Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Tumor antigens and antigen-presenting capacity in breast cancer.

AIMS: Cancer cells frequently express antigens capable of being recognized by the host immune system; however, any resultant immune response is often ineffective. This may be related in part to tumor-induced defects in antigen presentation. We screened for dendritic cell infiltration, tumor MHC II expression and associated lymphocytic reaction in the context of three established breast tumor antigens. METHODS: Forty primary breast tumors were evaluated by immunohistochemical techniques for expression of her2/neu, p53, and MUC1 and MHC class II molecules. Twenty-five samples were further analyzed for p53 mutations by PCR-SSCP analysis and DNA sequencing. The phenotype of tumor-infiltrating inflammatory cells was evaluated using the following markers: CD1a, MHC Class II, CD3, CD45, and CD45RO. RESULTS: Tumors with p53 mutations and overexpression, but not her2/neu or MUC1 overexpressing tumors, more frequently harbored marked CD1a+ dendritic cell infiltrates. An overall correlation between CD1a+ cell infiltrates and HLA class II expression on tumor cells (p = 0.0008) was also observed and these tumors had greater CD45RO+ lymphocytic infiltrates. CONCLUSIONS: In breast cancer, p53 mutations may present a more visible signal to the immune system and hence provide a better target for immunotherapy. Infiltrating CD1a positive cells are associated with a more dense tumor lymphocytic infiltrate and tumor cell expression of MHC II molecules.     

Cytokine induction of heat shock protein in human granulosa-luteal cells

The infiltration of leukocytes is a characteristic feature ofluteolysis in humans. Leukocytes are known to generate physiologicalinducers of cell stress such as cytokines which have been implicatedas mediators of functional luteal regression. In cells exposedto stress, a response characterized by an increase in heat shockprotein (HSP) synthesis occurs. Recently, the induction of HSP-70in rat luteal cells has been shown to inhibit luteinizing hormone(LH) and cAMP-sensitive progesterone production, possibly byinterfering with the translocation of cholesterol to the mitochondrialcytochrome P450_SCC. We therefore investigated whether HSP-70is induced in human granulosa-luteal cells and its relationshipto steroidogenesis. [³⁵S]Methionine labelling showed an increasein a 70 kDa protein after heat treatment which was demonstratedto be HSP-70 by Western analysis using monoclonal antibodiesagainst the constitutive and inducible forms of HSP-70. Inductionof HSP-70 in human granulosa-luteal cells was also seen withinterferon (IFN) (10 ng/ml), tumour necrosis factor (TNF)-     

Phase I and pharmacokinetic study of tiazofurin (TCAR,NSC 286193) administered by continuous infusion

Summary Tiazofurin (2--D-ribofuranosylthiazole-4-carboxamide, TCAR) is a synthetic C-nucleoside that demonstrated significant in vivo activity against a variety of animal tumors as well as in vitro activity against human tumor-derived cell lines. Thirteen patients were treated with TCAR administered as a 5-day continuous infusion in this Phase I trial. Seventeen complete cycles were administered in three dose levels ranging from 550 to 1450 mg/M². Dose-limiting toxicities were myelosuppression and neurotoxicity including severe lethargy. Other toxicities including superficial skin peeling, myalgias, and tearing were seen at all doses. One patient had chest pain on day 4 resulting in stopping the drug, however, there was no evidence of cardiac or pericardial disease. Uric acid levels rose within one day in the absence of allopurinol treatment. There were no treatment related deaths. HPLC measurement of drug levels demonstrated steady-state plasma levels during the infusion, and a half-life following the infusion of 7.7 ± 0.6 hours. Minor abnormalities in renal function were associated with dramatic changes in pharmacokinetics and toxicity. No clinical responses were observed in this trial.Abbreviations TCAR Tiazofurin - HPLC High performance liquid chromatography - IMPD Inosine monophosphate dehydrogenase - WBC white blood cell - CPK creatine phosphokinase - C_ss steady-state concentration     

Induction of rat liver microsomal epoxide hydrolase by thiazole and pyrazine: hydrolysis of 2-cyanoethylene oxide

