The association of an HPV16 oncogene variant with HLA-B7 has implications for vaccine design in cervical cancer.

HLA-restricted cytotoxic T-lymphocyte (CTL) recognition of human papillomavirus (HPV) oncogene products may be important in the control of the HPV infections associated with the development of cervical cancer. We have identified, in HLA-B7 individuals, a consistent variation in the HPV16 E6 oncoprotein sequence, which alters an HLA-B7 peptide binding epitope in a way likely to influence immune recognition by CTLs. These results illustrate a biologically relevant mechanism for escape from immune surveillance of HPV16 in HLA-B7 individuals. Thus, both HLA type and HPV16 strain variation need to be considered in the screening of at-risk individuals and for the rational design of anti-HPV vaccines.

The major histocompatibility complex-restricted response of recombinant inbred strains of mice to natural tick transmission of Borrelia burgdorferi.

The causative agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes ricinus complex. In this study, we report the antibody response of recombinant inbred strains of mice of the H-2, b, d, and k haplotypes, infected with B. burgdorferi as a result of exposure to infected I. dammini. The patterns of antibody response assayed by Western blot analysis indicate significant major histocompatibility complex (MHC) restriction to bacterial antigens within the first 2 mo of infection in mice. Other bacterial antigens induce a significant response across the MHC haplotypes tested when assayed on the same bacterial strain used to transmit the infection, but do not crossreact with the same proteins derived from heterologous strains of B. burgdorferi. No response to outer surface protein A was detected at any time during the 60-d period we analyzed this infection. A third group of bacterial antigens appear to generate a MHC-nonrestricted response, and this lack of restriction is maintained when assaying the crossreactivity of the response with other strains of B. burgdorferi. These proteins may provide more accurate diagnostic probes than those currently in use. Finally, there appears to be a significant difference in the expression of most bacterial antigens when the spirochete is cultured for many passages since the same strain of bacterium isolated from low-passage and high-passage preparations exhibit different banding patterns in Western blots when assayed with the same sera.

The association of the sodium "setpoint" to interdialytic weight gain and blood pressure in hemodialysis patients.

INTRODUCTION: Hemodialysis patients lack the normal mechanisms to regulate body water volume and osmolality. The dialysis treatment is expected to adequately regulate both body water volume and body Na+ content, which is the primary action determining body water osmolality. Data in subjects with normal renal function indicate that an individual has a specific osmolality value above which thirst is generated and fluid will be ingested. This specific osmolality value or "setpoint" varies among individuals, but is quite reproducible within an individual. It was postulated that hemodialysis patients also may have a Na+ 'setpoint', which if increased by the use of higher dialysate Na+ concentration, might be associated with increased interdialytic weight gain and blood pressure. METHODS: Monthly laboratory and treatment data were abstracted on 58 hemodialysis patients and included pre- and post-dialysis serum Na+ concentrations, interdialytic weight gain and blood pressure over 9 to 16 months. The Na+ concentrations were averaged to determine the individual Na+ 'setpoint' and the Na+ gradient (Dialysis Na+ concentration - mean Na+ concentration) was determined for each patient. RESULTS: Linear regression analyses showed that there was a statistically significant association between the magnitude of the Na+ gradient and interdialytic weight gain and blood pressure. CONCLUSIONS: These data suggest that interdialytic weight gain in individual patients may be associated with the use of dialysate Na+ concentration in excess of the patient's desired Na+ 'setpoint'. More individualization of dialysate Na+ concentration may be indicated.

HLA class II antigen expression in human papillomavirus-associated cervical cancer.

The observation that tumor cells of some neoplasms display major histocompatibility complex (MHC) class II molecules may be of functional significance, influencing the progression of malignancy by allowing the cancer cells to present antigen to the immune system. In the normal cervix, class II molecules are expressed by columnar but not squamous epithelium. The pattern of MHC class II expression in cervical carcinomas has been documented using immunohistochemical methods. Of 53 cervical squamous carcinomas examined for MHC class II expression, only 17% maintained a negative phenotype characteristic of the epithelium from which they were derived, while the remaining tumors exhibited either uniform (45%) or heterogeneous (38%) expression. Tumor areas which were class II positive also express class II associated invariant chain and the adhesion molecules lymphocyte function antigen 3 and intercellular adhesion molecule 1. The DR, DP, and DQ class II MHC subloci are differentially expressed, suggesting independent regulation. There is a trend for tumors with the uniform class II phenotype to predominantly express DR antigen, whereas tumors of the heterogeneous class II phenotype express with equal frequency either DR or DP antigens dominantly. There is no apparent influence of class II status on lymphocyte infiltration of the tumors. The presence of human papillomavirus 16 DNA in the cervical carcinoma specimens was analyzed by Southern blotting of restriction enzyme digested DNA and no correlation between the presence of human papilloma virus and MHC class II expression was found.

