Cytotoxic and antitumour activity from Bursera fagaroides ethanol extract in mice with L5178Y lymphoma

Chloroform extracts of Bursera fagaroides (Burseracea) have previously shown antitumour activity against the Walker carcinoma 256 tumour system (WA16). In the present work we have determined the cytotoxic and antitumour activity of the ethanol extract (70%) of the bark of B. fagaroides using the L5178Y lymphoma. The cytotoxic activity is expressed as the ED₅₀ of the L5178Y lymphoma cells in culture, (20 µg/mL) whilst the antitumour activity was shown via a tumour growing inhibition test, measuring survival of BALB/c mice (2 × 10⁴ cells L5178Y i.p.). 24 h after inoculation mice were treated with 50 or 100 mg/kg of extract daily, over 15 days in independent groups of 10, using two administration routes. We observed the tumour evolution with and without treatment. Oral administration resulted in 8% of mice being tumour free after 60 day whilst intraperitoneal administration showed 26% survived at a dose of 100 mg/kg/day for 15 days. A significant increase in the survival of the treated animals (at 50 mg/kg/day over 15 days) was found compared with those treated with placebo or without treatment. Copyright © 1998 John Wiley & Sons, Ltd.     

On the use of α-chloralose for repeated BOLD fMRI measurements in rats

Most animal experiments in neurosciences require anesthesia of the experimental animal. For fMRI experiments most neuroscientist would prefer (I) a recoverable anesthesia that would (II) allow strong BOLD responses which are (III) reproducibly obtained for the same animal in repetitive experiments. Strong BOLD responses are particularly needed if weak physiological stimuli like whisker deflection are used. A-chloralose, in contrast to isoflurane, fulfils the need for strong activation but has been traditionally considered as a terminal drug due to its negative physiological side effects. Here it is demonstrated, that new commercial α-chloralose in a careful application scheme allows for repeated fMRI studies on the same animal with similar responses after whisker stimulation. Only minor effects on the normal behavior were observed between drugs. These include a longer time to return to baseline values of food ingestion and slower tail reaction for the α-chloralose experiments.     

A reproducible method for the enumeration of functional (cytokine producing) versus non-functional peptide-specific cytotoxic T lymphocytes in human peripheral blood

One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme-linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample-specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen-presenting cell platform for flow cytometry-based quantification of the frequency and activation status of peptide-specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)-A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine-producing) and inactive tetramer-positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high-throughput applications and automation. The utility and application of this method are discussed.     

EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy

Background:

Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy.

Methods:

We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition.

Results:

Among several predicted peptide epitopes, EGFR_875–889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR_875–889 epitope could be detected in the blood of HNSCC patients. EGFR_875–889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR_875–889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells.

Conclusion:

We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR_875–889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.     

Chemotherapy enhances tumor cell susceptibility to CTL-mediated killing during cancer immunotherapy in mice

Cancer immunotherapy faces a serious challenge because of low clinical efficacy. Recently, a number of clinical studies have reported the serendipitous finding of high rates of objective clinical response when cancer vaccines are combined with chemotherapy in patients with different types of cancers. However, the mechanism of this phenomenon remains unclear. Here, we tested in mice several cancer vaccines and an adoptive T cell transfer approach to cancer immunotherapy in combination with several widely used chemotherapeutic drugs. We found that chemotherapy made tumor cells more susceptible to the cytotoxic effect of CTLs through a dramatic perforin-independent increase in permeability to GrzB released by the CTLs. This effect was mediated via upregulation of mannose-6-phosphate receptors on the surface of tumor cells and was observed in mouse and human cells. When combined with chemotherapy, CTLs raised against specific antigens were able to induce apoptosis in neighboring tumor cells that did not express those antigens. These data suggest that small numbers of CTLs could mediate a potent antitumor effect when combined with chemotherapy. In addition, these results provide a strong rationale for combining these modalities for the treatment of patients with advanced cancers.     

Validation of the INNO-LiPA HBV DR Assay (Version 2) in Monitoring Hepatitis B Virus-Infected Patients Receiving Nucleoside Analog Treatment

