A role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles

In the 1960s, a formalin-inactivated measles vaccine (FIMV) predisposed recipients to atypical measles, an immune complex-mediated disease. To identify characteristics of the immune priming that leads to atypical measles, responses of monkeys to FIMV were compared with responses to live attenuated virus (LAV) and hemagglutinin (H-DNA) vaccines that do not prime for atypical measles. Antibodies induced by FIMV were transient and avidity did not mature. Antibodies induced by LAV and H-DNA vaccines were sustained and avidity matured over time. After challenge with measles virus, FIMV and H-DNA recipients developed high titers of complement-fixing antibodies. In FIMV recipients, the antibodies were of low avidity, whereas in H-DNA vaccine recipients, the antibodies were of high avidity. Neutralizing capacity in B958 cells correlated with avidity. Only FIMV recipients had immune complex deposition. Failure of FIMV to induce affinity maturation results in anamnestic production of nonprotective, complement-fixing antibodies, immune complex deposition and atypical measles.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

Modulation of lymphocyte mitogen responses by cocultured fibroblasts

Immunological reactions in vivo occur in an environment rich in fibroblasts (FB) and other connective tissue cells. The possibility that FB might affect mononuclear cell proliferative responses to mitogens in vitro was examined in a microculture system. Human peripheral blood mononuclear cells (MC) were cocultured with mitomycin C-treated FB in the presence of phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Cocultured FB (at a 1:100 to 1:10 FB:MC ratio) suppressed the response of MC to PHA (by as much as 35%) but did not significantly affect PWM responses. Cocultures of FB and MC were characterized by 10- to 30-fold increases in prostaglandin E₂ concentrations compared to MC or FB cultured alone. Inhibition of prostaglandin synthesis with indomethacin or mefenamic acid significantly reversed the FB-mediated suppression of lymphocyte PHA responses. Prostaglandin-dependent FB suppression of lymphocyte PHA responses was seen only when FB:MC coculture was initiated at the onset of exposure of MC to PHA. When FB were added to MC after 24 hr of culture with PHA, no effect was seen. Addition at 48 or 66 hr resulted in prostaglandin-independent enhancement of lymphocyte proliterative responses to PHA. Thus in cocultures of FB and MC, MC reactivity to PHA may be influenced in part via alterations in FB prostaglandin metabolism. The interaction between FB and MC may be important in the modulation of immune responses in inflammatory lesions.     

Measles: immune suppression and immune responses

Measles is a highly contagious viral disease that remains the leading vaccine-preventable cause of child mortality worldwide. Deaths from measles are due largely to an increased susceptibility to secondary bacterial and viral infections, attributed to a prolonged state of immune suppression. Several abnormalities of the immune system have been described, including changes in lymphocyte number and function, shifts in cytokine responses, immunomodulatory effects of interleukin-10, down regulation of interleukin-12, impaired antigen presentation, and altered interferon alpha/beta signaling pathways. Although the current vaccine is very effective, knowledge of the molecular basis of the immune responses to measles virus could contribute to the development of a safer, more immunogenic measles vaccine. However, the safety of new measles vaccines must be carefully investigated, as two measles vaccines have resulted in unintended immunologic consequences: atypical measles following administration of the formalin-inactivated measles vaccine and increased mortality in girls following administration of high-titer measles vaccines.     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

Atypical measles infections in leukaemic children on immunosuppressive treatment.

One case of giant-cell pneumonia and two of encephalopathy, all due to measles infection in children in leukaemic remission on immunosuppressive treatment, were seen recently. The clinical syndromes were variable and atypical and the antibody responses unpredictable. Conventional doses of pooled immunoglobulin failed to protect the two children to whom it was given. Degeneration rather than inflammation seems to characterise the encephalopathy in immunosuppressed children infected with measles virus.     

Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody.

The severe disease atypical measles occurred when individuals immunized with a poorly protective inactivated vaccine contracted measles, and was postulated to be due to a lack of fusion-inhibiting antibodies. Here, rhesus macaques immunized with formalin-inactivated measles vaccine developed transient neutralizing and fusion-inhibiting antibodies, but no cytotoxic T-cell response. Subsequent infection with measles virus caused an atypical rash and pneumonitis, accompanied by immune complex deposition and an increase in eosinophils. Fusion-inhibiting antibody appeared earlier in these monkeys than in non-immunized monkeys. These data indicate that atypical measles results from previous priming for a nonprotective type 2 CD4 T-cell response rather than from lack of functional antibody against the fusion protein.     

