Studies on H-2 specificities on mouse tumour cells by a new microradioassay.

Seven mouse tumour cell lines of different aetiology and origin were tested for the expression of surface alloantigens using twenty-four well defined H-2 alloantisera and anti-Thy 1.2. We used a new radioassay that involves antibody-complement treatment of the tumour target cells followed by postlabeling the surviving tumour cells with 14C-thymidine. With a relatively high frequency the anti-H-2 sera were reacting differently with the tumour cells than with respective syngeneic lymphoid cells. Thirty-six anomalous reactions out of 129 investigated were detected. Absorbtion experiments performed with H-2 antigen positive or negative lymphoid cells revealed a striking similarity between these extra-specificities and H-2 specificities of foreign haplotypes. The results are discussed in relation to the biological importance of the extra-specificities, both with regard to origin (derepression) and function (transplantation antigen properties).     

Induction of T-suppressors during immunization with allogeneic spleen cells in the mouse H-2 system

The nature of suppressor cells contained in a suspension of splenic lymphocytes immunized with allogeneic spleen cells and inhibiting activation of DNA synthesis in mixed lymphocyte cultures was studied. The suppressor cells were resistant to mitomycin C and carrageenin, were not inactivated by treatment with rabbit anti-B- and anti-Ig or mouse antibodies (anti-Mls serum) against B-lymphocytes, in the presence of complement, but were eliminated by rabbit antilymphocytic globulin and also by antibodies against T-lymphocytes (rabbit ATG and anti--serum). The T-suppressors studied were concentrated in the large lymphocyte fraction in a ficoll gradient. Blocking of activation of DNA synthesis by these cells has a well-marked nonspecific component.Laboratory of Immunochemistry and Diagnosis of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 10, pp. 429–431, October, 1979.     

Immunogenicity of H-2 positive and H-2 negative clones of a mouse tumour, GR9

The GR9 tumour was induced with methylcholanthrene in a BALB/c mouse, adapted to tissue culture and cloned without any passage in vivo GR9 clones were typed for H-2 with three monoclonal antibodies that define H-2Kd + Dd, Kd and Dd antigens. A great heterogeneity of H-2d expression was found from clones which were Kd and Dd positive to clones Kd and Dd negative. These results were confirmed for A7 and B9 clones using immunoprecipitation with anti-H-2D.4 and anti-H-2K.31 alloantisera and SDS-PAGE analysis. In addition, the number of chromosomes per cell was heterogeneous amongst the clones, ranging from 38 +/- 2 to pseudotetraploid clones which have 75 +/- 2 chromosomes. GR9 clones were injected into syngeneic BALB/c mice to measure local tumour growth. We found that the growth correlated with the amount of H-2 antigen expressed, i.e. clones with low H-2d expression were highly malignant while clones with normal H-2d expression were highly immunogenic. Finally we found that BALB/c mice immunized against A7 (Kd, Dd-positive) protected against A7, as expected, but surprisingly also against B9 (Kd, Dd-negative).     

The role of H-2 and H-2-like determinants in syngeneic and allogeneic cytostasis.

Specific sensitization against H-2 determinants was affected by immunizing allogeneic mice with spleen and lymph node cells in H-2 congenic combinations. Lymph nodes from the sensitized and non-sensitized mice were respectively cultured together with H-2 syngeneic tumour cell lines. The growth and viability of the tumour cells was subsequently measured by the amount of radiothymidine incorporation. If the tumour cells incorporated less isotope when cultured with the immune cells than with the normal cells this was termed 'cytostasis'. To identify the H-2 genes controlling the sensitization phase in the cytostasis assay, we studied the effect on different transplantable tumour target cells of lymphoid cells from mice sensitized against different congenic spleen cells. The results suggest that the cytostasis assay can can measure an in vitro specific response to H-2-incompatible sensitizing antigens, and that I--B incompatibility, together with K and/or D, is essential to produce effectors. Furthermore, H-2 allogeneic sensitization could induce cytostasis even against tumour cells syngeneic for the H-2 halotype of the responder strain. The implications of these findings are discussed.     

Cytogenetic studies of blast cells in the cerebrospinal fluid in meningeal leukemia

Chromosome analysis can be performed by culturing cerebrospinal fluid from patients with CNS leukemia. The quality of the metaphase spreads are adequate for banding studies. This approach is useful, especially when the bone marrow is difficult to aspirate or when CNS involvement is the only manifestation of the leukemia.     

Inhibition of the growth of murine tumour cells in vitro by serum from non-immune syngeneic and allogeneic mice.