Hepatitis B virus (HBV) antiviral drug resistance mutations prevent successful outcome of treatment and lead to worsening of liver disease. Detection of its emergence permits opportune treatment with alternative drugs. Unfortunately, the use of newly approved antivirals, including adefovir dipivoxil, emtricitabine, and telbivudine, is also associated with the development of drug resistance, albeit to a lesser extent than the use of lamivudine. The objectives of this work were to assess the performance characteristics (sensitivity and accuracy) of an updated drug resistance test, the INNO-LiPA HBV DR v2, which includes detection of mutations associated with lamivudine, adefovir, emtricitabine, and telbivudine resistance, and to compare the results with consensus sequencing of serum samples from patients treated with HBV antivirals. Diagnostic sensitivity, defined as detection of a positive amplification line on the line probe assay (LiPA) strip, was 94.8% (95% confidence interval [CI], 89.7 to 97.9) after initial testing, increasing to 96.3% (95% CI, 91.6 to 98.8) after repeat test 1 and to 100% (95% CI, 97.3 to 100.0) after repeat test 2. In diagnostic accuracy determinations, full concordance was observed between sequencing and LiPA for 77.0% of the codons tested (620/805 codons [95% CI, 74.0 to 79.9]), whereas LiPA and sequencing were partially concordant 22% of the time (177/805 codons). In 167 out of 177 cases, LiPA detected a wild-type/mutant mixture whereas sequencing detected only one of the two results. Performance testing of the new LiPA test, the INNO-LiPA HBV DR v2, showed convincing diagnostic sensitivity and accuracy. The ability of the test to detect mixed infections and minority viral populations associated with resistance to the current generation of antivirals, including adefovir, emtricitabine, and telbivudine, makes it a useful tool for HBV therapy monitoring.For the 350 million persons chronically infected with hepatitis B virus (HBV), the two therapeutic approaches presently available to control infection and its sequelae are the use of immunomodulatory agents and/or antiviral chemotherapy. These treatments aim at interrupting the progression and clinical outcomes of the disease (cirrhosis, hepatocellular carcinoma) by stimulating the anti-HBV-specific host immune response or by markedly decreasing viral replication. Of these two approaches, antiviral therapy has been the more frequently chosen option for long-term treatment, especially because of its relative lack of side effects or when immunomodulation therapy has been unsuccessful.Unfortunately, the sustained efficacy of antiviral agents with respect to halting disease progression can readily be compromised by the frequent occurrence of viral mutations. In the course of treatment using approved antivirals such as lamivudine, adefovir, and emtricitabine, HBV mutants are often selected from the preexisting pool of circulating quasispecies. Over time, one or several of these mutants will become the dominant species as a result of a variety of factors (21). In particular, resistance to lamivudine, the most frequently prescribed antiviral, is practically unavoidable, and the inexorable development of lamivudine drug resistance has been a major clinical impediment to its extended use. In pooled results from four multicenter controlled trials involving patients receiving lamivudine monotherapy, HBV variants associated with drug resistance were detected in 24% of patients after 1 year, a proportion that rose to 42% after 2 years (4, 9, 10).In contrast, the emergence of mutants with resistance to adefovir has been somewhat slower in naïve compared to lamivudine-resistant patients. Adefovir has a low resistance profile, with 3%, 9%, 18%, and 28% of patients showing resistance after 2, 3, 4, and 5 years, respectively (6). For its part, emtricitabine is associated with a high rate of drug resistance, even in treatment-naïve patients, which tends to preclude its use as monotherapy (15). Finally, the use of telbivudine has also been associated with drug resistance for about 5% of patients after only 12 months of treatment (11). This is not surprising, given that it shares the same resistance profile as lamivudine, especially at rtM204I within the YMDD motif of the viral polymerase (24).Due to the increasing use and expanding repertoire of nucleotide or nucleoside analogues to treat HBV, drug resistance mutations are becoming more problematic clinically. Unfortunately, the emergence of drug resistance often leads to a worsening of liver disease (13). Furthermore, cases of severe hepatitis reactivation due to infections by drug-resistant virus, resulting in hepatic decompensation and even mortality, have been previously reported (5, 14, 17). Cases of severe reactivation after withdrawal of antiviral therapy have also been previously described (7). Such unsatisfactory outcomes of drug resistance could be prevented by early detection of its emergence, thereby permitting an opportune alteration of treatment with appropriate alternatives.The CE-marked INNO-LiPA HBV DR v2 (Innogenetics, Gent, Belgium) (see Fig. ​Fig.1)1) is an in vitro, reverse hybridization line probe assay (LiPA) used to detect the presence of different genetic variants of HBV in human serum or plasma samples. The test—an update of the INNO-LiPA HBV DR, which detected only lamivudine-related mutations—includes new and clinically relevant wild-type and mutant motifs for codons L80V/I, V/G173L, L180M, A181T/V, M204V/I/S, and N236T, located in the HBV polymerase protein, that confer resistance to lamivudine, adefovir dipivoxil, emtricitabine, and telbivudine.Open in a separate windowFIG. 1.Example of monitoring resistance with the INNO-LiPA HBV DR v2 strip. The patient was receiving lamivudine treatment and developed an M204V resistance mutation and later a compensatory L80V mutation. The patient was switched to adefovir therapy in month 36 and in week 60 developed a new adevofir resistance mutation (N236T).The need for new-generation assays to detect resistance mutations and tailor therapy with respect to virological status is clinically relevant, given the emergence of complex patterns of resistance mutations which may impact treatment decisions (2, 23, 22, 25). While the previous version of the test covered only mutations in the HBV polymerase B and C domains, some of the new mutations associated with recently approved antivirals are present in other HBV polymerase domains. Therefore, a new assay was developed, extending coverage to HBV polymerase domains A through F. Because of this change and the fact that nested PCR technology is less well accepted in the market, the new amplicon was developed for use in a single-round PCR. This assay has been extensively challenged during the development phase. A design verification procedure was performed with 100 HBV DNA positive clinical samples. All these samples were tested with LiPA and sequenced. Mixtures which were not confirmed by sequencing were investigated through a method called “reduced clonal analysis.” These samples were cloned, and two to four clones were picked up and plated several times to produce monoclonal clones. Each clone was sequenced, amplified two to four times, and tested on LiPA strips. The identities of the components of the mixture were considered to have been confirmed when all LiPA results had been confirmed by sequencing of the clones. Data of the design verification procedure are not shown, but the results of this study are described in the INNO-LiPA HBV DR v2 kit insert. We report here the performance characteristics of the INNO-LiPA HBV DR v2 and compare the results with those obtained using direct sequencing.