Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alphabeta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes. Here we describe a DNA plasmid encoding a polyepitope or "polytope" protein, which contained multiple contiguous minimal murine CTL epitopes. Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models. CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help. The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.     

Field cancerization: why late "recurrent" ovarian cancer is not recurrent

OBJECTIVE: Late "recurrence" of ovarian cancer may result from either regrowth of dormant tumor cells or from development of a new cancer caused by the phenomenon of field cancerization. Clinically, some recurrent ovarian cancers show the same therapeutic sensitivities to chemotherapy and surgery as did the primary disease, whereas others are refractory to all therapy. We hypothesize that recurrent ovarian cancers are distinguishable on the basis of a molecular genetic fingerprint and that some are actually new primary cancers of the peritoneum rather than recurrent ovarian cancer. STUDY DESIGN: We constructed molecular genetic fingerprints of 13 paired primary and late recurrent ovarian cancers to study their clonal relationships. The tumor pairs were analyzed for p53 mutations and allelotypes, patterns of X-chromosome inactivation, loss of heterozygosity, and microsatellite instability at 12 different loci on 6 different chromosomes. Techniques used included single-strand conformational polymorphism mutation screening and polymerase chain reaction-based sequence analysis of the p53 locus, restriction digestion of the androgen receptor locus to determine X-chromosome inactivation, and polyacrylamide gel electrophoresis of highly polymorphic dinucleotide, trinucleotide, and tetranucleotide repeats. RESULTS: The average age at initial diagnosis for this cohort was 54.7 years (range 45.3 to 65.5). Mean interval to recurrence was 42.7 months (range 28 to 62). Molecular fingerprints were characterized for 4 to 8 informative loci per tumor pair. The fingerprints of 10 (77%) differed significantly, strongly suggesting that a second primary cancer had developed. The remaining 3 tumor pairs demonstrated identical allelotypes consistent with regrowth of dormant tumor cells. CONCLUSION: Our results are consistent with the "field cancerization" hypothesis of ovarian carcinogenesis but could also be explained by a polyclonal tumor origin, which contrasts with the currently accepted monoclonal theory of ovarian carcinogenesis. Late development of a new primary cancer may herald the proband as a member of a familial cancer phenotype. These studies provide a molecular genetic rationale that both explains and prognosticates the clinical course of recurrent ovarian cancer.     

Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression

Extracellular single-cell recordings were made from the cerebellar thalamus (89 neurones) and the VPLc (53 neurones) of three conscious monkeys. The animals were trained to perform wrist movement paradigms including: (a) visually triggered skilled, voluntary movements; (b) 100-ms duration torque pulse perturbations applied during a hold period (termed Pa perturbations); (c) 100-ms perturbations that commenced 100 ms after the visual trigger but during preparation before a skilled, voluntary movement (termed Pb perturbations); and (d) 100-ms perturbations during the skilled, voluntary movement (termed Pm perturbations). These Pb and Pm perturbations were used to identify central and peripheral influences on patterns of neuronal discharge in the ventrolateral thalamus. There was no systematic difference between the responses to Pb and Pm perturbations of neurones in the cerebellar thalamus and those in VPLc. The responses of VPLc and cerebellar thalamic neurones to Pa perturbations were considered to represent transduction of peripheral afferent input, and these responses were compared with the responses to the other types of perturbations. Up to 40% of neurones in cerebellar thalamus and VPLc responded to Pb and Pm perturbations in a similar pattern to that which followed Pa perturbations, and therefore most likely represented faithful transduction of peripheral input. However, the response of over half the neurones in VPLc and cerebellar thalamus to Pb or Pm perturbations differed from Pa perturbations in a manner suggesting that central influences had gated the peripheral input. The short-latency response in cerebellar thalamus which was modified by central influences is appropriately timed to contribute to the "intended" response to perturbations of motor cortical neurones.     

Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: results in 183 patients

OBJECTIVES: Our aim was to identify prognostic variables for long-term postoperative survival in trimodality management of malignant pleural mesothelioma. METHODS: From 1980 to 1997, 183 patients underwent extrapleural pneumonectomy followed by adjuvant chemotherapy and radiotherapy. RESULTS: Forty-three women and 140 men (age range 31-76 years) had a median follow-up of 13 months. The perioperative mortality rate was 3.8% (7 deaths) and the morbidity, 50%. Survival in the 176 remaining patients was 38% at 2 years and 15% at 5 years (median 19 months). Univariate analysis identified 3 prognostic variables associated with improved survival: epithelial cell type (52% 2-year survival, 21% 5-year survival, 26-month median survival; P =.0001), negative resection margins (44% at 2 years, 25% at 5 years, median 23 months; P =.02), and extrapleural nodes without metastases (42% at 2 years, 17% at 5 years, median 21 months; P =.004). Using the Cox proportional hazards, the relative risk of death was calculated for nonepithelial cell type (OR 3.0, CI 2.0-4.5; P <.0001), positive resection margins (OR 1.7, CI 1.2-2.6; P =.0082), and metastatic extrapleural nodes (OR 2.0, CI 1.3-3.2; P =.0026). Thirty-one patients with 3 positive variables had the best survival (68% 2-year survival, 46% 5-year survival, median 51 months; P =.013). A previously published staging system using these variables stratified survival (P <.05). CONCLUSIONS: (1) Multimodality therapy including extrapleural pneumonectomy is feasible in selected patients with malignant pleural mesotheliomas, (2) pre-resectional evaluation of extrapleural nodes may select patients for radical therapy, (3) microscopic resection margins affect long-term survival, highlighting the need for further investigation of locoregional control, and (4) patients with epithelial, margin-negative, extrapleural node-negative resection had extended survival.     

