Natural killer (NK) cells were recently shown to play a relevant role in the process of dendritic cell (DC) maturation. This function is exerted either by direct DC stimulation or through killing those DCs that did not properly acquire a mature phenotype. While killing of immature DCs is dependent on the function of the NKp30 triggering receptor, the mechanism by which NK cells induce DC maturation is still unclear. In this study, we show that also the NK-mediated induction of DC maturation is dependent on NKp30. Upon NK/DC interaction, resulting in NKp30 engagement, NK cells produced tumor necrosis factor alpha (TNFalpha) (and interferon gamma [IFNgamma]) that, in turn, promoted DC maturation. Masking of NKp30 with specific monoclonal antibodies (mAbs) strongly reduced maturation of DCs cocultured with NK cells. In addition, supernatant from NK cells stimulated via NKp30 induced DC maturation, and this effect was neutralized by anti-TNFalpha antibodies (Abs). This NKp30 function is controlled by the HLA-specific inhibitory NK receptors. Accordingly, the ability to promote maturation was essentially confined to NK cells expressing the killer immunoglobulin-like receptor-negative (KIR-) NKG2A(dull) phenotype. Finally, the analysis of perforin-deficient NK cells allowed the dissection of the 2 NKp30-mediated NK-cell functions, since NKp30 could induce cytokine-dependent DC maturation in the absence of NK-mediated DC killing.

Magic-angle spinning solid-state NMR (SSNMR) studies of the beta1 immunoglobulin binding domain of protein G (GB1) are presented. Chemical shift correlation spectra at 11.7 T (500 MHz 1H frequency) were employed to identify signals specific to each amino acid residue type and to establish backbone connectivities. High sensitivity and resolution facilitated the detection and assignment of every 15N and 13C site, including the N-terminal (M1) 15NH3, the C-terminal (E56) 13C', and side-chain resonances from residues exhibiting fast-limit conformational exchange near room temperature. The assigned spectra lend novel insight into the structure and dynamics of microcrystalline GB1. Secondary isotropic chemical shifts report on conformation, enabling a detailed comparison of the microcrystalline state with the conformation of single crystals and the protein in solution; the consistency of backbone conformation in these three preparations is the best among proteins studied so far. Signal intensities and line widths vary as a function of amino acid position and temperature. High-resolution spectra are observed near room temperature (280 K) and at <180 K, whereas resolution and sensitivity greatly degrade substantially near 210 K; the magnitude of this effect is greatest among the side chains of residues at the intermolecular interface of the microcrystal lattice, which we attribute to intermediate-rate translational diffusion of solvent molecules near the glass transition. These features of GB1 will enable its use as an excellent model protein not only for SSNMR methods development but also for fundamental studies of protein thermodynamics in the solid state.

Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.

Much of T and B lymphocyte receptor diversity derives from the addition of nontemplated N regions at the junctions of receptor gene elements, although fetal T cells expressing gamma/delta receptors lack N regions. I have sequenced immunoglobulin H chain variable regions of PCR-amplified DNA and cDNA from fetal and newborn mouse liver and spleen cells. These sequences showed an absence of N regions. Only 1/87 DNA sequences and 17/146 RNA sequences contained N regions, in striking contrast to adult Ig sequences. These data show that N region insertion is a developmentally regulated process in B cells as well as in T cells, and demonstrate that receptor diversity in neonatal B cells is limited by the absence of N regions as well as by biased usage of Vh genes.

