Polyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the VH-diversity(D)-JH and V kappa-J kappa gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro C gamma l and C kappa human expression system. These mAbs are virtually identical in their VH and V kappa gene segment sequences. First, we expressed the VH-D-JH and V kappa-J kappa genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or kappa chain with the mAb13-derived kappa or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived kappa V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived kappa V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1)-FR3 and complementarity determining region 3 (CDR3) regions of the H and kappa chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived kappa chain FR1-FR3, but not that of the kappa chain CDR3.(ABSTRACT TRUNCATED AT 400 WORDS)

We present experimental data that offer, in part, a better understanding of the immunosuppression that accompanies measles virus infection. We note that measles virus "silently" infects human lymphocytes and that the infection does not alter lymphocyte survival in vitro. Yet such infected lymphocytes fail to generate natural killer (NK) cell activity or synthesize immunoglobulins (Ig). Thus, the presence of virus within lymphocytes impairs their specific immune functions in the absence of cytolysis. Influenza virus also infects human lymphocytes. In contrast to measles virus infection of resting lymphocytes in which viral antigen is rarely expressed, influenza virus infection of these cells yields viral antigens expressed in the cytoplasm and on the cell surface. Influenza virus-infected lymphocytes have normal NK cell activity but fail to synthesize IgG or IgM.

In patients with rheumatoid arthritis (RA), circulating CD5+ B lymphocytes, but not CD5- B lymphocytes, are increased in number and size, exist in an activated state, spontaneously proliferate, and secrete Ig that binds to the Fc fragment of IgG. By constructing continuous mAb-secreting cell lines from CD5+ B lymphocytes, the properties and dissociation constants (Kd) of these antibodies were determined. Two types of rheumatoid factors (RFs) with discrete reactivities were produced. The first type is polyreactive and binds with relatively low affinity (Kd, 10(-5) mol/liter) to the Fc fragment of IgG. These antibodies are similar to those produced by CD5+ B cells from healthy subjects. The second type of RF is monoreactive and binds with higher affinity (Kd, 10(-7) mol/liter) to the Fc fragment of IgG. These latter autoantibodies are produced by CD5+ B cells of RA patients, but not healthy subjects. Long-term longitudinal studies are needed to determine the role of these two types of RFs in the pathogenesis of RA.

Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte-rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity.

The CUORE experiment is a large bolometric array searching for the lepton number violating neutrino-less double beta decay ($0 u\beta\beta$) in the isotope $\mathrm{^{130}Te}$. In this work we present the latest results on two searches for the double beta decay (DBD) of $\mathrm{^{130}Te}$ to the first $0^{+}_2$ excited state of $\mathrm{^{130}Xe}$: the $0 u\beta\beta$ decay and the Standard Model-allowed two-neutrinos double beta decay ($2 u\beta\beta$). Both searches are based on a 372.5 kg$\times$yr TeO$_2$ exposure. The de-excitation gamma rays emitted by the excited Xe nucleus in the final state yield a unique signature, which can be searched for with low background by studying coincident events in two or more bolometers. The closely packed arrangement of the CUORE crystals constitutes a significant advantage in this regard. The median limit setting sensitivities at 90\% Credible Interval (C.I.) of the given searches were estimated as $\mathrm{S^{0 u}_{1/2} = 5.6 \times 10^{24} \: \mathrm{yr}}$ for the ${0 u\beta\beta}$ decay and $\mathrm{S^{2 u}_{1/2} = 2.1 \times 10^{24} \: \mathrm{yr}}$ for the ${2 u\beta\beta}$ decay. No significant evidence for either of the decay modes was observed and a Bayesian lower bound at $90\%$ C.I. on the decay half lives is obtained as: $\mathrm{(T_{1/2})^{0 u}_{0^+_2} > 5.9 \times 10^{24} \: \mathrm{yr}}$ for the $0 u\beta\beta$ mode and $\mathrm{(T_{1/2})^{2 u}_{0^+_2} > 1.3 \times 10^{24} \: \mathrm{yr}}$ for the $2 u\beta\beta$ mode. These represent the most stringent limits on the DBD of $^{130}$Te to excited states and improve by a factor $\sim5$ the previous results on this process.