The in vitro transformation of frozen-stored lymphocytes in the mixed lymphocyte reaction and in culture with phytohemagglutinin and specific antigens.

We have investigated the in vitro transformation of lymphocytes that have been frozen and stored at low temperatures. Cells frozen at different times and stored for various times do transform in a reproducible manner when placed in culture with PHA or as one population of the two-way or the one-way MLR. When frozen-stored lymphocytes are cultured with specific antigen or as both partners in the MLR the response is minimal. The freezing process destroys neutrophils, and the remaining population transforms in a manner similar to cultures of purified lymphocytes. Practical aspects of the technique are discussed and we have suggested possible practical applications for future investigation.

Elevated ribonuclease activity in the thymus and white blood cells of genetically cancer prone mice.

Ribonuclease activity in cell-free thymus homogenates was elevated for five strains of mice genetically predisposed toward leukemia or reticulum cell neoplasms (AKR, C58, PL, RF, and SJL). Such increased activity was directed against polyuridylic acid and was observed in 8-wk old mice, well before the onset of neoplastic transformation. Similarly, white blood cell ribonuclease activity was elevated in mice of the strains AKR, C2H/He, PL and RF. Statistical analysis indicated that such elevated activity in these strains related to their high incidence of spontaneous neoplastic disease. Elevated ribonuclease activity thus represents a new biochemical marker relating to the genetic propensity of some strains of mice to die prematurely of spontaneous neoplasia.

A proper sequence for the treatment of B16 melanoma: chemotherapy, surgery, and immunotherapy.

The therapeutic effectiveness of surgery, chemotherapy, and immunotherapy alone and in combination were studied in the B16 melanoma model. The sequence of chemotherapy and immunotherapy as adjuvants to surgery proved important: Chemotherapy was significantly better when given before surgery; immunotherapy was more effective when delivered after surgery. The most effective therapeutic regimen was a combination of all three modalities: a single course of chemotherapy preceding surgery followed by immunotherapy.

The dependence of successful immunotherapy on adequate tumor burden as shown by the treatment of AKR leukemia with poly A-poly U.

The therapeutic efficacy of polyadenlyic-polyuridylic acid (poly A-poly U) on the transplantable AKR leukemia varied with the dose of tumor cells implanted. The greater the number of AKR tumor cells injected into 8-week-old AKR mice free of clinical evidence of cancer, the greater the effect of poly A-poly U in mediating host immunologic control of the tumor. Poly A-poly U was either ineffective or could enhance tumor growth when smaller doses of tumor cells were transferred. The efficacy of an immune adjuvant depended on a tumor burden affording optimum host responsiveness. This does not necessarily arise in the host bearing minimal tumor burden.

A new apparatus for BCG scarification.

A procedure for administering BCG by scarification that allows for speed of application and standardization of technique was discribed.

A white blood cell RNase assay for the possible monitoring of malignancy.

The RNase activity observed in the sera of leukemic guinea pigs was compared to that observed in white blood cell (WBC) lysates of the same animals. The WBC-associated RNase activity directed against polyuridylic acid decreased with the progression of neoplastic disease, though serum RNase activity remained unchanged. With certain forms of cancer, therefore, variations in cell RNase may be more sensitive markers than changes in serum RNase for the evaluation of the progression or regression of disease.

Abnormal profile of human nucleolytic activity as a test for cancer.

Serum samples from patients with various malignancies including acute nonlymphocytic leukemia (ANLL), brain tumor (BT), Hodgkin's disease (HD), and non Hodgkin's lymphoma (NHL) were evaluated for nucleolytic activity against six synthetic polynucleotides: polyadenylic acid, polyuridylic acid, polycytidylic acid, polyguanylic acid, polyadenylic-polyuridylic acid, and polyguanylic-polycytidylic acid; The enzyme activity was determined spectrophotometrically by following the degradation of substrate to acid-soluble nucleotides. Most patients had elevated serum RNase activity at the 95% confidence level when compared to 30 controls. Included in this group were 67% of patients with ANLL, 46% of patients with BT, 73% of patients with HD, and 67% of patients with NHL. These data confirmed the earlier suggestion that elevated serum nuclease activity is found in patients with neoplastic disease. However, whether or not a serum was identified as abnormal depended on the substrate used in the assay; this underscored the need to test samples against a variety of polynucleotides. Alterations in serum nucleolytic activity represent an important marker of neoplastic disease and can serve as the basis for a useful clinical screening device.

