No description available.

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.

There is evidence that kinetics of tumour markers (TMs) CEA, CA15-3 and CA19-9 provide valuable information about disease state over time in patients with advanced breast and colorectal cancer but the literature contains differences in methodology so comparing findings is difficult. By modifying criteria developed by Rustin and colleagues [1-5] in ovarian carcinoma we have retrospectively identified a subset of patients (those with progressive (P) TMs) where survival is significantly reduced compared with those with responsive (R) TMs. This is true for CEA, CA15-3 and CA19-9 at the first chemotherapy given in advanced disease (chem1) (Hazard ratios (HR) = 9.99, 8.89, 5.75, P ¿ 0.001 in all cases) and CEA and CA19-9 at the second chemotherapy (chem2) (HR = 7.95, 9.00, P = 0.001 and 0.002 respectively) in patients with breast cancer. It is also true for CEA at chem1 in patients with colorectal cancer (HR = 2.51, P <0.001). Further studies are necessary to see if treatment directed by these criteria can influence survival. CEA and CA19-9 Rustin category in colorectal patients and CA15-3 Rustin category in breast patients correlated significantly with radiological category at chem1 and chem2 (CEA rs = 0.45 and 0.43, CA19-9 rs = 0.26 and 0.35, CA15-3 rs = 0.28 and 0.44). CA19-9 also correlates with radiological category at chem2 (rs = 0.38) in breast patients. This provides valuable information because RECIST criteria can delay radiological identification of disease progression compared with WHO criteria [6, 7] and new therapies may act to stabilise tumour growth rather than reduce it [8]. / Oncology Research Trust Fund at Airedale NHS Trust

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Antibodies specific for insulin and human immunoglobulin G (HlgG) were attached to controlled pore glass (CPG) particles which had been silanized with a diol-bearing silane. Up to 20 mg of antibody protein could be attached covalently to 1 gram of CPG. Such immobilized antibodies, or immunosorbents, would bind specific antigens, but not unrelated proteins, when used in a high pressure liquid chromatographic configuration. This technique was given the name "high performance immunoaffinity chromatography" (HPIC). The HPIC properties of these immunosorbents were evaluated by an equilibrium theory and were found to be comparable to batch values. An immunosorbent for HIgG antigen showed an HPIC association constant of 10⁷·⁶; the batch equilibrium constant for the same immunosorbent was 10⁷·⁸. Two different anti-insulin immunosorbents retained the intrinsic affinity (10⁶ and 10⁹) of the antibody used to make them. The total active antibody concentrations of these immunosorbents were evaluated by HPIC and batch methods with good agreement between the two. The immobilization reaction was seen to result typically in the loss of 90% of the original antibody activity. HPIC was shown to be applicable to the rapid analysis of antigens at levels as low as ng/mL. This was found to be possible in part because of the rapid forward kinetics which were assessed by HPIC. A forward rate constant of 3 X 10⁷ L·mol⁻¹·sec⁻¹ for the binding of insulin by a specific HPIC column could be determined. The possibility of HPIC fluorescence immunoassays was investigated using a highly sensitive fluorescence detector. An Eimac collimated xenon arc lamp provided sufficient power to detect picomolar levels of fluorescamine labeled insulin and other compounds. The limitations of HPIC in performing picomolar immunoassays were thus shown to be immunochemical rather than instrumental. The ability of immunoaffinity purifications to overcome these limitations was demonstrated.

No description available.

The antigens of Trichomonas vaginal is grown jn Diamond 1 s medium modified by Klass were analyzed by gel diffusion methods. Three methods were employed in the extraction of the antigens. They were Genetron extract, Borate Buffer Saline extract, and alcohol extract. The alcohol extract was made up of four fractions (TF1 , TF2, TF3, TF4). The antisera used as analytic agents were made in two rabbits obtained from the Center for Disease Control Breeding Colony. All the antigens were compared to show bands of identity, non-identity, and partial identity, and to determine the number of bands produced by each extract. Genetron extract antigen and Borate Buffer Saline extract antigen reacted strongly with the homologous antiserum made in rabbit 3 (R3), producing 4 bands each, while alcohol extract reacted minimally. Fractions TF1 and TF4 produced 3 bands each and fraction TFz and TF3 produced 2 bands each Two bands of precipitate were found to be common to all the antigens, suggesting the similarity of the antigens. In rabbit 4 (R4), 3 bands were produced by each antigen and they were all identical, confirming the similarity of the antigens. Rabbit 3 antiserum reacted more strongly than rabbit 4, even though all the rabbits were inoculated on the same dates with the same number of live Trichomonas vaginalis antigens under the same conditions.