Anti-alpha-galactosyl immunoglobulin G (anti-Gal) is a natural antibody present in unusually high amounts in human sera. It constitutes as much as 1% of circulating immunoglobulin G in humans and displays a distinct specificity for the carbohydrate epitope galactosyl alpha(1----3) galactosyl (Gal alpha 1----3Gal). Recently, it has been suggested by various investigators that anti-Gal may be related to some autoimmune phenomena, since marked elevation of its titer was found in sera of patients with autoimmune thyroid disorders, rheumatoid arthritis, glomerulonephritis, and Chagas' disease. In view of the ubiquitous presence of anti-Gal in high titers in humans, throughout life, we hypothesized that, analogous with synthesis of anti-blood group antibodies against bacterial antigens, bacteria within normal intestinal flora may provide constant antigenic stimulation for the synthesis of anti-Gal. This hypothesis would imply that anti-Gal may bind to a variety of bacterial strains of human flora. In the present study, the interaction between affinity chromatography-purified anti-Gal and various bacterial strains was studied. By the use of a direct immunostaining assay and an enzyme-linked immunosorbent assay, anti-Gal was found to interact with a variety of Escherichia coli, Klebsiella, and Salmonella strains, some of which were isolates from normal stool. Furthermore, the anti-Gal-binding sites in some strains were found to be present on the carbohydrate portion of bacterial lipopolysaccharides. It is thus suggested that Gal alpha 1----3Gal epitopes in the outer membranes of normal flora enterobacteria may provide a continuous source for antigenic stimulation. Since there is no immune tolerance to the Gal alpha 1----3Gal carbohydrate structure in humans, anti-Gal seems to be constantly produced in response to these enterobacteria. In addition, bacteria which express Gal alpha----3Gal epitopes and which may adhere to various cells mediated binding of anti-Gal to human cell lines. These findings raise the possibility that anti-Gal may damage normal human tissues via inflammatory processes facilitated by bacterial Gal alpha 1----3Gal epitopes.

We hypothesized that the presence of monoclonal free kappa or lambda immunoglobulin light chains in monoclonal gammopathy of undetermined significance (MGUS), as detected by the serum free light chain (FLC) assay increases the risk of progression to malignancy. Of 1384 patients with MGUS from Southeastern Minnesota seen at the Mayo Clinic from 1960 to 1994, baseline serum samples obtained within 30 days of diagnosis were available in 1148. At a median follow-up of 15 years, malignant progression had occurred in 87 (7.6%) patients. An abnormal FLC ratio (kappa-lambda ratio < 0.26 or>> 1.65) was detected in 379 (33%) patients. The risk of progression in patients with an abnormal FLC ratio was significantly higher compared with patients with a normal ratio (hazard ratio, 3.5; 95% confidence interval [CI], 2.3-5.5; P < .001) and was independent of the size and type of the serum monoclonal (M) protein. Patients with an abnormal serum FLC ratio, non-immunoglobulin G (non-IgG) MGUS, and a high serum M protein level >> or = 15 g/L) had a risk of progression at 20 years of 58% (high-risk MGUS) versus 37% with any 2 of these risk factors (high-intermediate risk), 21% with one risk factor (low-intermediate risk), and 5% when none of the risk factors were present (low risk).

NF-IL6 was originally identified as a DNA-binding protein responsible for IL-1-stimulated IL-6 induction. Direct cloning of NF-IL6 revealed its homology with C/EBP. C/EBP is expressed in liver and adipose tissues and is supposed to regulate several hepatocyte- and adipocyte-specific genes. In contrast, NF-IL6 is suppressed in normal tissues, but is rapidly and drastically induced by LPS or inflammatory cytokines such as IL-1, TNF, and IL-6. NF-IL6 can also bind to the regulatory region of various genes including IL-8, G-CSF, IL-1 and immunoglobulin genes. Furthermore, NF-IL6 is shown to be identical to IL-6DBP, a DNA-binding protein responsible for IL-6-mediated induction in acute-phase proteins, demonstrating that NF-IL6 is responsible for the genes regulated by IL-6. These results indicate that NF-IL6 may be a pleiotropic mediator of many inducible genes involved in acute, immune, and inflammatory responses, like NFkB. In this regard, it is noteworthy that both an NF-IL6 binding site and an NFkB binding site are present in the inducible genes such as IL-6, IL-8, and several acute-phase genes. On the other hand, accumulating evidence has revealed that overproduction of IL-6 may be responsible for the pathogenesis and/or several symptoms of a variety of diseases, including autoimmune diseases, malignancies, and viral diseases. At present, the molecular mechanisms of abnormal expression of the IL-6 gene are not known. Recently it has become evident that interplays between viral proteins and cellular proteins play an important role in viral oncogenesis and infection. The fact that NF-IL6 binds to the enhancer core sequences of various viruses strongly suggests a possible relationship of virus infection and IL-6 expression. In fact some evidence (Mahe et al. 1991, Spergel et al. 1992) indicates that NF-IL6 may interact with viral gene enhancers or viral products, although there are no definite data about the involvement of NF-IL6 in viral pathogenesis. Future studies will be required to clarify whether or not the interplay between NF-IL6 and viral infection is responsible for deregulation of the IL-6 gene.

