The sap from Euphorbia peplus is effective against human nonmelanoma skin cancers.

BACKGROUND: The sap from Euphorbia peplus, commonly known as petty spurge in the U.K. or radium weed in Australia, has been used as a traditional treatment for a number of cancers. OBJECTIVE: To determine the effectiveness of E. peplus sap in a phase I/II clinical study for the topical treatment of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and intraepidermal carcinomas (IEC). METHODS: Thirty-six patients, who had refused, failed or were unsuitable for conventional treatment, were enrolled in a phase I/II clinical study. A total of 48 skin cancer lesions were treated topically with 100-300 µL of E. peplus sap once daily for 3 days. RESULTS: The complete clinical response rates at 1 month were 82% (n = 28) for BCC, 94% (n = 16) for IEC and 75% (n = 4) for SCC. After a mean follow-up of 15 months these rates were 57%, 75% and 50%, respectively. For superficial lesions < 16 mm, the response rates after follow-up were 100% for IEC (n = 10) and 78% for BCC (n = 9). CONCLUSIONS: The clinical responses for these relatively unfavourable lesions (43% had failed previous treatments, 35% were situated in the head and neck region and 30% were > 2 cm in diameter), are comparable with existing nonsurgical treatments. An active ingredient of E. peplus sap has been identified as ingenol mebutate (PEP005). This clinical study affirms community experience with E. peplus sap, and supports further clinical development of PEP005 for the treatment of BCC, SCC and IEC.

Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge.

The development of prophylactic vaccines against retroviral diseases has been impeded by the lack of obvious immune correlates for protection. Cytotoxic T-lymphocyte (CTL), CD4-lymphocyteS, chemokine and/or antibody responses have all been associated with protection against HIV and AIDS; however, effective and safe vaccination strategies remain elusive. Here we show that vaccination with a minimal ovine CTL peptide epitope identified within gp51 of the retrovirus bovine leukemia virus (BLV), consistently induced peptide-specific CTLs. Only sheep whose CTLs were also capable of recognizing retrovirus-infected cells were fully protected when challenged with BLV. This retrovirus displays limited sequence variation; thus, in the relative absence of confounding CTL escape variants, virus-specific CTLs targeting a single epitope were able to prevent the establishment of a latent retroviral infection.

The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon alpha/beta priming.

The serine proteinase inhibitor (serpin) plasminogen activator inhibitor type 2 (PAI-2) is well characterized as an inhibitor of extracellular urokinase-type plasminogen activator. Here we show that intracellular, but not extracellular, PAI-2 protected cells from the rapid cytopathic effects of alphavirus infection. This protection did not appear to be related to an effect on apoptosis but was associated with a PAI-2-mediated induction of constitutive low-level interferon (IFN)-alpha/beta production and IFN-stimulated gene factor 3 (ISGF3) activation, which primed the cells for rapid induction of antiviral genes. This primed phenotype was associated with a rapid development of resistance to infection by the PAI-2 transfected cells and the establishment of a persistent productive infection. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations, together with the recently demonstrated PAI-2-mediated inhibition of tumor necrosis factor-alpha induced apoptosis, (a) illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and (b) demonstrate a novel activity for a eukaryotic serpin.

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta chains from HLA-B8-restricted, CD8+ cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved beta chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

SerpinB2 deficiency modulates Th1/Th2 responses after schistosome infection.

SerpinB2, also known as plasminogen activator inhibitor type-2, is a major product of macrophages and is upregulated during many infections. Although SerpinB2 inhibits urokinase plasminogen activator in vitro, evidence that this represents its physiological role in vivo is not compelling. We have recently shown that SerpinB2-/-mice generate enhanced Th1 responses after immunization with a Th1 immunogen. Herein,we show that Schistosoma japonicum granulomas induced liver SerpinB2 mRNA expression by >600-fold in wild-type mice. In SerpinB2-/- mice, worm and egg burden, and granuloma number and volume were unaffected. However, granulomas in these mice were associated with reduced fibrosis (as determined by Sirius red staining and image analysis) and increased iNOS, IL-6, IL-10 and TNFa and decreased Arg 1 and IL-13 mRNA expression. SerpinB2-/- mice immunized with soluble egg antigen (SEA) also showed reduced levels of SEA-specific IgG1. SerpinB2 deficiency thus promoted certain Th1 and reduced certain Th2 responses in response to this Th2 immunogen.