Objectives: A decrease in Ki67 during neoadjuvant therapy predicts response to tamoxifen. Previous trials have shown a decreased Ki67 in breast tumors with as little as two or more weeks of preoperative tamoxifen. Shortening the preoperative treatment time in window of opportunity clinical trials makes these trials more attractive to women. POWERPIINC examined the effect of 7 days of preoperative tamoxifen on breast tumor proliferation and patient symptoms. Methods: Women with untreated stage I/II, ER-positive, invasive breast cancer with no contraindications to tamoxifen were enrolled. Women received 20 mg of tamoxifen for 7 days up to the day of surgery. Proliferation was assessed by Ki67 immunohistochemistry before and after 7 days of tamoxifen. Symptoms and QOL were assessed by the FACT-ES and MENQOL. Adherence was measured by pill counts. Results: 52 women were enrolled, and 44 were evaluable for Ki67. The median age was 58.5 years, and the median tumor diameter was 1.2 cm. Most women (73%) were post-menopausal. Most tumors were PR positive (88%) and HER2-negative (92%). The Ki67 decreased by a geometric mean of 40% (95% CI 29%-63%), and 73% (95% CI 57%-85%) of women had tumors with decreased proliferation (p = 0.0001 by paired t-test). Adherence to taking tamoxifen during the preoperative period was 100%. Women reported minimal bother from psychosocial or physical symptoms at baseline or on the day of surgery. Conclusion: Seven days of tamoxifen showed a similar relative decrease in Ki67 as that reported for longer courses, was acceptable to women, and could be considered for window of opportunity studies.

Flukes of the genus Trichobilharzia are typical parasites using aquatic gastropods as intermediate hosts and birds as definitive hosts. However, infectious stages in open water are able to penetrate the skin of a mammal, including humans, and thereby induce a hypersensitivity reaction known as swimmer's itch. Infection with neurotropic species T. regenti in ducks induces an increase in level of IgY antibody which recognize certain parasitic antigens. In this work, these parameters in two other relative but visceral species, T. szidati and T. franki, and cross-reactivity of antibodies in serum of infected duck against heterologous parasitic antigen by ELISA and Western blot were characterized. Antibodies of ducks infected with T. szidati showed very low reactivity with all three parasitic antigens in compare to ducks infected with T. franki or T. regenti. The previously identified strongly immunogenic T. regenti antigens have been evaluated as species non-specific for all trichobilharzia species studied, but useful for avian fluke infection in wild individuals diagnosis.

Das Knochenmark (BM) beherbergt wesentliche Komponenten des adaptiven Immunsystems, die einen langfristigen Schutz gegen wiederkehrende Pathogene vermitteln können, sodass es sich als Reservoir für ein immunologisches Gedächtnis qualifiziert. Neben langlebiger Antikörper-produzierender Plasmazellen bleiben auch Antigen (Ag)-spezifische CD8+ und CD4+ T-Gedächtniszellen dauerhaft im Knochenmark erhalten, auch wenn sie in den sekundären lymphoiden Organen (SLOs) und im Blut abwesend sind. Es wird angenommen, dass diese T-Gedächtniszellen bei erneutem Kontakt mit den gleichen systemischen Pathogenen schnell reagieren können. Allerdings sind die biologischen Mechanismen für ihre langfristige Aufrechterhaltung immer noch umstritten und demnach ungeklärt. Unklar ist auch, wie die T-Gedächtniszellen des Knochenmarks bei erneuter Konfrontation mit demselben Antigen reagieren. Hier wird dieser Frage begegnet, indem durch klassiche Immunisierung mit definieren Antigenen eine stabile Population Ag-spezifischer CD8+ und CD4+ T-Gedächtniszellen im Knochenmark erzeugt wird. / The bone marrow (BM) harbors critical components of the adaptive immune system being able to provide long-lasting protection against previously encountered pathogens, thus qualifying as a reservoir of immunological memory. In addition to long-lived antibody producing plasma cells, antigen (Ag)-specific CD8+ and CD4+ memory T lymphocytes are maintained long-term in the BM even when they are absent from secondary lymphoid organs (SLOs) and blood. Those memory T cells are thought to respond fast upon re-encounter of systemic pathogens. However, the biological mechanisms behind their long-term maintenance in the BM are still a matter of debate and thus remain unclear. Similarly, it is also unclear how the memory T cells of the BM react to antigenic re-challenge. Here we address these issues by generating a stable pool of Ag-specific CD8+ and CD4+ memory T lymphocytes in the BM by classical immunizations with defined antigens.