Immunological and viral factors associated with the response of vulval intraepithelial neoplasia to photodynamic therapy.

Topical 5-aminolevulinic acid-based photodynamic therapy (PDT) has produced complete response rates of >90% for nonmelanoma skin carcinomas, which are mostly human papillomavirus (HPV) negative. Using a similar treatment protocol, we observed a short-term response in only one third (10 of 32) of high-grade vulval intraepithelial neoplasia (VIN 2-3) lesions. Unifocal lesions were found more responsive than multifocal and pigmented lesions. Animal model studies have suggested that long-term PDT response involves an immune reaction in which CTLs play a crucial role. In this study, we have assessed: (a) HPV infection; (b) HLA expression; and (c) immune infiltrating cells in VIN biopsies from responders and nonresponders to determine whether these factors may limit response to topical 5-aminolevulinic acid-based PDT. Tissues from normal vulva (n = 9), vulval carcinoma (n = 11), and VIN (32 patients from which 19 pre- and 43 post-PDT biopsies were taken) were investigated for immune cell infiltration and HLA class I expression by immunohistochemistry and HPV infection by PCR. There was a greater likelihood of HPV positivity associated with a lack of response of VIN to PDT (P = 0.002), and VIN nonresponders were more likely to show HLA class I loss compared with responders (P = 0.030). HLA class I down-regulation was significantly greater in the carcinomas (82%, total loss) than the VIN (28%, 19%, total loss; and 9%, allele loss; P = 0.004). None of the cases with class I down-regulation responded to PDT, whereas 3 of 6 (50%) of cases that showed total class I loss subsequently developed superficial invasion. Compared with normal vulval skin, VIN lesions showed increased infiltration by CD4 (T-helper) and CD68 (macrophages) but not CD1a (Langerhans cells) or CD8 (CTLs). There was, however, a significant increase of CD8 infiltration in posttreatment VIN responders compared with nonresponders (P = 0.0001). These data clearly support the contention that high-risk HPV infection and lack of cell-mediated immunity may play a role in the observed poor response of lower genital lesions to topical PDT.

Differential T helper cell responses to human papillomavirus type 16 E7 related to viral clearance or persistence in patients with cervical neoplasia: a longitudinal study.

T-cell-mediated immune responses against oncogenic human papillomaviruses (HPVs) are believed to play a role in the prevention of cervical carcinogenesis. The in vitro production of interleukin 2 by CD4+ T helper (Th) cells in response to overlapping 20-mer peptides covering the HPV-16 E7 oncoprotein sequence was determined in 72 women with cytological evidence of premalignant cervical intraepithelial neoplasia (CIN) who participated in a nonintervention follow-up (FU) study. In addition, 15 HPV-16 + cervical carcinoma patients were tested. Positive Th cell reactivity was restricted to patients infected by HPV-16 and related types and showed a strong association with viral persistence and disease progression, as evidenced by the high frequency of positive responders among women with persistent HPV-16 infections who ended FU with high-grade CIN III lesions [14 of 15 (93%)]. Women with cervical carcinoma showed responses at a significantly reduced rate [7 of 15 (47%); P = 0.014]. Over the FU period (10-34 months), the level of E7-induced interleukin 2 production from the lymphocytes of CIN patients who had cleared HPV-16 infection showed an inverse correlation with time relative to the last positive HPV DNA test, with 8 of 13 of these patients showing positive responses after clearance. By contrast, among women with persistent HPV-16 infections and developing CIN III lesions (n = 8), there was a rise in Th cell activity over the course of FU. The majority of women responded to an immunogenic region in the carboxyl terminus of the E7 protein (amino acids 67-98). The observed HPV-16 E7-specific Th cell responses may develop as a consequence of increased antigen availability resulting either from clearance or from progression of cervical lesions.

The role of protein kinase A and protein kinase C in prostanoid IP receptor desensitization in NG108-15 cells.

