the cells. Actin was found to be polarized in the area of attachment of the lymphocytes (but not of the target cells), whereas myosin remained evenly distributed in the cytoplasm. When living conjugates were brought to 37 degrees C to induce cytotoxicity, this pattern remained unchanged, but observation by interference reflexion microcinematography revealed a high motility of the lymphocytes in the contact area. This localized motility in the area of attachment associated with a peculiar actin polarization, which has no equivalent in any type of cell contact presently known, could represent a necessary step in the sequence of events leading to target cell killing by cytotoxic T lymphocytes.