The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.

OBJECTIVE

Helicobacter pylori may disappear spontaneously with progressing precancerous changes and invalidate serologic studies of its association with gastric cancer. We reestimated the strength of the H. pylori-gastric cancer relationship, using both conventional immunoglobulin (Ig) G enzyme-linked immunosorbent assay (ELISA) and immunoblot (against cytotoxin-associated antigen A [CagA] antibodies that prevail longer after eradication) to detect past H. pylori exposure more relevant for time at cancer initiation.

METHODS

In our population-based case-control study, the seroprevalence among 298 gastric adenocarcinoma cases was 72% (IgG ELISA) and 91% (immunoblot) vs. 55% and 56% among 244 controls frequency-matched for age and gender.

RESULTS

Using IgG ELISA only, the adjusted OR for noncardia gastric cancer among H. pylori-positive subjects was 2.2 (95% confidence interval [CI], 1.4-3.6). When ELISA-/CagA+ subjects (odds ratio [OR], 68.0) were removed from the reference, the OR rose to 21.0 (95% CI, 8.3-53.4) and the previous effect modification by age disappeared. ELISA+/CagA- subjects had an OR of 5.0 (95% CI, 1.1-23.6). There were no associations with cardia cancer.

CONCLUSIONS

The weaker H. pylori-cancer relationships in studies based on IgG ELISA rather than CagA may be caused by misclassification of relevant exposure. A much stronger relationship emerges with more accurate exposure classification. In the general Swedish population, 71% of noncardia adenocarcinomas were attributable to H. pylori.

The alpha3beta1 integrin forms complexes with other cell-surface proteins, including transmembrane-4 superfamily (TM4SF) proteins (e. g. CD9, CD53, CD63, CD81, and CD82). To identify additional cell-surface proteins associated with alpha3beta1 integrin, a monoclonal antibody selection protocol was developed. Mice were immunized with integrin alpha3beta1-containing complexes isolated from HT1080 fibrosarcoma cells, and then 712 hybridoma clones were produced, and 95 secreted antibodies that recognized the HT1080 cell surface. Among these, 12 antibodies directly recognizing integrin alpha3 or beta1 subunits were eliminated. Of the remaining 83, 16 co-immunoprecipitated proteins that resembled integrins under non-stringent detergent conditions. These 16 included 15 monoclonal antibodies recognizing EMMPRIN/basigin/OX-47/M6, a 45-55-kDa transmembrane protein with two immunoglobulin domains. The EMMPRIN protein associated with alpha3beta1 and alpha6beta1, but not alpha2beta1 or alpha5beta1, as shown by reciprocal immunoprecipitation experiments. Also, association with alpha3beta1 was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments. Importantly, EMMPRIN-alpha3beta1 complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-alpha3beta1 complexes.

Twenty-one patients treated for active tuberculosis were examined for immune reactivity to purified protein derivative (PPD) and to a purified 32-kDa protein antigen (P32) from Mycobacterium bovis, strain BCG. Lymphoproliferation of peripheral blood leucocytes to PPD and P32 was positive in 95% and 71% of the patients respectively. A positive IFN-gamma response was detected in 62% against PPD and in 48% against P32. Low blastogenesis and IFN-gamma production were observed, especially in patients with poor general health and advanced tuberculous lesions. Twelve out of twelve (100%) of the tuberculin-positive healthy volunteers responded to PPD and P32 with mean lymphoproliferation and IFN-gamma values that were higher than in the patient group. Twelve tuberculin-negative control subjects were completely unreactive to PPD and P32 antigen. On the other hand, IgG antibodies in the serum were detected in 95% of the patients against PPD, in 77% of the patients against P32 but in none of the tuberculin-positive or negative healthy volunteers. The highest IgG levels against PPD were found in those patients with the lowest in vitro lymphoproliferation and IFN-gamma production (r = -0.54; P less than 0.05). Nonspecific interferon production following induction with Newcastle disease virus, Corynebacterium parvum, or phytohaemagglutinin was comparable in the control and patient groups. Finally, low IFN-alpha titres were detected in the serum of about 50% of the patients.