The association of primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) is thought to be rare, and its optimal treatment is unknown. Of 130 consecutive patients with a diagnosis of PBC, we identified 12 cases (9.2%) of overlap syndrome (10 females, 2 males; median age, 50 years) strictly defined by the presence of at least two of the three recognized biochemical, serological, and histological criteria of each disease. One patient had initially pure PBC and developed AIH characterized by a flare of alanine transaminase (ALT) (1,330 IU/L; N < 35), elevated immunoglobulin G (IgG) (42 g/L; N < 14.0), and presence of anti-smooth muscle antibodies (ASMA) after 20 months of ursodeoxycholic acid (UDCA) therapy. A complete clinical and biochemical remission was achieved under combination of corticosteroids and UDCA. Eleven patients had features of both diseases at presentation: high serum levels of alkaline phosphatase (AP) (median: 280 IU/L; N < 100), ALT (140 IU/L), and IgG (30.8 g/L), presence of mitochondrial antibodies (n = 9) or ASMA (n = 9), florid bile duct lesions (n = 8), and moderate or severe periportal or periseptal lymphocytic piecemeal necrosis (n = 11). UDCA (13-15 mg/kg/d) given alone in 5 patients induced a significant decrease in biochemical cholestasis but not in ALT levels, and liver fibrosis progressed in 3 patients. Corticosteroids given alone in 6 patients induced a significant decrease in ALT, IgG, and AP levels, but none had a biochemical normalization. The patients with persistently abnormal liver tests under either UDCA or corticosteroids received both UDCA and corticosteroids. A further marked biochemical improvement was observed, and all patients became asymptomatic. We conclude that, in patients with PBC: 1) overlap syndrome with AIH is not rare; 2) flares of AIH may occur either spontaneously or under UDCA; and 3) combination of UDCA and corticosteroids is required in most patients to obtain a complete biochemical response. Overlap syndrome may represent an important and unrecognized cause of resistance to UDCA in patients with PBC.

The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to cholera enterotoxin. A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity. These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A1 by a variety of chemical or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A1 and A2 components of the A subunit. This mutant contains a single amino acid substitution within the disulfide subtended region joining A1 and A2. This mutant toxin, designated LT(R192G), is not sensitive to proteolytic activation, has negligible activity on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltransferase activity. Nonetheless, LT(R192G) retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone. Further, LT(R192G) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the presence of significant serum anti-LT IgG and mucosal anti-LT IgA antibodies in immunized mice.

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.

BACKGROUND

Despite effective antiretroviral therapy (ART), patients with chronic human immunodeficiency virus (HIV) infection have increased microbial translocation and systemic inflammation. Alterations in the intestinal microbiota may play a role in microbial translocation and inflammation.

METHODS

We profiled the fecal microbiota by pyrosequencing the gene encoding 16S ribosomal RNA (rRNA) and measured markers of microbial translocation and systemic inflammation in 21 patients who had chronic HIV infection and were receiving suppressive ART (cases) and 16 HIV-uninfected controls.

RESULTS

The fecal microbial community composition was significantly different between cases and controls. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, Erysipelotrichi, Erysipelotrichales, Erysipelotrichaceae, and Barnesiella was significantly enriched in cases, whereas that of Rikenellaceae and Alistipes was depleted. The plasma soluble CD14 level (sCD14) was significantly higher and the endotoxin core immunoglobulin M (IgM) level lower in cases, compared with controls. There were significant positive correlations between the relative abundances of Enterobacteriales and Enterobacteriaceae and the sCD14 level; the relative abundances of Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae and the interleukin 1β (IL-1β) level; the relative abundances of Enterobacteriales and Enterobacteriaceae and the interferon γ level; and the relative abundances of Erysipelotrichi and Barnesiella and the TNF-α level. There were negative correlations between endotoxin core IgM and IL-1β levels.

CONCLUSIONS

Patients who have chronic HIV infection and are receiving suppressive ART display intestinal dysbiosis associated with increased microbial translocation and significant associations between specific taxa and markers of microbial translocation and systemic inflammation. This was an exploratory study, the findings of which need to be confirmed.