Sera from DBA/2 and Quackenbush mice (which are non-immune for mastocytoma and Sarcoma 180 respectively) contain a heat-labile (56 degrees for 30 min) component(s) that inhibits the in vitro growth of DBA Mastocytoma P-815 X-2 and Sarcoma 180. Adsorption of the sera with tumour cells at 4 degrees did not eliminate the factor(s), suggesting that it is not an antibody. In liquid suspension cultures inhibitory activity was observed at concentrations of mouse serum of 10--20% and in semisolid agar clonogenic cell assays at concentrations as low as 1%. The influences of the inhibitor(s) for both tumours and in both culture systems were parallel. However, there was a quantitative difference in susceptibility to other environmental factors (FCS concentration, bicarbonate concentration, and O2 tension) between the two tumours. These results parallel the in vivo findings where intravenously injected mastocytoma cells produced more tumours than did Sarcoma 180.     

Histoenzymic studies in the myelinopathy provoked by the cerebrospinal fluid exchange.

A histochemical study was performed on the activity of several phosphatases, esterases and oxidoreductases in the spinal cord of cat in the course of the myelinopathy provoked by the cerebrospinal fluid (CSF) exchange. The following results were obtained: 1. Neuroglial cells of the spinal cord react with a slight, focal increase of TPP-ase activity already during the early stage of myelinopathy evoked by repeated withdrawal and reinjecting of CSF into the Cisterna magna of experimental cats. 2. During the late stage of this experimental disease-- the oligo-and astroglia of the spinal cord dtsplay considerably increased activities of ATP-ase, acP, and of various oxidoreductases. 3. The observed morphological and functional (increased enzymic activity) changes of the oligodendroglia as well as the demonstrable damage of myelin sheath, seem to be the results of vasogenous edema. 4. The results of investigations performed did not yield any arguments, which would speak in favour of the assumption, that the changes of the oligodendroglia cells function as the primary cause in the myelinopathy provoked by the CSF exchange, affecting secondary the myelin sheaths. 5. The decissively increased lysosomal acP activity in oligodendroglial cells of the spinal cord investigated during the early stage of this myelinopathy, accompanied by an evidently damaged Golgi apparatus, may be explained when the Golgi zone is not necessarily the only cellular site where acid phosphatase may be performed.     

Inhibitory effect of cerebrospinal fluid on the growth of meningococci and pneumococci.

Initial cerebrospinal fluid (CSF) samples obtained from patients with pneumococcal meningitis usually contain far greater numbers of bacteria than initial CSF samples obtained from patients with meningococcal meningitis. Normal CSF was found to have an inhibitory effect on the growth of group A meningococci but not on type 1 pneumococci. The inhibitory effect of normal CSF was abolished by dialysis, indicating that the inhibitory factor has a low molecular weight. Heating normal CSF to 62.5 degrees C for 30 min resulted in a considerable reduction in the inhibitory effect, indicating that the inhibitory factor is heat labile.     

Human dendritic cells stimulate allogeneic T cells in the absence of IL-1

Dendritic cells (DC), which express high-density HLA class II molecules, stimulate strong primary allogeneic T-cell responses via an interaction of the T-cell receptor with major histocompatibility complex (MHC) antigens (signal 1). It is not yet clear whether they also provide a second stimulus to the responding T cell in the form of the cytokine interleukin-1 (IL-1). To clarify this point, the ability of purified human tonsil DC to produce IL-1 and to stimulate allogeneic T cells was tested. No intracellular IL-1 alpha or beta was identified in DC comparable to that readily demonstrated in monocytes, and IL-1 release from lipopolysaccharide (LPS)-stimulated DC was not detected in either a biological assay for IL-1 or an ELISA assay for IL-1 beta. Furthermore, strong stimulation of allogeneic T lymphocytes by DC in the mixed leucocyte reaction (MLR) was noted to occur in the absence of IL-1 production, and this stimulation was not inhibited by polyclonal antisera to IL-1 alpha and IL-1 beta, which were known to inhibit IL-1-mediated thymocyte proliferation. Other HLA-class II-positive cell populations, namely peripheral blood monocytes and B cells, purified by methods which avoided DC contamination, were unable to stimulate allogeneic T cells with or without supplementary IL-1. We conclude that DC are very effective stimulators of T lymphocytes and that IL-1 is not required as a second signal for allogeneic T-cell responses.     