Suppression of bovine T- and B-lymphocyte responses by fetuin, a bovine glycoprotein

Fetuin, a bovine glycoprotein present in milligram amounts in fetal calf serum, suppressed lymphocyte responses to phytohemagglutinin (PHA) by 70–90%. T-lymphocyte-enriched populations were suppressed by fetuin in their response to PHA indicating direct T-cell regulation and suggesting that macrophages were not required for fetuin activity. Lymphocyte responses to the lipopolysaccharides (LPS) of Escherichia coli and Brucella abortus in the presence of fetuin were markedly reduced compared to cultures without fetuin. Fetuin also suppressed alloantigen recognition in 85% of the mixed-leukocyte responses, and primary in vitro antibody responses induced by the LPS of B. abortus. No toxicity to responding cells or inhibition of early cell division could be demonstrated. A mechanism of regulation appeared to be via suppressor cells. Lymphocytes maintained in fetuin for 48 hr, but not beyond 72 hr, exhibited suppressor cells which could modulate normal adult lymphocyte responses to PHA.     

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

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.     

In vivo and in vitro evaluation of cell-mediated immunity in patients with paracoccidioidomycosis

The cellular immune response to specific and nonspecific agents was investigated. both in vivo and in vitro, in 19 patients with paracoceidioidomycosis. In addition, the immunologic study of an investigator aceidentally inoculated with P. brasiliensis was included in this study. Nearly half of the patients showed depressed cell-mediated immune responses, as evaluated by intradermal tests with an antigenic preparation from P. brasiliensis (P.b.Ag.), ubiquitous antigens, and by the ability to develop sensitization to 2,4-dinitrochlorobenzene. A similar proportion of impaired responses was observed when the patients' lymphocytes were cultured with phytohemagglutinin (PHA). C'. albicans antigen and P.h.Ag. A factor was detected in the plasma of some patients which reduced the ability of patients' and normal lymphocytes to undergo blastic transformation. A positive correlation was found between the ability to develop delayed cutaneous hypersensitivity reactions to P.b.Ag. and other ubiquitous antigens, normal in vitro responsiveness to PHA and the absence of humoral blastogenic inhibitory factor. The inhibition of leukocyte migration, but not lymphocyte transformation, correlated positively with delayed hypersensitivity. The percentage of T lymphocytes was significantly reduced in the group of patients, being the absolute number and percentage of B cells bearing receptors tor complement normal. Two polar immunological patterns emerged. One characterized by positiveness in the skin test to P.b.Ag. and lack of significant abnormalities in cellular immunity, and another anergic to P.b.Ag., with cell mediated immunity severely depressed. Between the two polar groups, there were patients with intermediary patterns of immune response. This paper also includes the results obtained with the administration of transfer factor and levamisole to some of the patients.     

Measles Immunoglobulins in Subacute Sclerosing Panencephalitis

Normal responses of measles specific immunoglobulins M and G (IgM and IgG) were defined in 10 children with measles. Abnormal responses of measles IgM and IgG were found in both sera and cerebrospinal fluids from three cases of subacute sclerosing panencephalitis. In two patients the serum titres of measles IgM and IgG were abnormally high. The measles IgM was present during prolonged illnesses in serum and cerebrospinal fluid, which suggested a correlation with the known persistence of measles virus antigen in the brain of the three patients. It was concluded that both measles IgM and IgG may be produced within the central nervous system in subacute sclerosing panencephalitis.     

Comparison of the Immune Responses Induced by Chimeric Alphavirus-Vectored and Formalin-Inactivated Alum-Precipitated Measles Vaccines in Mice

A variety of vaccine platforms are under study for development of new vaccines for measles. Problems with past measles vaccines are incompletely understood and underscore the need to understand the types of immune responses induced by different types of vaccines. Detailed immune response evaluation is most easily performed in mice. Although mice are not susceptible to infection with wild type or vaccine strains of measles virus, they can be used for comparative evaluation of the immune responses to measles vaccines of other types. In this study we compared the immune responses in mice to a new protective alphavirus replicon particle vaccine expressing the measles virus hemagglutinin (VEE/SIN-H) with a non-protective formalin-inactivated, alum-precipitated measles vaccine (FI-MV). MV-specific IgG levels were similar, but VEE/SIN-H antibody was high avidity IgG2a with neutralizing activity while FI-MV antibody was low-avidity IgG1 without neutralizing activity. FI-MV antibody was primarily against the nucleoprotein with no priming to H. Germinal centers appeared, peaked and resolved later for FI-MV. Lymph node MV antibody-secreting cells were more numerous after FI-MV than VEE/SIN-H, but were similar in the bone marrow. VEE/SIN-H-induced T cells produced IFN-γ and IL-4 both spontaneously ex vivo and after stimulation, while FI-MV-induced T cells produced IL-4 only after stimulation. In summary, VEE/SIN-H induced a balanced T cell response and high avidity neutralizing IgG2a while FI-MV induced a type 2 T cell response, abundant plasmablasts, late germinal centers and low avidity non-neutralizing IgG1 against the nucleoprotein.     

