An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18.

ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.     

Expression of human papillomavirus type 16 E7 protein by recombinant baculovirus and use for the detection of E7 antibodies in sera from cervical carcinoma patients

Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins. © 1993 Wiley-Liss, Inc.     

T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16

The E6 oncoprotein of human papillomavirus type 16 (HPV16 E6) produced by tumor cells of HPV16-associated cervical carcinoma is poorly immunogenic in patients, but nonetheless is a tumor-specific antigen to which therapeutic vaccine strategies may be directed. To investigate the subunit immunogenicity of E6 protein at the T-helper cell level, we immunized mice with overlapping peptides spanning the entire 158 amino acid sequence. Two peptides recalled a proliferative response in lymph node cells (LNC) from C57BL/6 (H-2b)-immunized mice. One of these peptides also recalled proliferative responses in the context of 5/5 other major histocompatibility complex (MHC) class II haplotypes, indicating a "promiscuous" T-epitope. Minimal consensus motif analysis identified the epitopes as 60VYRDGNPYA68 and 98GYNKPLCDLL107. LNC from mice immunized with T-epitope proliferated in response to challenge with whole E6 protein. Immunization with E6 T-epitopes linked to B-epitopes of HPV16 E7 protein elicited specific antibody indicating that T-cells recognizing the T-epitopes provided cognate "help" for B-cells. LNC from mice co-immunized with E6 T-epitope and the major T-helper epitope of HPV16 E7 (48DRAHYNI54) proliferated comparably when challenged with the peptides individually indicating co-dominance of the two T-epitopes. The findings have implications for incorporation of E6 into a therapeutic vaccine.     

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.     

A human myeloma-produced monoclonal protein directed against the active subpopulation of von Willebrand factor

The authors present a study of a human myeloma-produced monoclonal protein (IgG-k) directed against von Willebrand factor that caused an acquired von Willebrand's disease (vWD)-like syndrome. The illness was characterized by upper gastrointestinal bleeding, prolonged bleeding time, decreased platelet adhesiveness, lack of platelet aggregation in response to ristocetin, and a qualitatively abnormal Factor VIII related antigen (vWF) by two-dimensional immunoelectropheresis. Patient plasma or IgG fraction mixed with normal platelet-rich plasma completely inhibited aggregation with ristocetin, but patient platelets resuspended in normal plasma aggregated normally with ristocetin. VWF was markedly elevated and the two-dimensional immunoelectropheresis of vWF revealed a vWD type II-like pattern with an absence of the higher molecular weight forms of the vWF. Marked inhibitory activity was observed in the ristocetin cofactor assay but disappeared at the highest dilutions of patient plasma used in the assay. Infusion of cryoprecipitate following plasmapheresis led to a correction of the bleeding time, improvement in platelet adhesiveness, transient disappearance of inhibitory activity in the Factor VIII ristocetin cofactor assay, and no significant normalization of two-dimensional immunoelectropheresis of vWF. This case demonstrated a myeloma-associated monoclonal antibody that interacted specifically with that part of the Factor VIII molecule necessary for Factor VIII ristocetin cofactor activity, normal platelet adhesiveness, and bleeding time.     

Potential strategies utilised by papillomavirus to evade host immunity

Summary: The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced lo PV antigens, resting keratinocytes (KC) appear resistant to interferon-γ-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.     

Differences in the effectiveness of delivery of B- and CTL-epitopes incorporated into the Hepatitis B core antigen (HBcAg) c/e1-region

Summary.  Work from a number of laboratories including our own has shown that foreign B-epitopes inserted into the c/e1-region of Hepatitis B core antigen (HBcAg) elicit powerful antibody responses when mice are immunised with the recombinant core particles. In the present study, we wished to take advantage of the immunodominance of the c/e1-region to deliver cytotoxic T-lymphocyte (CTL) epitopes as a recombinant HBcAg vaccine. Our results indicated that recombinant HBcAg containing CTL epitopes of the E7 protein of human papillomavirus failed to prime E7-directed CTL responses when used to immunise mice for antigen processing through either the endogenous pathway via a Salmonella typhimurium vector, or through the exogenous pathway by parenteral immunisation with recombinant core. Hydropathicity plots predict that the presumed surface location of the hydrophilic c/e1-region within the core particle may alter following insertion of hydrophobic residues constituting the CTL epitopes, thereby compromising their presentation to the afferent immune system. Our data indicate that while the c/e1-region has a powerful adjuvanting effect for inserted B-epitopes, it does not serve this function for inserted CTL epitopes. These findings have generic implications for the development of CTL inducing vaccines using HBcAg as a vaccine vehicle. Received December 15, 1998 Accepted March 9, 1999     

NEGATIVE THERAPEUTIC REACTION

ABSTRACT For the last 80 years the term 'negative therapeutic reaction' has been used by psychoanalysts and psychotherapists to define the phenomenon of improvement followed by deterioration of a client's psychological or symptomatic condition, apparently in response to their therapist's interventions. Negative therapeutic reaction has been the subject of many papers written by these practitioners, who in various ways have attempted to clarify and understand what goes wrong in such therapeutic encounters.
This paper summarises the ideas of many psychoanalytic writers and practitioners who have worked throughout the span of the twentieth century, and up to the present day. Various aspects of negative therapeutic reaction are explored, such as alternative definitions of the concept, possible origins of NTR, the impact of the therapeutic relationship, and treatment implications. These ideas are compared and discussed, drawing mainly on the object relations theories of Fairbairn (1943), and illustrated through examples from myth, literature, research and clinical vignettes.     

樟叶胡椒中新木脂素成分的研究

自胡椒科胡椒属植物樟叶胡椒(Piper polysyphorum C. DC)中分离到六个新木脂素(neolignans)类化合物,经光谱(UV,IR,MS,¹H-NMR,¹³C-NMR,2D-NMR,CD)分析及衍生物制备,确定Ⅱ为新化合物,即threo-△⁷-7-羟基-3,4,5,3′,5′-五甲氧基-8-O-4′-新木脂素,为一对对映体,命名为樟叶素(polysyphorin),Ⅰ,Ⅲ,Ⅳ为新的对映体,分別为(+)-virolongin,(+)-grandisin及(+)-lancifolin D.化合物Ⅴ为南藤素(wallichinine),Ⅵ为山蒟素D(hancinone D)。血小板活化因子(PAF)受体结合实验及PAF引起的血小板聚集实验证明化合物Ⅰ～Ⅴ具有明显的抑制活性。     

赛庚啶对氧自由基的清除作用

赛庚啶(Cyp)对Fenton反应生成的·OH有较强的直接清除作用(EC₅₀为54μmol/L),并明显抑制·OH的生成速率(IC₅₀为22 μmol/L),且作用明显比·OH特异性清除剂甘露醇强(其EC₅₀和IC₅₀分别为22.7 mmol/L和10.7mmol/L)。Cyp对大鼠腹腔多形核白细胞(PMNs)产生的O₂也有一定的清除作用,其IC₅₀为179 μmol/L。提示Cyp的抗心肌损伤作用可能至少部分与其清除氧自由基作用相关。