The age-dependent efficacy of polyadenylic-polyuridylic acid therapy upon the development of spontaneous leukemia in AKR mice.

The synthetic double-stranded polynucleotide, polyadenylic-polyuridylic acid, has been shown to increase the long-term survival of AKR mice. In order to determine whether this effect was age dependent, polyadenylic-polyuridylic acid was administered to AKR mice starting at 2, 4, 6, or 8 months of age. The best therapeutic effect was achieved when polyadenylic-polyuridylic acid treatments were begun at 2 months of age, and there was no beneficial effect when begun at 8 months of age.

Abnormalities of complement and its components in patients with acute leukemia, Hodgkin's disease, and sarcoma.

Whole complement and component titers were measured in patients with acute leukemia, Hodgkin's disease, and sarcoma. Serum samples were obtained from 42 consecutive patients and 11 healthy control subjects. Sera were frozen and maintained at -70 degrees until analyzed by hemolytic assay. Titers were normalized using a titer obtained from a single source of pooled human serum analyzed simultaneously with each patient sample to correct for day-to-day variation inherent in the assay technique. Significant elevations (p less than or equal to 0.05) of whole complement and C5, C8, and C9 were observed for each patient category, compared to controls. Forty-one of 42 patients had C9 titers greater than or equal to 2 S.D. above the mean titer for controls. Mean C3 and C7 titers were not elevated or depressed in any group. No clinical factors that correlated with abnormal complement or component titers were identified.

Alterations in ribonuclease activities in the plasma, spleen, and thymus of tumor-bearing mice.

Six transplantable murine tumor models were evaluated for changes in RNase activity. This study was conducted with spleen and thymus homogenates, as well as with plasma collected from tumor-bearing mice. Nuclease activity directed against the synthetic substrates, polyadenylic acid, polyuridylic acid, and polycytidylic acid, was measured and the data obtained for tumor-bearing animals were compared to their normal counterparts. Elevated activity against polyuridylic acid was observed in the plasma of all tumor-bearing mice. Although not as all inclusive, RNase levels in both the spleen and thymus were generally altered as well. The observance of unilateral changes in nuclease activity directed against the synthetic substrates demonstrated that, in most cases, two or more enzymes were being detected. The assay may have some eventual value in the monitoring of cancer

Enhancement of spontaneous C3H/HeJ mammary tumorigenesis by long-term polyadenylic-polyuridylic acid therapy.

Female C3H/HeJ mice received weekly s.c. injections of 2 mg polyadenylic-polyuridylic acid. Therapy was initiated at either 2 or 9 months of age. In both cases, poly-adenylic-polyuridylic acid-treated animals developed the spontaneous mammary carcinoma associated with this strain more rapidly. Because the opposite result was formerly observed for AKR spontaneous leukemia, the data indicate the polyadenylic-polyuridylic acid has no generalized antineoplastic effect upon spontaneous tumors genetically associated with specific murine strains.

Postinfectious leukoencephalitis complicating Mycoplasma pneumoniae infection.

Neuropathological findings in a patient with fatal neurological complications due to infection with Mycoplasma pneumoniae were similar to those seen in postinfectious encephalitis and acute hemorrhagic leukoencephalitis. This case supports the hypothesis that immune mechanisms play a role in the pathogenesis of neurological symptoms during mycoplasmal infections.

The demonstration of cell-associated immunity to viruses. In vitro lymphocyte responsiveness to Varicella-zoster antigen.

