[Relationship between serum brain-derived neurotrophic factor and clinical stage and dysmenorrhoea of enodmetriosis].

Objective: To explore the association between serum brain-derived neurotrophic factor and clinical stage and dysmenorrhoea of endometriosis. Methods: A total of 82 patients were studied with laparoscopically diagnosed endometriosis between June 2017 and June 2019, and 75 healthy women with reproductive age were selected as the control group during the same period. The endometriosis patients were scored by visual analogue scale(VAS)according to their preoperative dysmenorrhoea.And endometriosis was staged and scored according to the score of Revised American Fertility Society(r-AFS).Enzyme-linked immunosorbent assay (ELISA) was used to determine preoperative BDNF level in serum, and the correlation between BDNF level with clinical stage as well as dysmenorrhea of endometriosis were analysed. Results: The serum BDNF level in endometriosis patients was (1 082±43) ng/L, significantly higher than that in the normal control [(649±30) ng/L], there was statistical difference between the two groups(P<0.001). The BDNF expression in patients with r-AFS stage Ⅲ-Ⅳ was higher than that in patients with Ⅰ-Ⅱ stage [(1 164±389) ng/L vs (791±218)ng/L, P<0.001]. BDNF level in serum was closely correlated with the degree of dysmenorrhea (r=0.682), and the BDNF level in patients with moderate or severe dysmenorrhea was significantly higher than that in patients without dysmenorrhea and patients with mild dysmenorrhea [(1 292±43) ng/L vs(718±36) ng/L, P<0.001]. Conclusions: The serum BDNF level in endometriosis patients is positively correlated with clinical stage and dysmenorrhea.

Oral bisphosphonate use and lung cancer incidence among postmenopausal women.

Background: Bisphosphonates are common medications for the treatment of osteoporosis in older populations. Several studies, including the Women's Health Initiative (WHI), have found inverse associations of bisphosphonate use with risk of breast and endometrial cancer, but little is known about its association with other common malignancies. The objective of this study was to evaluate the association of bisphosphonate use on the incidence of lung cancer in the WHI. Patients and methods: The association between oral bisphosphonate use and lung cancer risk was examined in 151 432 postmenopausal women enrolled into the WHI in 1993-1998. At baseline and during follow-up, participants completed an inventory of regularly used medications including bisphosphonates. Results: After a mean follow-up of 13.3 years, 2511 women were diagnosed with incident lung cancer. There was no evidence of a difference in lung cancer incidence between oral bisphosphonate users and never users (adjusted hazard ratio = 0.91; 95% confidence intervals, 0.80-1.04; P = 0.16). However, an inverse association was observed among those who were never smokers (hazard ratio = 0.57, 95% confidence interval, 0.39-0.84; P < 0.01). Conclusion: In this large prospective cohort of postmenopausal women, oral bisphosphonate use was associated with significantly lower lung cancer risk among never smokers, suggesting bisphosphonates may have a protective effect against lung cancer. Additional studies are needed to confirm our findings.

Innate immunity drives xenobiotic-induced murine autoimmune cholangitis.

Although primary biliary cirrhosis (PBC) is considered a model autoimmune disease, it has not responded therapeutically to traditional immunosuppressive agents. In addition, PBC may recur following liver transplantation, despite the absence of major histocompatibility complex (MHC) matching, in sharp contrast to the well-known paradigm of MHC restriction. We have suggested previously that invariant natural killer T (iNK T) cells are critical to the initiation of PBC. In this study we have taken advantage of our ability to induce autoimmune cholangitis with 2-octynoic acid, a common component of cosmetics, conjugated to bovine serum albumin (2-OA-BSA), and studied the natural history of pathology in mice genetically deleted for CD4 or CD8 following immunization with 2-OA-BSA in the presence or absence of α-galactosylceramide (α-GalCer). In particular, we address whether autoimmune cholangitis can be induced in the absence of traditional CD4 and CD8 responses. We report herein that CD4 and CD8 knock-out mice immunized with 2-OA-BSA/PBS or 2-OA-BSA/α-GalCer develop anti-mitochondrial antibodies (AMAs), portal infiltrates and fibrosis. Indeed, our data suggest that the innate immunity is critical for immunopathology and that the pathology is exacerbated in the presence of α-GalCer. In conclusion, these data provide not only an explanation for the recurrence of PBC following liver transplantation in the absence of MHC compatibility, but also suggest that effective therapies for PBC must include blocking of both innate and adaptive pathways.