Increased human herpesvirus 8 seroprevalence in young homosexual men who have multiple sex contacts with different partners

The objective of this study was to evaluate the behavioral risks that are associated with human herpesvirus 8 (HHV-8) infection in a cohort of young homosexual men. Seventy-nine subjects (ages 22-33 years) who completed a questionnaire about their sexual and drug use behavior over the preceding year were recruited from the San Francisco Young Men's Health Study. Plasma samples were tested for anti-HHV-8 antibodies using an indirect IFA. Thirty-eight subjects (48.1%) were infected with HHV-8. HHV-8 infection was significantly linked to an increasing number of male sex partners (P=.025, Mantel-Haenszel chi2 test for trend), suggesting a strong association between HHV-8 infection and multiple homosexual contacts.     

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides

Whether P/Q-type voltage-gated calcium channel (VGCC) antibodies are present in the serum of patients with paraneoplastic syndromes other than the Lambert-Eaton myasthenic syndrome (LEMS) and tumors other than small-cell lung cancer (SCLC) is controversial. Using a commercially available radioimmunoprecipitation assay kit, we examined the sera of 93 patients with paraneoplastic syndromes of the central nervous system (CNS), including 27 patients with paraneoplastic cerebellar degeneration (PCD) associated with tumors other than SCLC and 66 SCLC patients with paraneoplastic encephalomyelitis and sensory neuronopathy (PEM/SN). All PCD sera from patients with tumors other than SCLC were negative for P/Q-type VGCC antibodies. Eight of 66 (12%) SCLC patients with PEM/SN had P/Q-type VGCC antibodies; 4 had LEMS and the other 4 had no symptoms of LEMS or they were overlooked and, therefore, not examined electrophysiologically. In patients with paraneoplastic syndromes of the CNS, the detection of P/Q-type VGCC antibodies supports the diagnosis of LEMS; in our series, only 6% of patients with SCLC and PEM/SN may have had a false positive antibody result, or undiagnosed LEMS.     

Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.     

Comparison of screening methods in the detection of bladder cancer

PURPOSE: We prospectively evaluate and compare the sensitivity and specificity of urine cytology, BTA stat, NMP22, fibrin/fibrinogen degradation products (FDP), telomerase, chemiluminescent hemoglobin and hemoglobin dipstick to detect bladder cancer. MATERIALS AND METHODS: Single voided specimens were obtained from 57 patients with bladder cancer, and 139 without evidence of bladder malignancy on cystoscopy or a negative biopsy of indeterminate lesions. A cytology report was available for 125 patients and interpreted independently. BTA stat, NMP22 and FDP were analyzed according to manufacturer specifications. The telomerase assay was performed on cells collected from urine by centrifugation in preparation for polymerase chain reaction based amplification using the telomeric repeat amplification protocol assay. The chemiluminescent screening assay for hemoglobin in urine uses the pseudoperoxidase activity of hemoglobin on hydrogen peroxide and subsequent oxidation of 7-dimethylaminonaphthalene-1,2-dicarbonic acid hydrazide to generate chemiluminescence emission. Hemoglobin dipstick was interpreted as positive if the hemoglobin content in the urine was trace or greater. RESULTS: Overall sensitivity with urine cytology, BTA stat, NMP22, FDP, telomerase, chemiluminescent hemoglobin and the hemoglobin dipstick was 44, 74, 53, 52, 70, 67 and 47%, respectively. Specificity with cytology, telomerase and FDP was high (95, 99 and 91%, respectively) but BTA stat, NMP22 (optimized), chemiluminescent hemoglobin (optimized) and the hemoglobin dipstick demonstrated lower specificity of 73, 60, 63 and 84%, respectively. Stepwise logistic regression analysis revealed that for all tumors, and within each tumor grade and stage telomerase had the strongest association with bladder cancer among all tests (69% overall concordance). Telomerase was also positive in 91% of the patients (10 of 11) with carcinoma in situ. CONCLUSIONS: Urinary telomerase had the highest combination of sensitivity and specificity (70 and 99%, respectively) for bladder cancer screening in these patients. It was the strongest predictor with superior accuracy in patients with grade 1 and noninvasive tumors (pTa), and extremely useful in patients with carcinoma in situ. Telomerase appears to be promising and outperformed cytology, BTA stat, NMP22, FDP, chemiluminescent hemoglobin and hemoglobin dipstick in the prediction of bladder cancer.     

Direct alloreactivity by human cytotoxic T lymphocytes can Be inhibited by altered peptide ligand antagonism

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.