Germfree BALB/c mice have been treated from birth with intraperitoneal injections of purified goat antibodies to mouse IgM. The treated mice, and controls which had received an equivalent amount of goat gamma-globulin, were sacrificed at 8 or 13 wk of age. Compared to controls, mice given anti-micro (a) had very few germinal centers in spleen and lymph node, (b) had decreased numbers of mature plasma cells synthesizing IgM and IgG1 in spleen, and virtual absence of IgA-synthesizing plasma cells in the gut, (c) had greatly diminished numbers of B lymphocytes bearing membrane-bound immunoglobulins of the IgM, IgG1, IgG2, and IgA classes in spleen, (d) had reduced synthesis of IgM, IgG2, and IgA by in vitro spleen cultures, and (e) had significant decreases in serum levels of IgM, IgG1, IgG2, and IgA. The treated animals failed to make antibodies to ferritin after hyperimmunization, and lacked natural antibodies to sheep erythrocytes. These results indicate that cells ultimately committed to synthesis of IgG1, IgG2, and IgA immunoglobulins are derived from cells which have expressed IgM determinants at an earlier stage of differentiation. They are consistent with a proposed two-stage model for plasma cell differentiation. The first stage is antigen independent, involves sequential activation of Cmicro, Cgamma, and Calpha genes by progeny of a single stem cell, and results in the formation of B lymphocytes bearing membrane-bound recognition antibodies of each class. The second, antigen-dependent, stage results in formation of mature plasmacytes and memory cells.

Previous studies from this laboratory showed that serum anti-neutrophil cytoplasmic antibodies, distinct from those associated with active Wegener's granulomatosis, are present in the majority of patients with ulcerative colitis. In this study, the specificity of anti-neutrophil cytoplasmic antibodies for ulcerative colitis as compared with other colitides and diarrheal illness was determined. In a blinded study, test samples of serum were screened for anti-neutrophil immunoglobulin G in a fixed neutrophil enzyme-linked immunosorbent assay. Levels of neutrophil binding by immunoglobulin G and titers of anti-neutrophil immunoglobulin G in sera from patients with ulcerative colitis and patients with ulcerative colitis post colectomy were each significantly greater than the levels and titers for normal controls and patients with a variety of other colitides and diarrheal illnesses. Levels of neutrophil binding for colonic Crohn's disease, bacterial/amoebic colitis, the irritable bowel syndrome with diarrhea, and other miscellaneous diarrheal illnesses were not significantly different than the levels for normal controls. Although the levels of binding for collagenous colitis were significantly less than the levels for ulcerative colitis, they were significantly greater than the levels for normal controls. Patterns of neutrophil binding by serum samples that were positive in the enzyme-linked immunosorbent assay were determined by indirect immunofluorescence. Perinuclear staining was the predominant pattern shown by sera from patients with ulcerative colitis. The combination of a positive value in the enzyme-linked immunosorbent assay and a perinuclear immunofluorescence pattern was 60% sensitive and 94% specific for ulcerative colitis. It is concluded that anti-neutrophil cytoplasmic antibodies in ulcerative colitis are not simply an epiphenomenon related to inflammation of the colon. Identification of the antigen(s) to which these autoantibodies are directed may facilitate understanding of the underlying immune response and may allow development of an assay that is more sensitive and specific for ulcerative colitis.

Helicobacter hepaticus has been associated with naturally occurring hepatitis in certain inbred strains of mice, and in A/JCr mice it has been linked to the development of hepatic adenomas and adenocarcinomas. H. hepaticus was orally inoculated into 30 axenic, outbred female mice, and the mice were studied longitudinally to fulfill Koch's postulates and to ascertain the pathogenic potential of the organism under defined germfree conditions. Ten cage contact mice were also housed in the same germfree isolator to study transmission patterns, and 10 germfree mice were maintained in separate isolators as controls. Mice serially euthanized from 3 weeks through 24 months postinoculation (p.i.) were surveyed by culture and PCR for H. hepaticus in liver and intestinal tissues. Tissues were analyzed for histopathological changes, and sera were assayed for the presence of immunoglobulin G antibody to H. hepaticus and changes in the liver enzyme alanine aminotransferase. Inoculated mice and cage contact mice were persistently infected with H. hepaticus as identified by culture and PCR, in both the intestine and, less frequently, the liver, for the duration of the 2-year study. Animals developed persistent chronic hepatitis, and in some animals enterocolitis was noted. Hepatocellular carcinoma was diagnosed in one H. hepaticus-infected mouse. The level of H. hepaticus serum antibody was highest in experimentally infected mice at 12 to 18 months p.i.; this corresponded in general to the time interval when the highest levels of alanine aminotransferase were recorded. Although cage contact mice became persistently infected with H. hepaticus, lesions were less severe and the levels of serological biomarkers utilized in the study were lower. The H. hepaticus-infected mouse will provide an ideal model to study putative bacterial virulence determinants and how they interact with the host to induce chronic inflammation and tumorigenesis.

