Monoclonal antibodies directed against rat kappa light chains have been generated by immunizing SJL/J mice with soluble rat immunoglobulin, followed by fusion of immune spleen cells with the P3-X63-Ag8 myeloma cell line. Monoclonal antibodies from three of these hybridoma cell lines, MAR 18.5, 80.2, and 103.6, have been extensively characterized. MAR 18.5, 80.2, and 103.6 antibodies are of the gamma 2a kappa isotype, and bind strongly to protein A, allowing easy purification. Monoclonal antibody from clone 18.5 binds equally well to Ig of both RI-1a and RI-1b allotypes, whereas 80.2 and 103.6 antibodies selectively bind to RI-1b. These monoclonal antibodies can be FITC conjugated for use as a second antibody in indirect immunofluorescence assays, or radiolabeled for use in radio immunoassays requiring a specific antirat kappa antibody. The antiallotype specific monoclonal antibodies also may be of use in the study of rat immunoglobulin genetics.

The Fc receptor on B lymphocytes, Fc gamma RIIB (beta 1 isoform), helps to modulate B-cell activation triggered by the surface immunoglobulin complex. Crosslinking of membrane immunoglobulin by antigen or anti-Ig F(ab')2 antibody induces a transient increase in cytosolic free Ca2+, a rise in inositol-3-phosphate, activation of protein kinase C, and enhanced protein tyrosine phosphorylation. Crosslinking Fc gamma RIIB with the surface immunoglobulin complex confers a dominant signal that prevents or aborts lymphocyte activation triggered through the ARH-1 motifs of the signal transduction subunits Ig-alpha and Ig-beta. Here we show that Fc gamma RIIB modulates membrane immunoglobulin-induced Ca2+ mobilization by inhibiting Ca2+ influx, without changing the pattern of tyrosine phosphorylation. A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB is both necessary and sufficient for this effect. Tyrosine at residue 309 in this motif is phosphorylated upon co-crosslinking with surface immunoglobulin; mutation of this residue aborts the inhibitory effect of Fc gamma RIIB. This inhibition is directly coupled to signalling mediated through Ig-alpha and Ig-beta as evidenced by chimaeric IgM/alpha and IgM/beta molecules. The 13-residue motif in Fc gamma RIIB controls lymphocyte activation by inhibiting a Ca2+ signalling pathway triggered through ARH-1 motifs as a result of recruitment of novel SH2-containing proteins that interact with this Fc gamma RIIB cytoplasmic motif.

The success or failure of pulmonary defense mechanisms largely determines the appearance of clinical lung disease. The lung is protected by interlucking systems of nonspecific and specific defenses. Inhaled substrances can be isolated by mechanical barriers or can be physically removed from the lung either by transport up the bronchial mucociliary escalator or by transport through interstitial and lymphatic channels leading to lymph nodes. Substances can be locally detoxified within the lung by interaction with secretory proteins, such as antibodies, or by neutralization and dissolution within phagocytic cells. The pulmonary alveolar macrophage is the central figure in the protection of the respiratory membrane, operating in all 3 of the nonspecific modes of defense and augmented by specific immunologic mechanisms as well. Alterations in macrophage function and physiology may be crucial in determining the effectiveness of pulmonary defense. Recent advances in the cell biology of the alveolar macrophage have led to a greater understanding of its complex funcition. The multiple origins of macrophages from local and circulating cell pools and the variability in their fate and lifespan reflect the multi-faceted role of this cell type. The importance of the interactions between macrophages, orther lung cells, and other defense mechanisms has become increasingly clear. As well as functioning as resident defender of the alveolus, the macrophage is an important effector of the pulmonary immune response and plays a key role in the pathogenesis of a wide variety of inflammatory, destructive, and fibrotic lung diseases. Humoral and cell-mediated immune responses amplify and direct lung defenses against infection and may also participate in protection against other agents. Immunoglobulin A and G, microbial neutralizing and opsonizing anti-bodies, and macrophage-stimulating T lymphocytes are the major immunospecific forms of lung defense. Infectious agents, cigarette smoke, air pollutants, industrial dusts, and a spectrum of coexistent disease states may impair pulmonary defense mechanisms and increase susceptibility to asute and chronic respiratory diseases. A thorough understanding of the ways in which the lung protects itself against the daily assault of infectious, toxic, and immunogenic materials should lead to a beter understanding of pathogenesis and consequences of lung disease and to better clinical care of the patient with respiratory disease.