Pretreatment of NG108-15 cells for 1 h with 1 microM phorbol 12-myristate,13-acetate produced no significant effect on the subsequent stimulation of adenylate cyclase activity by the IP receptor agonist, iloprost, the adenosine A2 receptor agonist, N-ethylcarboxamidoadenosine (NECA), or sodium fluoride, suggesting that protein kinase C activation does not produce desensitization in this system. Pretreatment of cells with 10 microM iloprost or forskolin for 17 h produced a decrease in the specific binding of [3H]iloprost, consistent with a decrease in IP receptor number. Iloprost pretreatment produced a decrease in responses to iloprost, NECA and sodium fluoride, whereas forskolin pretreatment produced a decrease in subsequent responsiveness to iloprost and NECA, but the response to sodium fluoride remained unaffected. The desensitization produced by forskolin could be completely inhibited by the inhibitor of protein kinase A and protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), but H7 had no effect on the desensitization produced by iloprost.

Inhibition of prostacyclin release from cultured endothelial cells by nitrovasodilator drugs.

Pretreatment (18 h) of the bovine aortic endothelial cell line AG4762 to 500 microM sodium nitroprusside (SNP), glyceryl trinitrate (GTN) or 3-morpholino-sydnonimine (SIN-1) significantly inhibited 100 nM bradykinin-stimulated prostacyclin (PGI2) release. SIN-1 produced the greatest reduction (67 +/- 6%), followed by SNP (47 +/- 12%) and GTN (45 +/- 9%). Only SIN-1 and GTN inhibited basal PGI2 release where again the effect of SIN-1 (66 +/- 6%) was greater than that of GTN (31 +/- 15%). There was no effect of SNP on basal PGI2 release. We have demonstrated this inhibition of bradykinin-stimulated PGI2 release is not the result of cell death. In addition, 8-bromo-cyclic GMP, whilst having no effect on basal PGI2 release, demonstrated a small but significant inhibition (15 +/- 6%) of the enhanced response to 100 nM bradykinin. These studies may reflect a mechanism by which the release of vasodilators from endothelial cells is altered during therapy with nitrovasodilators and thus may contribute to the development of tolerance to these drugs.

Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function.

Prostacyclin and adenosine A2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of Gs and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A2 receptor agonist) was reduced by 30-40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827-3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309-316) suggesting that cAMP may not play a major role in agonist mediated desensitization.

Testing models of agonism for G protein-coupled receptors.

Despite much debate as to the validity of different models of agonism, there is apparently very little to choose between them in practice. However, most experiments to test the various models have used classical isolated tissue systems. In these systems, the response that is measured is far removed from receptor activation, and it is likely that the properties of the events at the receptor may be masked by more distal processes. In this article, Mary Keen examines the effects of agonists on responses that follow, very closely, activation of G protein-coupled receptors. The data appear to be inconsistent with any of the current models of agonism.

Different Toll-like receptor agonists induce distinct macrophage responses.

We previously reported that gram-negative bacterial lipopolysaccharide (LPS) activates cells via Toll-like receptor (TLR) 4, whereas the mycobacterial cell wall glycolipid lipoarabinomannan (LAM) activates cells via TLR2. We also identified a secreted TLR2 agonist activity in short-term culture filtrates of Mycobacterium tuberculosis bacilli, termed soluble tuberculosis factor (STF). Here we show that STF contains mannosylated phosphatidylinositol (PIM) and that purified PIM possesses TLR2 agonist activity. Stimulation of RAW 264.7 macrophages by LPS, LAM, STF, and PIM rapidly activated nuclear factor (NF)-kappaB, activator protein-1 (AP-1), and mitogen-activated protein (MAP) kinases. These TLR agonists induced similar levels of NF-kappaB and AP-1 DNA-binding activity, as well as trans-activation function. Unexpectedly, these TLR agonists induced tumor necrosis factor alpha secretion, whereas only LPS was capable of inducing interleukin-1beta and nitric oxide secretion. Thus, different TLR proteins are still capable of activating distinct cellular responses, in spite of their shared capacities to activate NF-kappaB, AP-1, and MAP kinases.

Measurement of cartilage volumes in rheumatoid arthritis using MRI.

MRI is a valuable imaging modality for assessment of the articular cartilage in rheumatoid arthritis (RA) and is potentially of use in monitoring disease progression and response to therapy. In this study, we investigated the sources of error in volume measurements obtained by segmentation of MR images of knee cartilage in patients with RA and followed cartilage volume in a group of RA patients for 12 months. 23 RA patient volunteers were recruited for knee imaging. Six subjects were imaged at baseline only, six were imaged at baseline and again within an hour in the same imaging session, six subjects were imaged at baseline and 7 days, and 17 subjects were imaged at baseline, 4+/-2 months and 12 months. Imaging was performed at 1.0 T using a three-dimensional spoiled gradient-echo sequence with fat-suppression. Manual image segmentation was performed once or twice on the lateral tibial, medial tibial, patellar and femoral compartment by either one or two segmenters. Coefficients of variation (CoV) for repeated volume measurement of total cartilage were 2.2% (same segmenter, same scan), 5.2% (different segmenter, same scan), 4.9% (same segmenter, different scan, same session), and 4.4% (same segmenter, different scan, different session). Over the 12 month duration of the study there was no significant change in total cartilage volume, nor were there significant changes in volume in any individual compartment. This measurement technique is reproducible, but any net change in cartilage volume over 1 year is very small.