Liver microsomal epoxide hydrolase (mEH) is active in the detoxificationof epoxide-containing carcinogens. The effects of thiazole andpyrazine, constituents of tobacco and tobacco smoke as wellas of a variety of foods, on the expression and regulation ofmEH were examined in rats (200 mg/kg body wt/day, i.p., 1/emdash3 days). Immunoblot analyses using rabbit anti-rat mEH antibodyrevealed a significant increase in mEH levels in hepatic microsomesisolated from either thiazole- or pyrazine-treated animals.Another protein (43 kd) cross-reacting with polyclonal mEH antibodywas found to be increased concomitantly following pyrazine treatment.Northern and slot blot analyses showed substantial increasesin mEH mRNA following either thiazole or pyrazine treatment.The level of mEH mRNA increased 17-fold at 24 h following thiazoletreatment, relative to control. Approximately 20- and 16-foldincreases in mEH mRNA were also observed at 48 and 72 h respectivelyfollowing treatment with pyrazine. The level of polymerase chainreaction (PCR)-amplified mEH DNA derived from poly(A)+ RNA wasclearly elevated following either thiazole or pyrazine treatmentrelative to that from untreated animals. Both sense and antisensestrands of PCR-amplified mEH DNA were cloned into an M13mpl9phage vector in order to examine the nucleotide sequences ofPCR-amplified mEH DNA derived from the poly(A)+ RNA isolatedfrom thiazole- or pyrazine-treated animals. Sequence analysesrevealed that the sequence of PCR-amplified DNA from the inducedmRNA was identical to that published for mEH cDNA. Epoxide hydrolaseactivity toward the hydrolysis of 2-cyanoethylene oxide (CEO),the epoxide metabolite of the rat carcinogen acrylonitrile,was not significant in hepatic microsomes from untreated rats,but was substantially induced by treatment with thiazole orpyrazine. Microsomal hydrolysis activity was heat-sensitiveand potently inhibited by l, l, l-trichloropropene-2, 3-oxide,indicating that mEH was the catalyst. The V_max for the hydrolysisof CEO by hepatic microsomes from thiazole-treated rats (13.4nmol/min/mg protein) was 1.5-fold greater than that with microsomesfrom pyrazine-treated rats, whereas similar K_m values ( 1 mM)were observed for both microsomal preparations. These kineticdata correlate well with the increases in mEH mRNA observedafter administration of thiazole or pyrazine to rats. Theseresults provide evidence that administration of thiazole orpyrazine induces mEH with a large increase in mEH mRNA, andthat the induced mEH catalyzes the hydrolysis of CEO.     

Telomerase and telomere inhibitors in preclinical development

Telomerase is an enzyme required by actively dividing cells to maintain the ends of chromosomes (telomeres). It is present in germline tissue, stem cells and cancer cells, but is repressed in somatic cells. Efforts are underway to exploit this selective expression of telomerase in cancer therapeutics. This review describes the status of telomerase research, which although at present predominantly preclinical, has the potential to enter clinical research.     

Can parturients distinguish between intravenous and epidural fentanyl?

We tested the hypothesis that the sedative, euphoric, and analgesic effects of intravenous fentanyl would distinguish intravenous from epidural administration. One hundred ASA I and II labouring parturients received 100 μg fentanyl either iv or via an epidural catheter in a double-blind, randomized, crossover fashion. Nineteen anaesthetists (8 staff and 11 residents) participated and correctly guessed the route of administration of the fentanyl in 61/66 intravenous doses and in 69/75 epidural doses yielding a sensitivity of 92.4%, a specificity of 92.0%, a positive predictive value of 91.0%, and a negative predictive value of 93.2%. Of the 41 patients that were crossed over, 38 (92.7%) were able to detect a difference between the routes of administration. Most patients experienced prompt, short-lived symptoms with iv fentanyl but no important differences in fetal heart rate pattern or in maternal desaturation were seen between the groups. This study suggests that subjective symptoms will accurately distinguish intravenous from epidural fentanyl administration in labouring parturients (P < 0.001), and should serve as a safe and reliable intravenous test dose for epidural anaesthesia in the obstetric population.