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.

Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway hyperresponsiveness in vivo by genetic immunization.

The efficacy of an "allergen-gene immunization" protocol in altering allergic response was examined. Intramuscular injection of rats with a plasmid DNA encoding a house dust mite allergen into the muscle results in its long-term expression and the induction of specific immune responses. Significantly, this approach prevents the induction of immunoglobulin E synthesis, histamine release in bronchoalveolar fluids, and airway hyperresponsiveness in rats challenged with aerosolized allergen. Furthermore, this suppression is persistent and can be transferred into naive rats by CD8+ T cells from gene-immunized rats. These findings suggest that allergen-gene immunization is effective in modulating allergic responses, and may provide a novel therapeutic approach for allergic diseases.

The differential ability of human IgG1 and IgG4 to activate complement is determined by the COOH-terminal sequence of the CH2 domain.

Using domain switch chimeric antibodies, we confirm the important role of CH2 in complement activation. In addition, we demonstrate that the structures responsible for the differential ability of human IgG1 and IgG4 to activate complement are located at the COOH-terminal part (from residue 292 to 340) of the CH2 domain. The amino acids in CH2 that might be involved in complement interaction are discussed. While CH3 contributes to efficient complement activation, CH3 from IgG2 and CH3 IgG3 are equally effective.

Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation.

Although very similar in sequence, the four subclasses of human immunoglobulin G (IgG) differ markedly in their ability to activate complement. Glu318-Lys320-Lys322 has been identified as a key binding motif for the first component of complement, C1q, and is present in all isotypes of Ig capable of activating complement. This motif, however, is present in all subclasses of human IgG, including those that show little (IgG2) or even no (IgG4) complement activity. Using point mutants of chimeric antibodies, we have identified specific residues responsible for the differing ability of the IgG subclasses to fix complement. In particular, we show that Ser at position 331 in gamma 4 is critical for determining the inability of that isotype to bind C1q and activate complement. Additionally, we provide further evidence that levels of C1q binding do not necessarily correlate with levels of complement activity, and that C1q binding alone is not sufficient for complement activation.

Specificity and variable region cDNA sequence of an isogeneic monoclonal antiidiotype to an anti-alpha(1----6)dextran.

We have characterized a monoclonal isogeneic antiidiotype, IdB5.7, from a BALB/c mouse immunized with the anti-alpha(1----6)dextran C57BL/6 45.21.1. It defined a hapten-inhibitable idiotope expressed on four of the 2 myeloma and 37 hybridoma anti-alpha(1----6)dextrans tested. Sequence comparison of Id+ and Id- anti-alpha(1----6)dextrans suggested that two extra amino acids at VH 100A and 100B and different residues at VH 101 abolish the expression of the idiotope in the Id- anti-alpha(1----6)dextrans. Sequence analysis of the VH of IdB5.7 showed a CDR1 longer than usual and a D segment in CDR3 formed by the fusion of two D minigenes. The IdB5.7 V kappa uses the V kappa 1 germline gene K5.1 with a few substitutions. The D-D fusion in VH CDR3 is a feature which has been reported in several other antiidiotypic antibodies.

Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: electron microscopic and hydrodynamic characterization.

The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).

Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy.

It has been well demonstrated that interleukin-12 (IL-12) could be useful to defend against a variety of pathogens, to suppress tumor growth and metastasis, and even to be employed as an adjuvant of vaccines to enhance beneficial type 1 T helper (Th1) cell response over detrimental type 2 T helper (Th2) cell responses. To apply IL-12 genes in gene therapy such as a DNA vaccine, a pIL-12 vector was constructed that contained two cytomegalovirus (CMV) promoters to drive the expression of p35 and p40 subunits, respectively. In addition, a pscIL-12 vector was designed with a linker to fuse p35 cDNA with p40 cDNA to produce a single-chain IL-12 protein, ensuring not only that the expression of p35 and p40 subunits was equally expressed, but also that no free p40 subunits interfered with IL-12 activity. The data suggested pIL-12 could produce a rather high level of biologically active IL-12 after transfection of COS cell lines as well as C2C12 muscle cell lines, as measured by both concanavalin A blast proliferation assay and enzyme-linked immunosorbent assay. Interestingly, the pscIL-12 vector could also express a bioactive murine IL-12 fusion protein in vitro. Furthermore, in vivo functional studies also demonstrated that mice co-immunized with a pS vector expressing the major envelope protein of hepatitis B virus (HBV) and IL-12 vectors encoding native IL-12 or single-chain IL-12 fusion protein elicited higher levels of IgG2a anti-HBs antibody and of Th1-related cytokine. Because p35 and p40 genes can be expressed in a vector by using a single promoter, pscIL-12 should be useful in future applications for nucleic acid vaccination or for gene therapy against diseases.

Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.

We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of immune responses to DNA vaccines by codelivery of cytokine genes.

DNA vaccines containing genes for antigenic portions of viruses have recently been developed as a novel vaccination technology. Direct injection of plasmid DNA in vivo results in prolonged expression of viral proteins and may, thus, mimic the action of attenuated vaccines. An important advantage of this vaccination method is that in vivo-synthesized viral proteins can enter both major histocompatibility complex (MHC) class I and class II antigen-processing pathways to activate specific immunization. In many animal models for infectious diseases, DNA vaccines induced a broad range of immune responses, including antibody, CD8+ cytotoxic T lymphocytes (CTL) and CD4+ helper T (Th) lymphocyte responses, and protective immunity against challenge with the pathogen. The magnitude and nature of these immune responses to DNA vaccines can be further manipulated by codelivery of cytokine genes. Summarizing the many studies reported to date, we can draw conclusions regarding the adjuvant effects of these cytokine genes on DNA vaccines. Coadministration of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2 genes induces higher antibody titers and T-cell proliferation responses than other cytokine genes tested to date. In contrast, the CTL activity is only modestly increased by the GM-CSF and IL-2 genes. The IL-12 gene polarizes the immune responses to DNA vaccines toward Th1 cell development and stimulates the strongest CTL activity. In contrast, co-injection of the IL-4 gene promotes the development of Th2 cells and increases production of antibodies, but suppresses CTL activity. Thus, the immune responses to DNA vaccines can be engineered by co-injection of an appropriate cytokine gene to favor the formation of either CTL or neutralization antibodies and, therefore, provide the best protection against a particular pathogen.

Exposures in early life: associations with DNA promoter methylation in breast tumors.

There is evidence that epigenetic changes occur early in breast carcinogenesis. We hypothesized that early-life exposures associated with breast cancer would be associated with epigenetic alterations in breast tumors. In particular, we examined DNA methylation patterns in breast tumors in association with several early-life exposures in a population-based case-control study. Promoter methylation of E-cadherin, p16 and RAR-ß2 genes was assessed in archived tumor blocks from 803 cases with real-time methylation-specific PCR. Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation. We found no differences in the prevalence of DNA methylation of the individual genes by age at menarche, age at first live birth and weight at age 20. In case-case comparisons of premenopausal breast cancer, lower birth weight was associated with increased likelihood of E-cadherin promoter methylation (OR = 2.79, 95% CI, 1.15-6.82, for ⩽2.5 v. 2.6-2.9 kg); higher adult height with RAR-ß2 methylation (OR = 3.34, 95% CI, 1.19-9.39, for ⩾1.65 v. <1.60 m); and not having been breastfed with p16 methylation (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. Being other than first in the birth order was inversely associated with likelihood of ⩾1 of the three genes being methylated for premenopausal breast cancers, but positively associated with methylation in postmenopausal women. These results suggest that there may be alterations in methylation associated with early-life exposures that persist into adulthood and affect breast cancer risk.

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA.

RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.

A case-control study in Shanghai of fruit and vegetable intake and endometrial cancer.

In a population-based case-control study of 832 incident endometrial cancer cases and 846 frequency-matched controls among Chinese women in Shanghai, using a validated food-frequency questionnaire, dietary habits were estimated by in-person interviews. Total vegetable consumption was inversely associated with endometrial cancer risk (highest quartile vs lowest: OR=0.69, 95% CI 0.50-0.96). The risk was reduced with increasing intake of dark green/dark yellow vegetables (trend test, P=0.02), fresh legumes (trend test, P<0.01), and allium vegetables (trend test, P=0.04). Fruit consumption was unrelated to risk. These results suggest that high consumption of certain vegetables may reduce the risk of endometrial cancer.

Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region.

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.

Idiotype/granulocyte-macrophage colony-stimulating factor fusion protein as a vaccine for B-cell lymphoma.