Comparison of the responses of density gradient separated lymphocytes to phytohemagglutinin and histocompatibility antigens

Rat peripheral blood leukocytes were fractionated into 5–9 subpopulations by centrifugation on discontinuous density gradients of bovine serum albumin. Responses of the various fractions to phytohemagglutinin (PHA) in vitro were compared with their responses to alloantigens in the mixed lymphocyte interaction in vitro and in a graft versus host reaction in vivo. The results showed that: (a) Cells from each of the gradient fractions responded to alloantigens both in vitro and in vivo, (b) Only cells of intermediate density responded vigorously to PHA at a concentration which optimally stimulated unfractionated cells, (c) Low density lymphocytes could be stimulated by 3–9-fold lower concentrations of mitogen. (d) Cells from low and high density fractions, which alone responded poorly to PHA, showed enhanced responses when mixed. These findings may have a significant bearing on the use of the in vitro response to PHA for evaluating the overall function of thymus derived cells in clinically related studies.     

Clinical and endocrine characteristics in atypical and classical growth hormone insensitivity syndrome

OBJECTIVE: Classical growth hormone insensitivity syndrome (GHIS) comprises a dysmorphic phenotype, extreme short stature (height SDS < 3), normal GH and low IGF-I and IGFBP-3. Wide clinical variation is recognised with classical and atypical forms. We aimed to delineate features of the milder "atypical" GHIS phenotype, and to determine whether this correlates with milder auxological and biochemical features. METHODS: Fifty-nine patients from a European series of 82 patients with GHIS, with strict diagnostic criteria of GHIS, were studied and assigned to classical or atypical GHIS groups according to facial phenotype, i.e. "classical" required 2 of 3 recognized GHIS features (frontal bossing, mid-facial hypoplasia and depressed nasal bridge), "atypical" required 0 or 1 of these facial features. Classical and atypical GHIS groups were compared in terms of (1) phenotypic features, including high-pitched voice, sparse hair, blue sclera, hypoglycaemia, microphallus, (2) birth length, height SDS, and (3) basal IGF-I, IGF-II, IGFBP-1, IGFBP-3, GHBP and increase in IGF-I on IGF-I generation testing. RESULTS: Fifty patients [24 males, 26 females, aged 8.6 +/- 4.6 years (mean +/- SD)] had "classical GHIS", 9 patients (7 males, 2 females, aged 7.8 +/- 4.1 years) had "atypical GHIS", 7 with normal facies. Atypical GHIS patients had lesser height deficit (Ht SDS -4.0 +/- 1.4) compared to classical GHIS (-6.7 +/- 1.4), less reduction in IGFBP-3 SDS (atypical -5.5 +/- 3.3; classical -8.6 +/- 2.4), and more had normal GHBP (>10% binding). Other variables were also less frequent in atypical GHIS patients: high-pitched voice 11% (70% classical), sparse hair 11% (42% classical), blue sclera 0% (38% classical), hypoglycaemia 11% (42% classical), and microphallus 14% (1 of 7 males), compared to 79% of classical (19 of 24 males). CONCLUSIONS: Atypical GHIS patients, with relatively normal facial appearance, demonstrate less height defect and biochemical abnormalities compared to classical patients. GH insensitivity may be present in children with short stature and an otherwise normal appearance.     

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.     

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

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 ³H-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 host's cellular immune response to viruses that can grow by cell-to-contiguous cell spread.     

Immunologic response in vitro after thymechtomy in patients with myasthenia gravis.

Thymectomy in adult animals impairs immune functions such as lymphocyte response to phytohemagglutinin (PHA) and to allogeneic cells. The responses of lymphocytes from 18 myasthenia gravis patients, 12 of whom had undergone thymectomy, were studied; the interval between thymectomy and investigation ranged from 1 month to 26 years (mean, 9.5 years). Peripheral blood lymphocytes were cultured in autochthonous plasma or homologous AB serum. In vitro responses to stimulation with PHA, concanavalin A and allogeneic monomuclear cells were within the 95% range of normal responses in all patients. Because our findings contrast with the definite immune defects resulting from thymectomy found in adult animals, longer follow-up is needed.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.