The demonstration of in vitro lymphocyte responsiveness to common pediatric viruses has previously been fraught with many technical and conceptual problems. Based upon our prior experience in demonstrating cell-associated immunity to mumps, rubella and measles viruses we illustrate our methodology and conceptual framework by documenting in vitro lymphocyte responsiveness to the Varicella-zoster virus, another ubiquitous virus of childhood. We discuss our approach to the problems of reactivity, specificity and reliability in the use of membrane-associated viral antigens.

Light transmission analysis of lymphocyte activation by mitogens: a new technique.

In vitro lymphocyte transformation was evaluated by laser light transmission cytometry. Criteria are given for optimal application of this system to assessment of total cells and proportion of lymphoblasts in cultures. The light transmission studies revealed that cell numbers in mitogen stimulated cultures increase steadily, reaching a plateau. The percentage of lymphoblasts reached a peak after 4 days culture. The kinetics of [3H]-thymidine incorporation are similar. The increase in total cell number concomitant with decrease in [3H]thymidine incorporation at the fifth and sixth days indicates discordance between the cell proliferation and utilization of exogenous thymidine. The kinetics of total cell numbers and percentage of lymphoblasts suggest that in vitro reversion of lymphoblasts to small lymphocytes occurred. In addition to examining directly the cellular events during blastogenesis with ease, the current method appeared to be more reproducible than the [3H]thymidine incorporation assay.

Herpes zoster and impaired cell-associated immunity to the varicella-zoster virus in patients with Hodgkin's disease.

Thirty-two patients with Hodgkin's disease and 12 normal donors were studied for their in vitro lymphocyte responsiveness to a membrane-associated varicella-zoster (VZ) antigen. When compared to the normal donors, patients with Hodgkin's disease in whom radiotherapy was recently completed and those with active, recurrent disease had markedly impaired cell-associated immunity to VZ antigen. In addition, there was a suggestion that patients in long-term remission who had received primary combined modality therapy (radiotherapy plus chemotherapy) had an impaired response when compared to normal persons or to patients who had received single modality therapy. Newly diagnosed, untreated patients with Hodgkin's disease did not differ significantly from normal persons as a group but two of six were unresponsive to the VZ antigen whereas all normal subjects were responsive. Most patients in remission for at least one year following therapy had normal in vitro responsiveness. In two patients herpes zoster developed after the demonstration of absent in vitro lymphocyte reactivity to the VZ antigen.

The synergistic effect of synthetic polynucleotides and mercaptoethanol in amplifying the murine mixed lymphocyte reaction.

Tritiated thymidine incorporation of one-way murine mixed lymphocyte reactions was compared in the presence of mercaptoethanol and/or poly AU. Each of these agents amplified specific responsiveness in this system without altering reaction kinetics and the combined use of these agents enhanced specific thymidine incorporation beyond that achieved by either alone. These agents may be useful in those situations where increased sensitivity of murine mixed lymphocyte reactions is required.

Application of laser cytometry to the analysis of immunologically induced in vitro lymphocyte responsiveness.

This study defines an assay (laser analysis) that is a significant advance in our ability to quantitate and analyze immunologically induced in vitro lymphocyte responsiveness. Laser analysis is demonstrated to parallel radionucleotide incorporation (3H-thymidine) in terms of kinetic pattern, dose response characteristics, and statistical accuracy while exeeding radionucleotide incorporation in sensitivity. Direct quantitation of lymphocyte responsiveness, in terms of cellular proliferation, disclosed that substantial numbers of small lymphocytes were produced during in vitro stimulation with mitogen (concanavalin A) or antigen (streptokinase-streptodornase) in addition to the expected increase in lymphoblasts. The magnitude of this "total cellular response" (lymphocytes plus lymphoblasts) was found to be similar for antigen and mitogen stimulation, a finding not suggested by routine radionucleotide incorporation or morphological assays.