Uptake of Leishmania major by dendritic cells (DCs) results in activation and interleukin (IL)-12 release. Infected DCs efficiently stimulate CD4- and CD8- T cells and vaccinate against leishmaniasis. In contrast, complement receptor 3-dependent phagocytosis of L. major by macrophages (MPhi) leads exclusively to MHC class II-restricted antigen presentation to primed, but not naive, T cells, and no IL-12 production. Herein, we demonstrate that uptake of L. major by DCs required parasite-reactive immunoglobulin (Ig)G and involved FcgammaRI and FcgammaRIII. In vivo, DC infiltration of L. major-infected skin lesions coincided with the appearance of antibodies in sera. Skin of infected B cell-deficient mice and Fcgamma-/- mice contained fewer parasite-infected DCs in vivo. Infected B cell-deficient mice as well as Fcgamma-/- mice (all on the C57BL/6 background) showed similarly increased disease susceptibility as assessed by lesion volumes and parasite burdens. The B cell-deficient mice displayed impaired T cell priming and dramatically reduced IFN-gamma production, and these deficits were normalized by infection with IgG-opsonized parasites. These data demonstrate that DC and MPhi use different receptors to recognize and ingest L. major with different outcomes, and indicate that B cell-derived, parasite-reactive IgG and DC FcgammaRI and FcgammaRIII are essential for optimal development of protective immunity.

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from protein gels was developed. The method employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 microg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein when produced in the presence of a glucosidase I inhibitor. Furthermore, since heavy and light chains of IgG became separated by SDS-PAGE, it could be established that most glycans were associated with the heavy chains.

The complete DNA sequence of rhesus cytomegalovirus (RhCMV) strain 68-1 was determined with the whole-genome shotgun approach on virion DNA. The RhCMV genome is 221,459 bp in length and possesses a 49% G+C base composition. The genome contains 230 potential open reading frames (ORFs) of 100 or more codons that are arranged colinearly with counterparts of previously sequenced betaherpesviruses such as human cytomegalovirus (HCMV). Of the 230 RhCMV ORFs, 138 (60%) are homologous to known HCMV proteins. The conserved ORFs include the structural, replicative, and transcriptional regulatory proteins, immune evasion elements, G protein-coupled receptors, and immunoglobulin homologues. Interestingly, the RhCMV genome also contains sequences with homology to cyclooxygenase-2, an enzyme associated with inflammatory processes. Closer examination identified a series of candidate exons with the capacity to encode a full-length cyclooxygenase-2 protein. Counterparts of cyclooxygenase-2 have not been found in other sequenced herpesviruses. The availability of the complete RhCMV sequence along with the ability to grow RhCMV in vitro will facilitate the construction of recombinant viral strains for identifying viral determinants of CMV pathogenicity in the experimentally infected rhesus macaque and to the development of CMV as a vaccine vector.

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

Surveillance for Nipah virus (NV) was conducted in Thailand's bat population. Immunoglobulin G antibodies to NV were detected with enzyme immunoassay in 82 of 1,304 bats. NV RNA was found in bat saliva and urine. These data suggest the persistence of NV infection in Thai bats.

Receptors for the Fc domain of immunoglobulins play an important role in immune defense. There are two well-defined functional classes of mammalian receptors. One class of receptors transports immunoglobulins across epithelial tissues to their main sites of action. This class includes the neonatal Fc receptor (FcRn), which transports immunoglobulin G (IgG), and the polymeric immunoglobulin receptor (pIgR), which transports immunoglobulin A (IgA) and immunoglobulin M (IgM). Another class of receptors present on the surfaces of effector cells triggers various biological responses upon binding antibody-antigen complexes. Of these, the IgG receptors (Fc gamma R) and immunoglobulin E (IgE) receptors (Fc epsilon R) are the best characterized. The biological responses elicited include antibody-dependent, cell-mediated cytotoxicity, phagocytosis, release of inflammatory mediators, and regulation of lymphocyte proliferation and differentiation. We summarize the current knowledge of the structures and functions of FcRn, pIgR, and the Fc gamma R and Fc epsilon RI proteins, concentrating on the interactions of the extracellular portions of these receptors with immunoglobulins.

A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a beta-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein <em>G</em> affinity chromatography. The purified antibodies to CMH (mainly <em>immunoglobulin</em> <em>G</em>1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the beta-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal beta-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.