Quantitative and electron microscopic studies of sensory ganglion cells of the Sprawling mouse

Summary The L4–6 sensory root ganglia of young and adult Sprawling (Swl) and normal mice were studied. Cell counts showed a great reduction in the total number of ganglion cells inSwl. Cell degeneration was observed in youngSwl animals but not in normal littermates. Most of the remaining ganglion cells showed morphological abnormalities very similar to those seen in chromatolytic neurons — enlarged nucleolus, eccentric nucleus with an infolded nuclear membrane, loss of juxtanuclear Nissl bodies and an increase in neurofilaments, Golgi membranes, autophagic vacuoles, and dense bodies. In contrast to the classical changes of chromatolysis the abnormalities inSwl neurons persisted throughout the lifespan of the animal. Reconstructions from serial sections showed that ganglion cells inSwl were highly irregular in shape.     

Quantitative changes in the amyloid beta A4 precursor protein in Alzheimer cerebrospinal fluid.

The major three secretory isoforms of Alzheimer beta A4 amyloid precursor protein (APP) were quantified in cerebrospinal fluid (CSF) using (1) a newly developed enzyme-linked immunosorbent assay (ELISA) and (2) densitometric analysis of CSF Western blots. The protease inhibitor-containing APP751/770 isoforms represented an average of 10.5% of total APP in CSF of patients with Alzheimer's disease (AD, n = 22), multi-infarct dementia (MID, n = 5) and normal controls (n = 10). APP levels in CSF did not depend on total CSF protein. Both findings are inconsistent with a hematogeneous origin of APP in CSF and suggest an intracerebral source. Total APP, APP695 and APP751/770 were significantly decreased in the AD and in the MID groups, but were not correlated to the ages of patients or controls.     

Identification of Ehrlichia chaffeensis morulae in cerebrospinal fluid mononuclear cells.

We report a case of ehrlichiosis in a 72-year-old man who developed extreme lethargy, acute renal failure requiring hemodialysis, and respiratory insufficiency requiring intubation. Lumbar puncture performed on the second day of hospitalization revealed significant cellular pleocytosis. Ehrlichia morulae were tentatively identified in mononuclear cells in routinely processed Wright-stained cytospin preparations of cerebrospinal fluid (CSF). Identification was confirmed by a specific immunocytochemical staining procedure. Subsequent identification specifically as Ehrlichia chaffeensis morulae was established by polymerase chain reaction analysis, which revealed E. chaffeensis-specific DNA in CSF, bone marrow, and blood samples; by indirect fluorescent-antibody analysis, the patient developed an antibody titer of 32,768 against E. chaffeensis antigen. The patient responded to intravenous therapy with doxycycline and dexamethasone. Subsequently, neurologic, hematologic, renal, and pulmonary status had returned to baseline at follow-up 12 weeks after admission. To our knowledge, this is the first identification of E. chaffeensis morulae in CSF cells in an infected patient.     

The effect of glutaraldehyde fixation on the immunogenicity of allogeneic lymphoid and tumour cells.

When CBA mice were injected with allogeneic (DBA/2) lymph node cells treated with glutaraldehyde at concentrations of 0.13% and 0.013% they failed to produce a primary cytotoxic antibody response; cells fixed with 0.0013% glutaraldehyde only provoked the slightest of antibody responses. No significant secondary response was provoked by cells fixed with 0.013% glutaraldehyde in mice primed 8 weeks earlier with normal lymphoid cells. As it is well established that such cells can stimulate a secondary mixed lymphocyte reaction and have ben reported to induce a secondary haemagglutinin response their assumed antigenicity in these experiments was checked. It was found that fixed cells did not have measurable antigenicity as assessed by ability to absorb anti-H2 antibody. The organ localization of chromium-labelled glutaraldehyde-fixed lymph node cells showed a lack of localization in lymph nodes at all levels of fixation, though localization in the spleen of cells fixed with 0.0013% glutaraldehyde was very variable, consistent with the borderline immunogenicity of such cells. Mitomycin treatment only modestly reduced the immunogenicity of lymph node cells and did not affect their organ localization. When CBA mice were injected with allogeneic (DBA/2) tumour cells, P 815, fixed with 0.13% or 0.013% glutaraldehyde, no cytotoxic antibody was produced and cells fixed with 0.0013% glutaraldehyde stimulated an erratic low response again suggesting a borderline level of activity. However P 815 cells fixed with 0.13% glutaraldehyde retained their antigenicity as assessed by absorption. Mitomycin treatment of P 815 cells had only a modest deleterious effect of their immunogenicity. These differences in immunogenicity are discussed in relation to the viability of cells required to stimulate an allo-cytotoxic antibody response.