Cardiovascular regulation during sleep quantified by symbolic coupling traces.

Sleep is a complex regulated process with short periods of wakefulness and different sleep stages. These sleep stages modulate autonomous functions such as blood pressure and heart rate. The method of symbolic coupling traces (SCT) is used to analyze and quantify time-delayed coupling of these measurements during different sleep stages. The symbolic coupling traces, defined as the symmetric and diametric traces of the bivariate word distribution matrix, allow the quantification of time-delayed coupling. In this paper, the method is applied to heart rate and systolic blood pressure time series during different sleep stages for healthy controls as well as for normotensive and hypertensive patients with sleep apneas. Using the SCT, significant different cardiovascular mechanisms not only between the deep sleep and the other sleep stages but also between healthy subjects and patients can be revealed. The SCT method is applied to model systems, compared with established methods, such as cross correlation, mutual information, and cross recurrence analysis and demonstrates its advantages especially for nonstationary physiological data. As a result, SCT proves to be more specific in detecting delays of directional interactions than standard coupling analysis methods and yields additional information which cannot be measured by standard parameters of heart rate and blood pressure variability. The proposed method may help to indicate the pathological changes in cardiovascular regulation and also the effects of continuous positive airway pressure therapy on the cardiovascular system.

A Kunjin replicon vector encoding granulocyte macrophage colony-stimulating factor for intra-tumoral gene therapy.

We have recently developed a non-cytopathic RNA replicon-based viral vector system based on the flavivirus Kunjin. Here, we illustrate the utility of the Kunjin replicon system for gene therapy. Intra-tumoral injections of Kunjin replicon virus-like particles encoding granulocyte colony-stimulating factor were able to cure >50% of established subcutaneous CT26 colon carcinoma and B16-OVA melanomas. Regression of CT26 tumours correlated with the induction of anti-cancer CD8 T cells, and treatment of subcutaneous CT26 tumours also resulted in the regression of CT26 lung metastases. Only a few immune-based strategies are able to cure these aggressive tumours once they are of a reasonable size, illustrating the potential of this vector system for intra-tumoral gene therapy applications.

Modeling the cardiovascular system using a nonlinear additive autoregressive model with exogenous input.

The parameters of heart rate variability and blood pressure variability have proved to be useful analytical tools in cardiovascular physics and medicine. Model-based analysis of these variabilities additionally leads to new prognostic information about mechanisms behind regulations in the cardiovascular system. In this paper, we analyze the complex interaction between heart rate, systolic blood pressure, and respiration by nonparametric fitted nonlinear additive autoregressive models with external inputs. Therefore, we consider measurements of healthy persons and patients suffering from obstructive sleep apnea syndrome (OSAS), with and without hypertension. It is shown that the proposed nonlinear models are capable of describing short-term fluctuations in heart rate as well as systolic blood pressure significantly better than similar linear ones, which confirms the assumption of nonlinear controlled heart rate and blood pressure. Furthermore, the comparison of the nonlinear and linear approaches reveals that the heart rate and blood pressure variability in healthy subjects is caused by a higher level of noise as well as nonlinearity than in patients suffering from OSAS. The residue analysis points at a further source of heart rate and blood pressure variability in healthy subjects, in addition to heart rate, systolic blood pressure, and respiration. Comparison of the nonlinear models within and among the different groups of subjects suggests the ability to discriminate the cohorts that could lead to a stratification of hypertension risk in OSAS patients.

The fate of the circumsporozoite antigens during the exoerythrocytic stage of Plasmodium berghei.