To produce a vaccine against cancer, antigens must be found that are preferentially expressed by tumour cells and can induce an immune response against the tumour. The variable regions of the immunoglobulin molecules expressed on malignant B cells (idiotypes) are tumour-specific, but are weak immunogens. To induce an immune response in animals or humans, the idiotypic protein has therefore to be chemically coupled to a strongly immunogenic protein and mixed with an adjuvant. The resulting response can protect animals from subsequent tumour challenge, and cure animals with established tumours in combination with chemotherapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) augments antigen presentation in a variety of cells. Here we show that by fusing a tumour-derived idiotype to GM-CSF, it can be converted into a strong immunogen capable of inducing idiotype-specific antibodies without other carrier proteins or adjuvants and of protecting recipient animals from challenge with an otherwise lethal dose of tumour cells. This approach may be applicable to the design of vaccines for a variety of other diseases.

Idiotype-cytokine fusion proteins as cancer vaccines. Relative efficacy of IL-2, IL-4, and granulocyte-macrophage colony-stimulating factor.

Idiotypic determinants, antigenic sites expressed on the variable region of Ig molecules of malignant B cells, represent tumor-specific Ags but are weak immunogens. We have previously shown that the immunogenicity can be dramatically increased by fusing tumor Id to granulocyte macrophage (GM)-CSF. Here, we demonstrate that fusion proteins with IL-2 or IL-4 can also be highly immunogenic. Co-immunization of these fusion proteins with another Id demonstrated the importance of physical linkage between the cytokine and relevant Ag for this enhancement. All three fusion proteins are capable of eliciting significant levels of specific Abs against the Id without the use of carrier proteins or adjuvants, although the GM-CSF fusion protein appeared to be unique in its ability to induce higher titers of anti-Id Abs in the primary response. Furthermore, the Id-IL-2 fusion protein induced high titers of IgG2a and IgG3 anti-Id Abs, whereas the Id-IL-4 and Id-GM-CSF fusion proteins did not. Despite the differences, tumor protection was comparable in all mice having significant titers of anti-Id Abs, regardless of the fusion protein used. We concluded that Id-cytokine fusion proteins are potent immunogens that can elicit significant antitumor immunity. The general approach of fusing a cytokine to a potential Ag may be applicable to the design of vaccines for immunotherapy of other types of tumors as well as for other pathogens and disease states.

Protective mechanisms induced by a Japanese encephalitis virus DNA vaccine: requirement for antibody but not CD8(+) cytotoxic T-cell responses.

We have previously shown that a plasmid (pE) encoding the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection against a lethal viral challenge. In the present study, we used adoptive transfer experiments and gene knockout mice to demonstrate that the DNA-induced E-specific antibody alone can confer protection in the absence of cytotoxic T-lymphocyte (CTL) functions. Plasmid pE administered by either intramuscular or gene gun injection produced significant E-specific antibodies, helper T (Th)-cell proliferative responses, and CTL activities. Animals receiving suboptimal DNA vaccination produced low titers of anti-E antibodies and were only partially or not protected from viral challenge, indicating a strong correlation between anti-E antibodies and the protective capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to sublethally irradiated hosts conferred protection against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protective immunity in Igmu(-/-) and I-Abeta(-/-) mice, whereas in CD8alpha(-/-) mice the pE-induced antibody titers and protective rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4(+) T cells but independent of CD8(+) T cells.

Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure.

The presence of N-linked carbohydrate at Asn58 in the VH of the antigen binding site of an antibody specific for alpha(1----6)dextran (TKC3.2.2) increases its affinity for dextran 10- to 50-fold. Site-directed mutagenesis has now been used to create novel carbohydrate addition sequences in the CDR2 of a non-glycosylated anti-dextran at Asn54 (TST2) and Asn60 (TSU7). These antibodies are glycosylated and the carbohydrates are accessible for lectin binding. The amino acid change in TSU7 (Lys62----Thr62) decreases the affinity for antigen; however, glycosylation of TSU7 increased its affinity for antigen 3-fold, less than the greater than 10-fold increase in affinity seen for glycosylated TKC3.2.2. The difference in impact of glycosylation could result either from the position of the carbohydrate or from its structure; unlike the other antibodies, TSU7 attaches a high mannose, rather than complex, carbohydrate in CDR2. In contrast, glycosylation of TST2 at amino acid 54 inhibits dextran binding. Thus slight changes in the position of the N-linked carbohydrate in the CDR2 of this antibody result in substantially different effects on antigen binding. Unlike what was observed for the anti-dextrans, a carbohydrate addition site placed in a similar position in an anti-dansyl is not utilized.