B cells use translesion DNA synthesis (TLS) to introduce somatic mutations around genetic lesions caused by activation-induced cytidine deaminase. Monoubiquitination at lysine(164) of proliferating cell nuclear antigen (PCNA(K164)) stimulates TLS. To determine the role of PCNA(K164) modifications in somatic hypermutation, PCNA(K164R) knock-in mice were generated. PCNA(K164R/K164R) mutants are born at a sub-Mendelian frequency. Although PCNA(K164R/K164R) B cells proliferate and class switch normally, the mutation spectrum of hypermutated immunoglobulin (Ig) genes alters dramatically. A strong reduction of mutations at template A/T is associated with a compensatory increase at G/C, which is a phenotype similar to polymerase eta (Poleta) and mismatch repair-deficient B cells. Mismatch recognition, monoubiquitinated PCNA, and Poleta likely cooperate in establishing mutations at template A/T during replication of Ig genes.

Cancer vaccines based on human tumor-associated antigens (TAA) have been tested in patients with advanced or recurrent cancer, in combination with or following standard therapy. Their immunogenicity and therapeutic efficacy has been difficult to properly evaluate in that setting characterized by multiple highly suppressive effects of the tumor and the standard therapy on the patient's immune system. In animal models of human cancer, vaccines administered in the prophylactic setting are most immunogenic and effectively prevent cancer development and progression. We report results of a clinical study that show that in patients without cancer but with a history of premalignant lesions (advanced colonic adenomas, precursors to colon cancer), a vaccine based on the TAA MUC1 was highly immunogenic in 17 of 39 (43.6%) of vaccinated individuals, eliciting high levels of anti-MUC1 immunoglobulin G (IgG) and long-lasting immune memory. Lack of response in 22 of 39 individuals was correlated with high levels of circulating myeloid-derived suppressor cells (MDSC) prevaccination. Vaccine-elicited MUC1-specific immune response and immune memory were not associated with significant toxicity. Our study shows that vaccines based on human TAAs are immunogenic and safe and capable of eliciting long-term memory that is important for cancer prevention. We also show that in the premalignant setting, immunosuppressive environment (e.g., high levels of MDSC) might already exist in some individuals, suggesting an even earlier premalignant stage or preselection of nonimmunosuppressed patients for prophylactic vaccination.

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca(2+) homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.

The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections.

BACKGROUND

Septicemia is a major antecedent of morbidity and mortality in very low birth weight (501- to 1500-g) infants. Our purpose was to determine prospectively the incidence, clinical presentation, laboratory features, risk factors, morbidity and mortality associated with late onset septicemia in infants 501 to 1500 g.

METHODS

Clinical data were prospectively collected for 2416 infants enrolled in a multicenter trial to determine the efficacy of intravenous immunoglobulin in preventing nosocomial infections. Septicemia was confirmed by positive blood culture in 395 symptomatic infants. Multivariate analyses of factors associated with septicemia were performed.

RESULTS

Sixteen percent of VLBW infants developed septicemia at a median age of 17 days. Factors associated with septicemia by logistic regression included male gender, lower gestational age and birth weight and decreased baseline serum IgG concentrations. Increasing apnea (55%), feeding intolerance, abdominal distension or guaiac-positive stools (43%), increased respiratory support (29%), lethargy and hypotonia (23%) were the dominant presenting features of septicemia. An abnormal white blood cell count (46%), unexplained metabolic acidosis (11%) and hyperglycemia (10%) were the most common laboratory indicators. Septicemic infants, compared with nonsepticemic infants, had significantly increased mortality (21% vs. 9%), longer hospital stay (98 vs. 58 days) and more serious morbidity, including severe intraventricular hemorrhage, bronchopulmonary dysplasia and increased ventilator days (P < 0.001).

CONCLUSIONS

Late onset septicemia is common in very low birth weight infants, and the rate is inversely proportional to gestational age and birth weight. Septicemia is more common in males and those with low initial serum IgG values. A set of clinical signs (apnea, bradycardia, etc.) and laboratory values (leukocytosis, immature white blood cells and neutropenia) increase the probability of late onset sepsis, but they have poor positive predictive value.

Tumor infiltrating lymphocytes (TILs) derived from tumor-bearing patients recognize tumor-associated antigens presented by major histocompatibility complex (MHC) class I molecules. The infusion of TIL586 along with interleukin (IL) 2 into an autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was recently isolated and shown to encode gp75. Here we report that an antigenic peptide, MSLQRQFLR, recognized by TIL586 was not derived from the normal gp75 protein. Instead, this nonamer peptide resulted from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a tumor rejection antigen recognized by T cells. This represents the first demonstration that a human tumor rejection antigen can be generated from a normal cellular gene using an open reading frame other than that used to encode the normal protein. These findings revealed a novel mechanism for generating tumor antigens, which may be useful as vaccines to induce tumor-specific cell-mediated immunity against cancer.