There has been considerable interest in the circumsporozoite proteins due to their potential use in anti-malarial vaccines. Previous authors have shown that these proteins persist from the invading sporozoite throughout the growing exoerythrocytic or liver stage. We show that the different distributions of these proteins seen during the development of the exoerythrocytic parasite of Plasmodium berghei closely follow morphological changes, which can be recognized under the light microscope. At the end of the exoerythrocytic cycle, the majority of the remaining circumsporozoite proteins were associated with the spongy stroma in which the emerging exoerythrocytic merozoites lay. Cell-mediated immunity originally directed against sporozoites might recognize the stroma as a second target resulting in the indirect destruction of the exoerythrocytic merozoites.

BLT esterase activity as an alternative to chromium release in cytotoxic T cell assays.

Granules released by cytotoxic T cells (CTL), during recognition and killing of target cells, contain granule enzyme A. This serine protease has an esterase activity, which is easily measured using the substrate benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). BLT activity, routinely used as an assay for granule release, provides an alternative to the standard chromium release assay as a measure of CTL-mediated killing. The two methods were highly comparable when either exogenous synthetic peptide or endogenously produced epitopes were used as targets and human CTL clones acted as effectors. The advantages of the BLT assay are that it uses inexpensive non-radioactive reagents, the assay can be run over any period between 4 and 30 h and can be performed with as few as 10(4) CTLs if synthetic peptide epitopes are used.

Inhibition of HLA B8-restricted recognition by unrelated peptides: evidence for allosteric inhibition.

A panel of synthetic peptides representing human lymphocyte antigen (HLA) B8, other class I and class II restricted T cell epitopes and two B cell epitopes, were all able to compete with recognition of a HLA B8 restricted epitope by a cytotoxic T cell clone. Competition was obtained when the competitor peptides were added either before or after the target epitope. The target epitope also had a slow off rate, implicating allosteric inhibition. The presence of non-specific, allosteric binding sites may interfere with experiments attempting to define immunologically relevant MHC binding specificities.

Delayed emergence of bovine leukemia virus after vaccination with a protective cytotoxic T cell-based vaccine.

In a previous study eight MHC class I-matched sheep were vaccinated with a minimal cytotoxic T lymphocyte (CTL) peptide epitope vaccine and were challenged with the retrovirus, bovine leukemia virus (BLV). Half the vaccinated animals remained PCR negative after challenge, whereas the remaining half and the placebo group became PCR positive within 4 weeks postchallenge (Hislop AD, Good MF, Mateo L, Gardner J, Gatei MH, Daniel RCW, Meyers BV, Lavin MF, and Suhrbier A: Nat Med 1998;4:1193). Here we show that neither epitope mutations nor processing differences explained why half the peptide-vaccinated animals failed to resist the BLV challenge. However, in these animals the development of BLV-induced lymphosarcomas was significantly delayed compared with the placebo group, suggesting a role for CTLs in preventing retrovirus-induced cancers. Importantly, two of the initially protected animals become PCR positive after approximately 1.5 years, indicating extended suppression but not elimination of challenge virus by vaccine-induced CTLs. The late emergence of virus could not be explained by epitope escape mutations or the loss of memory CTL responses. We speculate that high levels of effector CTL may be needed to protect animals from a postchallenge viremia and maintenance of such effector CTLs, rather than memory CTLs, may be required to prevent subsequent emergence of virus from latent pools.

Expression of the precursor of the major merozoite surface antigens during the hepatic stage of malaria.

The precursor of major merozoite surface antigens (PMMSA) and its proteolytic products are candidates for an asexual blood stage vaccine. Previous authors have shown that PMMSA epitopes are expressed in the liver or exoerythrocytic (EE) stage of malaria. Using Plasmodium berghei, we show that the molecular weight of the liver stage PMMSA is similar to that of the blood stage and that both EE and blood stage proteins are similarly processed. In the EE stage, it was synthesized toward the end of schizogony and appeared first to localize to the rough endoplasmic reticulum and then, as the cytomeres began to form, to the parasite plasmalemma. The EE and blood stage merozoites expressed similar amounts of this antigen as determined by indirect immunofluorescence.