T cell immunoglobulin-3 (Tim-3) has been identified as a marker of differentiated interferon-γ-producing CD4(+) T helper type 1 and CD8(+) T cytotoxic type 1 cells. The interaction of Tim-3 with its ligand, galectin-9 (Gal-9), induces cell death, and in vivo blockade of this interaction results in exacerbated autoimmunity and abrogation of tolerance in experimental models, establishing Tim-3 as a negative regulatory molecule. Recent studies have uncovered additional mechanisms by which Tim-3 negatively regulates T cell responses, such as promoting the development of CD8(+) T cell exhaustion and inducing expansion of myeloid-derived suppressor cells. In contrast to this inhibitory effect on T cells, Tim-3-Gal-9 interaction promotes macrophage clearance of intracellular pathogens. Here, we focus on the emerging role for Tim-3 in tumor and antimicrobial immunity.

We describe human immunodeficiency type 2 (HIV-2) strains which induce cell-to-cell fusion and infect certain CD4- human cell lines. Soluble CD4 (sCD4) induces or enhances fusion by most HIV-2 strains tested. Soluble CD4-immunoglobulin G chimeras and conjugates of sCD4 and antibody to the third domain of CD4 block HIV-2 fusion of CD4- cells. We conclude that HIV-2 can enter CD4- cells via an alternative cell surface receptor to CD4. While some strains entered efficiently, others retained a dependency on an interaction with sCD4 to initiate changes in the virion envelope required for membrane fusion.

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) takes place between large switch (S) regions that precede exons of the constant region. The precise functions of the S region are controversial, although transcription of the S region targets CSR. We have tested the effects of deletion, inversion and replacement of the endogenous 12-kilobase S(gamma1) region on CSR in vivo. Here we show that S(gamma1) is required for CSR, that CSR is effected by a 1-kilobase sequence that generates a G-rich transcript, and that inversion of S(gamma1) or the G-rich sequence decreases CSR. We conclude that S(gamma1) function is dependent on orientation and lacks an absolute requirement for common S region motifs. We propose that single-stranded DNA stabilized by transcription-dependent, higher order structures is a primary substrate of CSR.

The CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]/endothelial cell adhesion molecule [endoCAM]) molecule expressed on leukocytes, platelets, and endothelial cells is postulated to mediate adhesion to endothelial cells and thereby function in immunity, inflammation, and wound healing. We report the following novel features of CD31 which suggests a role for it in adhesion amplification of unique T cell subsets: (a) engagement of CD31 induces the adhesive function of beta 1 and beta 2 integrins; (b) adhesion induction by CD31 immunoglobulin G (IgG) monoclonal antibodies (mAbs) is sensitive, requiring only bivalent mAb; (c) CD31 mAb induces adhesion rapidly, but it is transient; (d) unique subsets of CD4+ and CD8+ T cells express CD31, including all naive (CD45RA+) CD8 T cells; and (e) CD31 induction is selective, inducing adhesive function of beta 1 integrins, particularly very late antigen-4, more efficiently than the beta 2 integrin lymphocyte function-associated antigen-1. Conversely, CD3 is more effective in inducing beta 2-mediated adhesion. Taken together, these findings indicate that unique T cell subsets express CD31, and CD31 has the capacity to induce integrin-mediated adhesion of T cells in a sensitive and selective fashion. We propose that, in collaboration with other receptors/ligands, CD31 functions in an "adhesion cascade" by amplifying integrin-mediated adhesion of CD31+ T cells to other cells, particularly endothelial cells.

Plasmodium falciparum parasites that bind to chondroitin sulphate A (CSA) express unique variant surface antigens that are involved in the placental sequestration that precipitates pregnancy-associated malaria (PAM). Two var gene subfamilies, var1csa and var2csa, have been associated with CSA binding. We show here that placental P. falciparum isolates highly transcribed var2csa but not var1csa. var2csa was not transcribed or was only minimally transcribed by parasites isolated from nonpregnant women. Placental parasites that effectively bound to placental chondroitin sulphate proteoglycans transcribed higher levels of var2csa. In pregnant women, levels of var2csa transcription and plasma anti-VAR2CSA immunoglobulin G were associated. These findings support the idea that VAR2CSA plays a crucial role in PAM and strengthen the rationale for the development of VAR2CSA-based vaccines.