Proteinuria is consequence of two mechanisms: the abnormal transglomerular passage of proteins due to increased permeability of glomerular capillary wall and their subsequent impaired reabsorption by the epithelial cells of the proximal tubuli. In the various glomerular diseases, the severity of disruption of the structural integrity of the glomerular capillary wall correlates with the area of the glomerular barrier being permeated by "large" pores, permitting the passage in the tubular lumen of high-molecular-weight (HMW) proteins, to which the barrier is normally impermeable. The increased load of such proteins in the tubular lumen leads to the saturation of the reabsorptive mechanism by the tubular cells, and, in the most severe or chronic conditions, to their toxic damage, that favors the increased urinary excretion of all proteins, including low-molecular-weight (LMW) proteins, which are completely reabsorbed in physiologic conditions. Recent clinical studies showed that in patients with glomerular diseases the urinary excretion of some HMW proteins [immunoglobulins G and M (IgG and IgM)] and of some LMW proteins, alpha1-microglobulin, beta2-microglobulin, correlates with the severity of the histologic lesions, and may predict, better than the quantity of proteinuria, the natural course, the outcome, and the response to treatment. It is suggested that some patients have already, at the time of clinical presentation, a structural damage of the glomerular capillary wall (injury of podocytes) and of the tubulointerstitium, the severity and scarce reversibility of which are reliably indicated by an elevated urinary excretion of HMW and LMW proteins.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgammagammagamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

The immunohistological localization of gammaA, secretory "piece" (SP), and lactoferrin (LF) in the mucosae of a variety of normal human tissues was investigated using specific fluoresceinated antisera. gammaA staining was localized in the apical portion of the mucosal epithelium, intercellular spaces, basement membrane area, and plasma cells of the interstitium or lamina propria of a number of normal human tissues. SP was ubiquitous in the mucosal epithelium of all tissues studied which included parotid and submaxillary glands, bronchi, pancreas, GI tract, sweat glands, kidney, and gall bladder. In addition, SP staining was localized in the intercellular spaces and on the surface of the epithelial cells lining the lumen of the secretory glands. No SP staining was observed in the plasma cells of the interstitium or lamina propria surrounding the secretory glands in these tissues, and no SP staining was observed in sections of normal spleen or lymph node tissue. SP staining was observed in the sweat glands, pancreas, and kidney in the absence of gammaA staining. LF was much less ubiquitous in the epithelial cells of the various tissues studied and appeared to be restricted primarily to the acinar epithelium of the bronchial mucosae, parotid, and submaxillary salivary glands, and was also found in renal tubular cells. A hypothetical model for the transport of gammaA and SP across mucosal membrane epithelium is presented.

OBJECTIVE

Natural killer (NK) cells are essential early after infection, not only for viral containment but also for timely and efficient induction of adaptive responses. An inhibitory effect of hepatitis C virus (HCV)-E2 proteins on NK cells has been reported, but the features of NK cell responses in the acute phase of hepatitis C are still largely undefined. Therefore, the aim of this study was to characterize the function and phenotype of NK cells in the acute phase of infection and compare individuals with chronic and self-limited outcomes.

METHODS

Twenty-two individuals with acute HCV infection, 14 with chronic evolution, and 8 with self-limited infection, were studied using NK phenotypic and functional assays.

RESULTS

An increased expression of NKG2D on both CD56(bright) and CD56(dim) NK cells was detected in patients with acute HCV, irrespective of the outcome, as compared with healthy controls. Also, interferon gamma production and cytotoxicity by NK cells were higher in individuals with acute HCV infection than in healthy controls. Subset analysis showed increased interferon gamma production in both NK cell subsets carrying group 1 and group 2 HLA-C-specific killer cell immunoglobulin-like receptors. However, increased CD107a was noted only on NK cells expressing the group 1 HLA-C-specific killer cell immunoglobulin-like receptor and was maximal in self-limited infection.

CONCLUSIONS

Our data show that in the acute phase of HCV infection, NK cells are activated regardless of outcome, with no evidence of a suppressive effect of HCV on NK cell function.