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin G (IgG) to the bloodstream of the newborn. FcRn is structurally similar to class I major histocompatibility complex (MHC) molecules, despite differences in the ligands they bind (the Fc portion of IgG and antigenic peptides, respectively). A low-resolution crystal structure of the complex between FcRn and Fc localizes the binding site for Fc to the side of FcRn, distinct from the tops of the alpha 1 and alpha 2 domains which serve as the peptide and T-cell receptor binding sites in class I molecules. FcRn binds to Fc at the interface between the Fc CH2 and CH3 domains, which contains several histidine residues that could account for the sharply pH-dependent FcRn/IgG interaction. A dimer of FcRn heterodimers observed in the co-crystals and in the crystals of FcRn alone could be involved in binding Fc, correlating with the 2:1 binding stoichiometry between FcRn and IgG (ref. 4) and suggesting an unusual orientation of FcRn on the membrane.

Indirect evidence implicates gamma delta T cells in the cross-regulation of CD4 alpha beta T cell responses. Adoptive transfer of small numbers of gamma delta T cells from ovalbumin (OVA)-tolerant mice selectively suppressed TH2-dependent immunoglobulin E (IgE) antibody production without affecting parallel IgG responses. Challenge of these gamma delta T cells in vitro with specific antigen resulted in production of high levels of interferon gamma. The effects of the gamma delta T cells may be mediated by direct inhibition of OVA-specific CD4+ TH2 cell proliferation or selection for specific CD4 TH2 cells.

The development of immune deposits on the subepithelial surface of the glomerular capillary wall was studied in isolated rat kidneys perfused at controlled perfusion pressure, pH, temperature, and flow rates with recirculating oxygenated perfusate containing bovine serum albumin (BSA) in buffer and sheep antibody to rat proximal tubular epithelial cell brush border antigen (Fx1A). Control kidney were perfused with equal concentrations of non-antibody immunoglobulin (Ig)G. Renal function was monitored by measuring inulin clearance, sodium reabsorption, and urine flow as well as BSA excretion and fractional clearance. Perfused kidneys were studied by light, immunofluorescence, and electron microscopy. All kidneys perfused with anti-Fx1A developed diffuse, finely granular deposits of IgG along the glomerular capillary wall by immunofluorescence. Electron microscopy revealed these deposits to be localized exclusively in the subepithelial space and slit pores. Similar deposits were produced in a nonrecirculating perfusion system, thereby excluding the formation of immune complexes in the perfusate caused by renal release of tubular antigen. Control kidneys perfused with nonantibody IgG did not develop glomerular immune deposits. Renal function and BSA excretion were the same in experimental and control kidneys. Glomerular deposits in antibody perfused kidneys were indistinguishable from deposits in rats injected with anti-Fx1A or immunized with Fx1A to produce autologous immune complex nephropathy. These studies demonstrate that subepithelial immune deposits can be produced in the isolated rat kidney by perfusion with specific antibody to Fx1A in the absence of circulating immune complexes. In this model deposits result from in situ complex formation rather than circulating immune complex deposition.

Cognate interactions between antigen-presenting B and T cells play crucial roles in immunologic responses. T cells that have been activated via the crosslinking of CD3 are able to induce B cell proliferation and immunoglobulin secretion in a major histocompatibility complex-unrestricted and contact-dependent manner. We find that such activated human CD4+ T cells, but not control Ig-treated T cells, may induce normal or leukemic B cells to express B7/BB1 and significantly higher levels of CD54 intercellular adhesion molecule 1 via a process that also requires direct cell-cell contact. To discern what cell surface molecule(s) may be responsible for signalling B cells to express B7/BB1, we added various monoclonal antibodies (mAbs) specific for T or B cell accessory molecules or control mAbs to cocultures of alpha-CD3-activated T cells and resting B cells. We find that only alpha-CD40 mAbs can significantly inhibit the increased expression of B7/BB1, suggesting that the ligand for CD40 expressed on activated T cells may be an important inducer of B7/BB1 expression. Subsequent experiments in fact demonstrate that alpha-CD40 mAbs, but not control mAbs, induce changes in B cell phenotype similar to those induced by activated T cells when the mAbs are presented on Fc gamma RII (CDw32)-expressing L cells. These phenotypic changes have significant effects on B cell function. Whereas chronic lymphocytic leukemia (CLL) B cells normally are very poor stimulators in allogeneic mixed lymphocyte reactions (MLRs), CLL-B cells preactivated via CD40 crosslinking are significantly better presenters of alloantigen, affecting up to 30-fold-greater stimulation of T cell proliferation than that induced by control treated or nontreated CLL-B cells. Similarly, the MLR of T cells stimulated by allogeneic nonleukemic B cells can be enhanced significantly if the stimulator B cells are preactivated via CD40 crosslinking. The enhanced MLR generated by such preactivated B cells may be inhibited by blocking B7/BB1-CD28 interaction with CTLA4Ig. These studies demonstrate a novel, CD40-dependent pathway for inducing B cell expression of B7/BB1 and enhancing B cell antigen-presenting cell activity that can be initiated via cell-cell contact with alpha-CD3-stimulated CD4+ T cells.