Expression and desensitisation of A2 purinoceptors on cultured bovine aortic endothelial cells.

Purine nucleotides and their metabolites are important mediators of vascular tone. Adenosine promotes relaxation of vascular smooth muscle, acting on the A2 subclass of purinoceptors. There is some evidence that these receptors are also present on vascular endothelial cells. We have therefore examined cultured aortic endothelial cells for adenosine (A2) sensitivity. Bovine aortic endothelial cells (AG4762) were obtained from the Institute of Aging cell repository (USA), and cultured in monolayer flasks. Adenylate cyclase activity in homogenates of AG4762 cells was increased by 5'-(N-ethylcarboxamido)-adenosine (NECA) greater than 2-chloroadenosine greater than adenosine. NECA dependent activation of adenylate cyclase was inhibited by 3-isobutyl-l-methylxanthine greater than theophylline greater than caffeine. The rank order of potency of these compounds identified the responses as mediated by A2 purinoceptors. Prolonged exposure of AG4762 cells to NECA in culture resulted in loss of A2 purinoceptor responsiveness, and purinoceptor activity could be restored to non-dividing densensitized cells by further culture in the absence of the desensitising agent. The cyclic AMP dependent phosphorylation of specific sites in endothelial cells may trigger a number of important biological events which are yet to be determined.

Soluble and membrane-bound muscarinic acetylcholine receptors.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

Papillomavirus vaccines.

The considerable morbidity and mortality associated with certain human papillomaviruses (HPV) has provided the impetus for HPV vaccine development. The design of such vaccines has evolved from an understanding of the nature of HPV infections and their consequences, together with evaluation of the efficacy of different approaches to vaccination in animal models. These studies have culminated in the production of several different vaccine preparations which are currently undergoing Phase I and II clinical trials. The justification for the widespread implementation of prophylactic HPV vaccines will depend on the outcome of larger scale studies of vaccine efficacy that take into account the epidemiology of HPV infections and associated disease. The usefulness of therapeutic HPV vaccines will require evidence that they can substantially augment or substitute for the effectiveness of currently available treatments.

ELISA measurement of IgG subclass production in culture supernatants using monoclonal antibodies.

Specific and sensitive ELISA to quantitate the human IgG subclasses in cell culture supernatants are described. These assays detect a minimum of 5 ng/ml IgG1, 90 ng/ml IgG2, 8 ng/ml IgG3 and 8 ng/ml IgG4 and can generally measure IgG subclasses in lymphocyte cultures containing a minimum of 200 ng/ml of total IgG. The isotype specificity of these ELISA is demonstrated and each individual ELISA shown to react with a number of paraproteins of the relevant subclass independently of their light chain type or their (major Caucasian) allotype. These assays have been used to determine the IgG subclass response of normal human lymphocytes to pokeweed mitogen in vitro.

NaF and guanine nucleotides modulate adenylate cyclase activity in NG108-15 cells by interacting with both Gs and Gi.

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.

The binding of pirenzepine to digitonin-solubilized muscarinic acetylcholine receptors from the rat myocardium.

The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4 degrees C. Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD approximately 3 X 10(-8)M) to the soluble myocardial receptors could be monitored directly using [3H]-pirenzepine. [3H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]-pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5'-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding.

Segregation of discrete GS alpha-mediated responses that accompany homologous or heterologous desensitization in two related somatic hybrids.

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.

Solubilization and characterization of guanine nucleotide-sensitive muscarinic agonist binding sites from rat myocardium.

Muscarinic receptors from rat myocardial membranes may be solubilized by digitonin in good yield at low temperatures in the presence of Mg2+. Under these conditions, up to 60% of the soluble receptors show high affinity binding for the potent agonist [3H]-oxotremorine-M (KA = 10(9)M-1), which is inhibited by 5'-guanylylimidodiphosphate. The muscarinic binding site labelled with [3H]-oxotremorine-M has a higher sedimentation coefficient (13.4 s) than sites labelled with a 3H antagonist in the presence of guanylylimidodiphosphate (11.6 s) and probably represents a complex between the ligand binding subunit of the receptor and a guanine nucleotide binding protein.