Whether serum autoantibodies to neutrophil cytoplasmic components, previously found in ulcerative colitis, are also associated with primary sclerosing cholangitis was determined. In an enzyme-linked immunosorbent assay for immunoglobulin G neutrophil antibodies, neutrophil binding by primary sclerosing cholangitis sera was significantly greater than that for primary biliary cirrhosis, chronic hepatitis B, and chronic non-A, non-B hepatitis. Similar differences were seen when sera from patients with primary sclerosing cholangitis without evidence for ulcerative colitis were compared with sera from liver disease controls. Perinuclear immunofluorescence staining of neutrophils was exhibited by the majority of ulcerative colitis, primary sclerosing cholangitis, and primary sclerosing cholangitis without ulcerative colitis sera. The combination of elevated immunoglobulin G neutrophil antibodies and a perinuclear pattern was 65% sensitive and 100% specific for primary sclerosing cholangitis compared with the liver disease control sera. It is concluded that neutrophil cytoplasmic antibodies in ulcerative colitis and primary sclerosing cholangitis may be markers of shared underlying immunopathogenic mechanisms. Identification of the target antigen(s) may facilitate understanding of the underlying immune response and development of an improved disease marker assay.

Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes. We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation. In Msh6(-)/- and Msh3(-)/-/Msh6(-)/- mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2(-)/- mice. In contrast, Msh3(-)/- mice show no differences from their littermate controls. These findings indicate that the MSH2-MSH6 heterodimer, but not the MSH2-MSH3 complex, is responsible for modulating Ig hypermutation.

The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.

An enzyme-linked immunosorbent assay microplate method was used for measuring levels of antibody specific for the oral serotype of Bacteroides asaccharolyticus (Bacteroides gingivalis) in serum samples obtained from umbilical cords, infants, children, periodontally normal adults, and edentulous adults. Serum from patients with various periodontal diseases, including adult periodontitis, localized juvenile periodontitis, generalized juvenile periodontitis, post-localized juvenile periodontitis, and acute necrotizing ulcerative gingivitis, were also studied. A positive correlation between increase in age and increase in both prevalence and level of specific antibody in the G, A, and M classes of immunoglobulins was observed. This indicates that antibodies reactive with oral B. asaccharolyticus found in up to 84% of normal adults are natural antibodies, presumably with a protective role. Among the patient groups, those with adult periodontitis were found to have levels of immunoglobulin G antibodies to oral B. asaccharolyticus that were five times higher than the antibody levels found in control subjects. The levels of IgG antibodies to this organism in the other patient groups were comparable to the levels found in the control group. However, 50% of the individuals in the generalized juvenile periodontitis group had high levels of immunoglobulin G antibodies to B. asaccharolyticus, suggesting heterogeneity with respect to immune response in these patients. These results indicate that antibodies to oral B. asaccharolyticus (B. gingivalis) occur at low levels in most normal children and adults and that the rise in titer of the specific antibodies of each major class of immunoglobulins parallels the ontogenic change in serum levels of that isotype. In contrast, there is a marked increase in titer of immunoglobulin G antibodies to oral B. asaccharolyticus in the group of patients with adult periodontitis and in patients with the generalized form of juvenile periodontitis.

Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the growth-promoting action of both peptides is mediated by the MSA receptor or other growth factors.

Little effort has been made to quantitate adverse outcomes of monoclonal gammopathy of undetermined significance (MGUS) of the immunoglobulin M (IgM) class, which progresses to lymphoma or Waldenström macroglobulinemia, whereas IgA and IgG MGUS progress to multiple myeloma, primary amyloidosis (AL), or a related plasma cell disorder. From 1960 to 1994, IgM MGUS was diagnosed in 213 patients in southeastern Minnesota. The end point was progression to lymphoma or a related disorder, as assessed with the Kaplan-Meier method. The 213 patients were followed up for 1567 person-years (median, 6.3 years per patient). Lymphoma developed in 17 patients (relative risk [RR], 14.8), Waldenström macroglobulinemia in 6 (RR, 262), primary amyloidosis in 3 (RR, 16.3), and chronic lymphocytic leukemia in 3 (RR, 5.7). The relative risk of progression was 16-fold higher in the patients with IgM MGUS than in the white population of the Iowa Surveillance, Epidemiology, and End Results Program. Cumulative incidence of progression was 10% at 5 years, 18% at 10 years, and 24% at 15 years. On multivariate analysis, the serum monoclonal protein and serum albumin concentrations at diagnosis were the only risk factors for progression to lymphoma or a related disorder. Risk for progression to lymphoma or a related disorder at 10 years after the diagnosis of MGUS was 14% with an initial monoclonal protein concentration of 0.5 g/dL or less, 26% with 1.5 g/dL, 34% for 2.0 g/dL, and 41% for more than 2.5 g/dL.