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (approximately 70-80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-gamma, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4(+)CD25(+)FoxP3(+)CD127(low) regulatory T cells, contraction of CD4(+) effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Serological parameters were compared in 15 cases of Coxiella burnetii infection comprising 5 cases each of primary Q fever, chronic granulomatous hepatitis, and endocarditis. The diagnosis was made on the basis of clinical history and serology and on the isolation of C. burnetii phase I from biopsy specimens of liver and bone marrow from two patients with granulomatous hepatitis and from the aortic valve vegetations of five patients with endocarditis. The temporal sequences of immunoglobulin levels, rheumatoid factor, and specific antibody responses to phase II and phase I antigens of C. burnetii were evaluated as predictive correlates of the three Q fever entities. Serum levels of immunoglobulin classes G, M, and A were variable in all the entities of Q fever. Increased mean levels (in milligrams per deciliter) of immunoglobulin G (IgG) and IgA were noted with chronic disease in the sera of some patients, whereas IgM levels were not significantly different from normal values. Rheumatoid factor was significantly elevated in chronic disease but not in primary Q fever. The temporal sequence of C. burnetii phase II and phase I antibodies were compared by microagglutination, complement fixation, and indirect microimmunofluorescence tests. All of these serological tests were useful in distinguishing primary from chronic disease. Thus, the ratio of anti-phase II to anti-phase I antibodies was greater than 1, greater than or equal to 1, and less than or equal to 1 for primary Q fever, granulomatous hepatitis, and Q fever endocarditis, respectively. Moreover, the high phase-specific IgA antibody titers in the indirect microimmunofluorescence test were diagnostic for endocarditis.

Recurrent attacks of optic neuritis and myelitis are the hallmarks of both neuromyelitis optica (NMO) and multiple sclerosis (MS). NMO immunoglobulin G (NMO-IgG), which recognizes astrocytic aquaporin-4 (AQP4) water channels, is a specific serum autoantibody that distinguishes NMO from MS. The pathogenic role of the anti-AQP4 antibody (AQP4-Ab, NMO-IgG) in NMO has been speculated based on several studies in vitro. The aim of this study was to demonstrate the pathogenicity of AQP4-Ab in vivo. We obtained IgG from patients who underwent therapeutic plasmapheresis, and developed an animal model by passive transfer of IgG to rats. The active lesions of the rats exhibited pathological characteristics strikingly similar to those of NMO, marked by astrocytic loss and perivascular deposition of immunoglobulin and complements. These findings provide the first evidence of the pathogenicity of AQP4-Ab in vivo and support the therapeutic efficacy of eliminating the antibodies by plasmapheresis.

The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in most mammalian species and limits the utility of small animal models for studies of disease pathogenesis, therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34(+) hematopoietic stem cells in BALB/c-Rag2(-/-)gamma(c)(-/-) mice. Intrapartum busulfan administration followed by irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG production, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5-coreceptor utilizing HIV-1(ADA) and subtype C 1157 viral strains elicited productive viral replication and lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2(-/-)gamma(c)(-/-) mice represent a unique and valuable resource for HIV-1 pathobiology studies.

BACKGROUND

Protein-carbohydrate interactions are believed to be important in many biological processes that involve cell-cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein.

RESULTS

We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc alpha 2-->3Gal beta 1-->3(4)GlcNAc or NeuAc alpha 2-->3Gal beta 1-->3GalNAc, myelin-associated glycoprotein recognizes only NeuAc alpha 2-->3Gal beta 1-->3GalNAc and CD22 binds specifically to NeuAc alpha 2-->6Gal beta 1-->4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes.

CONCLUSIONS

Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family.

Choline, although not a nutritional requirement for Haemophilus influenzae, is taken up from the growth medium and incorporated into its lipopolysaccharide (LPS). Incorporated choline is in the form of phosphorylcholine (ChoP) based on the reactivity with the monoclonal antibody with specificity for this structure, TEPC-15. Incorporation of [3H]choline from the growth medium and expression of the TEPC-15 epitope undergo high-frequency phase variation, characteristic of other LPS structures in this species. The expression and phase variation of ChoP require a previously identified locus involved in LPS biosynthesis, lic1. The first gene in lic1, licA, contains a translational switch based on variation in the number of intragenic tandem repeats of the sequence 5'-CAAT-3'. The full-length LicA polypeptide resembles choline kinases of eucaryotes, suggesting that the pathway for choline incorporation into the H. influenzae glycolipid has similarities to the pathway for choline incorporation in eucaryotic lipid synthesis. The display of ChoP, a host-like structure, renders the organism more rather than less susceptible to the bactericidal activity of human serum. The increased serum sensitivity of variants with ChoP correlates with higher serum immunoglobulin G titers to LPS containing this structure. ChoP appears to be a cell surface feature common to a number of pathogens of the human respiratory tract, including Streptococcus pneumoniae and mycoplasmas. In the case of H. influenzae, its primary contribution to pathogenesis does not appear to be antigenic variation to evade host humoral clearance mechanisms.