Use of recombinant vaccinia to restimulate antigen specific human peripheral blood cytotoxic T lymphocytes.

A simple method is described for using recombinant vaccinia virus for restimulating human peripheral blood mononuclear cells (PBMC) to generate antigen specific CD8 + alpha beta cytotoxic T cell (CTL) effector populations. Effector PBMC were restimulated in vitro with a subpopulation of PBMC, which had been infected with recombinant vaccinia at very low multiplicities of infection in the absence of serum. This protocol avoided the vaccinia mediated overt cytopathic effects on the effector PBMC and generated bulk CTL cultures, which could be used for the identification of epitopes recognised by antigen specific CTL.

Complete removal of mycoplasma from viral preparations using solvent extraction.

Mycoplasma contamination of two alphavirus isolates and adenovirus 5 was completely removed using a very simple, rapid protocol based on solvent extraction. Treated virus preparations remained free of mycoplasma after 6 months in culture.

The complete development in vitro of the vertebrate phase of the mammalian malarial parasite Plasmodium berghei.

All three 'vertebrate' stages of the rodent malarial parasite Plasmodium berghei berghei were grown in vitro in the absence of the vertebrate host. The parasite was introduced into culture from infected mosquitoes and 2 in vitro culture methods were used sequentially to complete the 'vertebrate' phases of development in hepatoma and erythrocyte host cells. The resultant blood infection produced mature schizonts and male and female gametocytes. The protocol, which is now being extended to the human pathogen P. falciparum, may assist future studies on this important group of parasites.

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta.

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.

Effect of CPAP therapy on daytime cardiovascular regulations in patients with obstructive sleep apnea.

Obstructive sleep apnea (OSA) is a sleep disorder with a high prevalence that causes pathological changes in cardiovascular regulation during the night and also during daytime. We investigated whether the treatment of OSA at night by means of continuous positive airway pressure (CPAP) improves the daytime consequences. Twenty-eight patients with OSA, 18 with arterial hypertension, 10 with normal blood pressure, were investigated at baseline and with three months of CPAP treatment. Ten age and sex matched healthy control subjects were investigated for comparisons. We recorded a resting period with 20min quiet breathing and an exercise stress test during daytime with ECG and blood pressure (Portapres). The bicycle ergometry showed a significant reduction of the diastolic blood pressure at a work load of 50W and 100W (p<0.05 and p<0.01, respectively) and a decrease of the heart rate recovery time after the stress test (p<0.05). These results indicate a reduction of vascular resistance and sympathetic activity during daytime. The coupling analysis of the resting periods by means of symbolic coupling traces approach indicated an effect of the CPAP therapy on the baroreflex reaction in hypertensive patients where influences of the systolic blood pressure on the heart rate changed from pathological patterns to adaptive mechanisms of the normotensive patients (p<0.05).

Symbolic coupling traces for causality analysis of cardiovascular control.

Directional coupling analysis of time series is an important subject of current research. In this paper, a method based on symbolic dynamics for the detection of time-delayed coupling in biosignals is presented. The symbolic coupling traces, defined as the symmetric and diametric traces of the bivariate word distribution, allow for a more reliable quantification of coupling and are compared with established methods like mutual information and cross recurrence analysis. The symbolic coupling traces method is applied to appropriate model systems and cardiological data which demonstrate its advantages especially for nonstationary and noisy data. Moreover, the method of symbolic coupling traces is used to analyze and quantify time-delayed coupling of cardiovascular measurements during different sleep stages. Significant different regulatory mechanisms are detected not only between the deep sleep and the other sleep stages but also between healthy subjects and patients. The proposed method may help to indicate pathological changes in cardiovascular regulation and also effects of continuous positive airway pressure therapy on the cardiovascular system.