During a sublethal murine infection with Listeria monocytogenes cells, tumor necrosis factor (TNF) activity was detectable in neither sera nor spleen homogenates at any stage of the infection when a bioassay with L-929 cells (less than 4 U/ml) was used. However, injecting the mice with an immunoglobulin fraction obtained from a rabbit hyperimmunized with recombinant murine TNF-alpha resulted in acceleration of listeriosis. When 1 mg of anti-TNF antibody was injected per mouse, all the mice died from listeriosis, even though the infectious dose was sublethal for the untreated controls. The antigen-specific elimination of the bacterium from the spleens and livers of anti-TNF antibody-treated mice was delayed, depending on the dose of the antibody injected. Endogenous TNF seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-TNF antibody was given between day zero and day 2 of infection. The effect of endogenous TNF on antilisterial resistance was due to neither regulation of alpha interferon (IFN-alpha) and IFN-gamma production nor induction of IFN-beta subtype 1 (IFN-beta 1), because anti-TNF antibody treated-mice produced normal levels of IFN-alpha and IFN-gamma in the bloodstream during infection and administration of monoclonal anti-murine IFN-beta 1 antibody had no effect on the development of listeriosis. Alternatively, the listericidal activity of peritoneal macrophages of L. monocytogenes-infected mice could be abrogated by injection of anti-TNF antibody in vivo. These results suggest that the lower level of TNF is produced endogenously in mice that received L. monocytogenes infection and that it plays an essential role in the host defense against L. monocytogenes infection.

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

To investigate the role of splicing in the regulation of gene expression, we have generated transgenic mice carrying the human histone H4 promoter linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), with or without a heterologous intron in the transcription unit. We found that CAT activity is 5- to 300-fold higher when the transgene incorporates a hybrid intron than with an analogous transgene precisely deleted for the intervening sequences. This hybrid intron, consisting of an adenovirus splice donor and an immunoglobulin G splice acceptor, stimulated expression in a broad range of tissues in the animal. Although the presence of the hybrid intron increased the frequency of transgenics with significant CAT activity, it did not affect the integration site-dependent variation commonly seen in transgene expression. To determine whether the enhancement is a general outcome of splicing or is dependent on the particular intron, we also produced equivalent transgenics carrying the widely used simian virus 40 small-t intron. We found that the hybrid intron is significantly more effective in elevating transgene expression. Our results suggest that inclusion of the generic intron in cDNA constructs may be valuable in achieving high levels of expression in transgenic mice.

The herpes simplex virus 1 open reading frames UL26 and UL26.5 are 3' coterminal. The larger, UL26 open reading frame encodes a protein approximately 80,000 in apparent molecular weight and contains the promoter and coding sequence of the UL26.5 gene, which specifies a capsid protein designated infected cell protein 35. The larger product contains in its entirety the amino acid sequence of the smaller protein. We report that the UL26 gene encodes a protease which catalyzes its own cleavage and that of the more abundant product of UL26.5. By inserting the coding sequence of an epitope to a cytomegalovirus monoclonal antibody and homologs of the immunoglobulin G binding domain of staphylococcal protein A into the 3' termini of the coding domains of the two open reading frames, we identified both products of the cleavage and determined that the cleavage site is approximately 20 amino acids from the carboxyl termini of both proteins.