Sonicated Borrelia burgdorferi was previously reported to possess both B-cell mitogenic and interleukin-6 (IL-6) stimulatory activities. In this report, two outer surface lipoproteins, OspA and OspB, were purified from B. burgdorferi and assessed for the presence of these functions. OspA was purified from two strains, an OspB-deficient variant of HB19 and N40, while OspB was purified from the N40 strain. All lipoprotein preparations were free of endotoxin contamination, and polymyxin B failed to inhibit responses, indicating that media contamination was not contributing to biological assays. All three preparations were able to stimulate proliferation of mononuclear cells from naive C3H/HeJ and BALB/c mice. Depletion experiments indicated that the responding cells were B lymphocytes and not T lymphocytes. Purified OspA and OspB stimulated immunoglobulin M production by splenocyte cultures from naive mice, a property also previously attributed to sonicated B. burgdorferi. OspA and OspB also stimulated the production of IL-6 and tumor necrosis factor alpha by bone marrow-derived macrophages from BALB/c and C3H/HeJ mice. Cytokine production was enhanced by the presence of gamma interferon in the cultures, indicating that the magnitude of responses to these lipoproteins may be modulated by cytokines in the microenvironment of infected tissues. Human endothelial cells produced IL-6 when incubated with OspA and OspB, indicating that non-hematopoietic lineage cells can respond to the lipoproteins. Purified OspA and OspB had approximately equal activity, with responses detected in the range of 10 ng of lipoprotein per ml to 1 microgram of lipoprotein per ml. Comparison with published dose responses for lipoproteins purified from Escherichia coli indicates that OspA and OspB purified from B. burgdorferi are much more potent. The high potency of the B. burgdorferi lipoproteins and the ability of the spirochete to invade tissues and persist argue that they could be important in the localized events contributing to the pathology of Lyme disease.

BACKGROUND

Measuring serum cytokines, pituitary hormones, or acute phase proteins during or after surgery is not an optimal method for quantifying the impact of surgical procedures. In an effort to assess surgical stress by means of the immune response, we focused on changes in cell-mediated and antibody-mediated immunity as illustrated by the type 1/type 2 T-helper (Th1/Th2) cell balance. The sensitivity of this approach was evaluated by comparing laparoscopic and conventional cholecystectomy (LCE, CCE).

METHODS

In a pragmatic prospective study 43 patients with symptomatic cholelithiasis were operated on either by LCE (n = 25) or CCe (n = 18). Blood sampling was done 24 hours before surgery, immediately before incision, and 2, 24, and 48 hours after surgery. Cell surface markers and cytokine production were used to characterize the Th1/Th2 balance and were measured by means of flow cytometry and enzyme-linked immunosorbent assay techniques.

RESULTS

Activation of Th2 cells evokes the production and secretion of interleukin-4 (IL-4), which up-regulates the expression of immunoglobulin E receptors (Fo epsilon RII, CD23) on B cells. Phytohemagglutinin-induced IL-4 production in freshly isolated peripheral blood mononuclear cells from patients increased more after CCE than LCE (IL-4, +41% versus +17%; p < 0.05). Also the expression of CD23 on B cells was higher after CCE than LCE (+146% versus +63%; P < 0.01). CD30, a membrane molecule that belongs to the tumor necrosis factor receptor superfamily and probably is an important indicator of Th2 activity, was more evaluated on T cells from patients who underwent CCE. The Th1 response, characterized by phytohemagglutinin-induced IFN-gamma secretion in peripheral blood mononuclear cells and up-regulation of human leukocyte antigen-DR expression on monocytes, was lower after CCE than after LCE.

CONCLUSIONS

This study shows that surgical stress induces a shift in the Th1/Th2 balance toward Th2, suggesting that cell-mediated immunity is down-regulated and antibody-mediated immunity is up-regulated after surgery. The evaluation of this shift may be clinically meaningful and help quantify even less invasive surgical procedures. When comparing CCE and LCE in this not strictly randomized study, we found LCE to be the less stressful procedure.