Michael R. Mardiney

Additional evidence that the cell-associated immune system is the primary host defense against measles (rubeola)

Indirect evidence, such as the ability of agammaglobulinemic children to respond normally to measles and the inability of thymic-deficient children to survive measles led Burnet to postulate that the cell-associated immune system is the primary host defense against measles. Using a classical leukocyte culture system, which measures 3 H-thymidine incorporation as its endpoint, we have recently described an in vitro assay for lymphocyte responsiveness to measles complement fixation antigen (CFA) that has allowed us to study this question directly. Two pediatric residents who had negative HIA titers against measles and who had frequent exposure to measles were tested using this assay. Neither resident has had clinical measles or atypical measles syndrome. Both subjects displayed strong cellular responsiveness to measles in this system. We feel that the lymphocyte responsiveness to measles CFA seen in these residents is the in vitro correlate of their clinical protection against measles and offers additional evidence for the fundamental role of cell-associated immunity in the hosts response to common viral infections.

Cell-associated immunity to measles (rubeola): The demonstration of in vitro lymphocyte tritiated thymidine incorporation in response to measles complement fixation antigen☆

Abstract Lack of a reliable in vitro assay for lymphocyte responsiveness to measles (rubeola) has hampered our understanding of the cell-associated response in diseases caused by, or related to, the measles virus. We report a reliable and reproducible system for demonstrating specific lymphocyte incorporation of 3 H-thymidine in response to measles complement fixation antigen (CFA). Seventeen patients with positive histories of measles as children demonstrated a dose-response curve that varied between individuals but was constant for each individual. Kinetic data disclosed maximal responsiveness on day 7, and viral inactivation experiments disclosed that live virus was neither necessary for nor inhibitory to the reaction. The implications of this assay in terms of our understanding of the cell-associated response to measles virus in clinical measles and SSPE are discussed. The concept is explored that membrane-associated antigen is crucial in demonstrating the hosts cellular immune response to viruses that can grow by cell-to-contiguous cell spread.

The relationship between rubella hemagglutination inhibition antibody (HIA) and rubella induced in vitro lymphocyte tritiated thymidine incorporation

Abstract The parameters of lymphocyte, rubella virus interaction were studied by means of tritiated thymidine incorporation and compared with the rubella HIA level. Stimulation of lymphocyte DNA synthesis was found to depend on antigen concentration and to be independent of the presence of viable virus. When compared to HIA titer, lymphocyte transformation was found to be detectable in the absence of antibody. It is suggested that lymphocyte transformation is a sensitive method to detect both prior exposure and cellular immune reactivity to rubella virus.

The characteristics of lymphocyte tritiated thymidine incorporation in response to mumps virus

Abstract The parameters of immune lymphocyte responsiveness to mumps virus were studied by evaluation of tritiated thymidine incorporation. Inactivated virus was found to be a potent stimulus and response was found to be dependent on antigen concentration, and a radioresistant, adherent cell population. Measurable lymphocyte transformation after primary exposure to this Measurement lymphocyte transformation after primary exposure to this virus was found to persist for up to 40 years, and to correlate well with delayed skin sensitivity. The results demonstrate that lymphocyte transformation is a sensitive in vitro assay of cellular reactivity to this virus if used in an inactivated form. It is suggested that it may be a useful method to study the course of cellular reactivity after viral exposure as well as a means to detect previous exposure to other viruses.

An improved system for controlled rate cooling of biological material.

Abstract The unit in present operation at the Baltimore Cancer Research Center has afforded far greater accuracy than freezing control units now commercially available. Its versatility in allowing direct dialing of the cooling rate and feedback control from the sample phantom allows for a completely automated cooling system which will take the specimen smoothly through the heat of fusion while maintaining a constant predetermined slope. An example of a typical freezing curve depicting lymphocytes frozen in 1640 media containing 10% DMSO as previously described (1–3) is shown in Fig. 2. The present unit is wired to function with the Linde BF4-1 and BF4-2 freezing chambers; however, any similar chamber could be used by providing the proper electrical connections and sample sensor. The cost of the controllers component parts will vary according to the number of features desired but for the unit described here it is approximately

Mycoplasma contamination of membrane associated measles antigens. Inability to demonstrate in vitro lymphocyte responsiveness to measles

350.00.

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

Abstract Mycoplasma contamination of tissue cultures used in the preparation of membrane associated measles antigen is demonstrated to result in diminishment of measles antigenicity with subsequent inability to demonstrate in vitro lymphocyte responsiveness to measles. Electron microscopic studies disclose the absence of measles antigen in mycoplasma containing specimens and suggest that the effect of mycoplasma is to block in vitro proliferation of measles virus during antigen preparation. Mycoplasma contamination per se has no consistent effect on spontaneous lymphocyte reactivity.

The prevention of clumping of frozen-stored leukocyte populations by EDTA

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.

Suppression of in vitro lymphocyte responsiveness to purified protein derivative by measles virus. A reexploration of the phenomenon.

Abstract The clumping of cell suspensions observed after thawing of frozen-stored leukocyte suspensions was investigated. The clumping, which was secondary to the aggregation of dead granulocytes and viable lymphocytes, could be virtually eliminated by the use of purified mononuclear cell preparations or by the addition of EDTA to media prior to freezing and during thawing.

In vivo abrogation of serum C3 and C5 by administration of cobra venom factor and heterologous anti-C3

Abstract Clinical measles and measles vaccination have classically been associated with transient in vivo impairment of delayed hypersensitivity-type responses, especially skin test reactivity to purified protein derivative (PPD). In vitro data appeared to substantiate this in vivo observation by the demonstration of suppression of lymphocyte responsiveness to PPD by measles. Utilizing a measles preparation which has been recently demonstrated to elicit specific blastogenesis of sensitized human lymphocytes in vitro , we have reexplored the question of in vitro suppression of lymphocyte responsiveness to PPD by this virus. In contrast to previous reports, this study demonstrates that the addition of both measles and PPD to lymphocyte cultures can have a variable effect on lymphocyte responsiveness to PPD alone. This effect varies from marked inhibition to enhancement beyond a summation effect. The response is different for each lymphocyte donor and is dose related but cannot be predicted on the basis of combinations of high or low concentrations of either antigen. Purified, attenuated measles virus (Enders strain), which uniformly suppressed in vitro lymphocyte reactivity when tested alone also demonstrated a significant dose related enhancement of the response to PPD alone. The present data suggest a reconsideration of the supposed importance of transient diminution of skin test reactivity during measles infection or immunization.