A murine respiratory infection model was used to study the mechanism of protective immunity to Bordetella pertussis. We found that nude mice, which are deficient in T cells, developed a persistent infection and failed to clear the bacteria after aerosol inoculation. In contrast, normal adult nonimmune mice cleared a respiratory infection approximately 35 days after challenge. Before bacterial clearance, antipertussis antibody levels in serum were low or undetectable, whereas consistent antigen-specific T-cell responses were demonstrated throughout the course of infection. The in vitro responses detected in immune spleen cells were mediated by a population of CD4+ major histocompatibility complex class II-restricted Th1-like cells that secreted interleukin-2 and gamma interferon but not interleukin-4. Adoptive transfer of immune spleen cells into nude or sublethally irradiated immunosuppressed mice before challenge resulted in bacterial clearance within 14 to 21 days. In contrast, injection of serum from convalescent mice before challenge only marginally reduced the bacterial load early in the course of infection. Furthermore, transfer of enriched T cells or purified CD4+ T cells but not CD8+ T cells from immune mice conferred a high level of protection. Recipients of CD4+ T cells cleared the bacteria from the lungs within 20 days of challenge, at which time B. pertussis-specific antibodies in the serum were undetectable. Although we do not rule out a contribution of mucosal immunoglobulin A, our findings suggest that cellular responses mediated by CD4+ Th1 cells play an important role in protective immunity to B. pertussis.

Colonization of the nasopharynx is the initial step in all infections caused by Streptococcus pneumoniae. The antibody response to carriage was examined in an experimental model of human colonization in healthy adults. Asymptomatic colonization was detected in 6/14 subjects and continued for up to 122 d. Susceptibility to carriage did not correlate with total serum immunoglobulin (Ig)G to the homotypic capsular polysaccharide. All of the colonized subjects, in contrast, developed a serum IgG and secretory IgA response to a 22 kD protein, whereas 7 of 8 subjects who did not become colonized had preexisting antibody to this protein. Analysis of the 22 kD protein identified it as the NH(2)-terminal region of pneumococcal surface protein A (PspA). Our findings provide evidence for the role of antibody to this protein fragment in preventing pneumococcal carriage by humans.

Immune responses dominated by interleukin-4 (IL-4)-producing T helper type 2 (TH2) cells or by interferon gamma (IFN-gamma)-producing T helper type 1 (TH1) cells express distinctive protection against infection with different pathogens. Interleukin-4 promotes the differentiation of naïve CD4+ T cells into IL-4 producers and suppresses their development into IFN-gamma producers. CD1-specific splenic CD4+NK1.1+ T cells, a numerically minor population, produced IL-4 promptly on in vivo stimulation. This T cell population was essential for the induction of IL-4-producing cells and for switching to immunoglobulin E, an IL-4-dependent event, in response to injection of antibodies to immunoglobulin D.

BACKGROUND

Common variable immunodeficiency disorders (CVIDs) are the most common forms of symptomatic primary antibody failure in adults and children. Replacement immunoglobulin is the standard treatment, although there are few consistent data on optimal dosages and target trough IgG levels required for infection prevention.

OBJECTIVE

To provide data to support the hypothesis that each patient requires an individual dose of therapeutic immunoglobulin to prevent breakthrough infections and that efficacious trough IgG levels vary between patients.

METHODS

Data, collected prospectively from a cohort of 90 patients with confirmed CVIDs from 1 center over a follow-up period of 22 years, was validated and analyzed. Immunoglobulin doses had been adjusted in accordance with infections rather than to achieve a particular trough IgG level. Doses to achieve infection-free periods were determined and resultant trough levels analyzed. A smaller group of patients with X-linked agammaglobulinemia was analyzed for comparison.

RESULTS

Patients with a CVID had a range of trough IgG levels that prevented breakthrough bacterial infections (5-17 g/L); viral and fungal infections were rare. Doses of replacement immunoglobulin to prevent breakthrough infections ranged from 0.2 to 1.2 g/kg/mo. Those with proven bronchiectasis or particular clinical phenotypes required higher replacement doses. Patients with X-linked agammaglobulinemia showed a similar range of IgG levels to stay infection-free (8-13 g/L).

CONCLUSIONS

These data offer guidance regarding optimal doses and target trough IgG levels in individual patients with CVIDs with or without bronchiectasis and for particular clinical phenotypes. The goal of replacement therapy should be to improve clinical outcome and not to reach a particular IgG trough level.