A longitudinal analysis of cytotoxic T lymphocyte precursor frequencies to the hepatitis B virus in chronically infected patients

Individuals with acute hepatitis B virus (HBV) infection characteristically mount a strong, multispecific cytotoxic T lymphocyte (CTL) response that is effective in eradicating virus. In contrast, this response in chronic carriers is usually weak or undetectable. Since it is generally acknowledged that HBV pathogenesis is immune-mediated, the occurrence of episodes of active liver disease in many carriers suggests that these individuals can mount active CTL responses to HBV. To see whether the detection of circulating CTLs is related to these flare episodes, we have determined the CTL precursor (CTLp) frequencies to HLA-A2-restricted viral peptides in seven patients over a 12-24-month period of their disease. Limiting dilution analyses (LDA) were performed longitudinally to five epitopes comprising the viral capsid (HBc), envelope (HBs) and polymerase (pol) proteins. Assays were performed against a mixture of peptides, or against each individual peptide, to measure overall CTL activity and the multispecificity of the responses, respectively. Since two of the patients were treated with recombinant human interleukin-12 (rHuIL-12) at the time, with one individual achieving complete disease remission a year later after being treated with interferon-alpha, we were also able to examine the effects of these cytokines on HBV cytotoxicity. Our results indicate that weak but detectable CTL responses do occur in chronic carriers which are generally associated with disease flares, although CTLps were also seen occasionally during minimal disease activity. The range of specificities varied between individuals and within each individual during the course of the disease. Finally, we also provide evidence that CTL reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration.

Cytotoxic T cell polyepitope vaccines delivered by ISCOMs

CD8 alpha beta cytotoxic T lymphocyte (CTL) polyepitope or polytope vaccines have traditionally been delivered using recombinant vector or DNA based delivery modalities. Here we show the delivery of polytope vaccines in the form of either synthetic polypeptides or recombinant polytope proteins by ImmunoStimulatory COMplexes (ISCOMs (R)). Induction of multiple protective CTL responses by these polytope-ISCOM formulations were comparable to viral vector or DNA based delivery modalities as assessed by IFN gamma ELISpot, chromium release and viral challenge assays. Measurement of CTL responses specific for the different epitopes revealed imunodominance patterns, which were largely independent of the vaccine vector or the order of the epitopes in the polytope. ISCOMs thus emerge as a viable human delivery modality for protein-based polytope vaccines. (C) 2001 Elsevier Science Ltd. All rights reserved.

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 similar to1.5 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.

Interferon-gamma enhances cytotoxic T lymphocyte recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide

Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV-mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL-sensitive E7-expressing cell lines but are not susceptible to CTL-mediated lysis in vitro . E7 transgenic and non-transgenic KC are susceptible to conventional mechanisms of CTL-mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL-4), despite large differences in H-2D(b) expression at the cell surface. Furthermore, exposure of KC to IFN-gamma increased H-2D(b) expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN-gamma. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Noninvasive measurement of cerebral bioimpedance for detection of cerebral edema in the neonatal piglet

The association of sustained cerebral edema with poor neurological outcome following hypoxia-ischaemia in the neonate suggests that measurement of cerebral edema may allow early prediction of outcome in these infants. Direct measurements of cerebral impedance have been widely used in animal studies to monitor cerebral edema, but such invasive measurements are not possible in the human neonate. This study investigated the ability of noninvasive cerebral impedance measurements to detect cerebral edema following hypoxia-ischaemia. One-day-old piglets were anaesthetized, intubated and ventilated. Hypoxia was induced by reducing the inspired oxygen concentration to 4-6% O-2. Noninvasive cerebral bioimpedance was measured using gel electrodes attached to the scalp. Cerebral bioimpedance was also measured directly by insertion of two silver-silver chloride electrodes subdurally. Noninvasive and invasive measurements were made before, during and after hypoxia. Whole body impedance was measured to assess overall fluid movements. Intracranial pressure was measured continuously via a catheter inserted subdurally, as an index of cerebral edema. There was good agreement between noninvasive and invasive measurements of cerebral impedance although externally obtained responses were attenuated. Noninvasive measurements were also well correlated with intracranial pressure. Whole body impedance changes did not account for increases in noninvasively measured cerebral impedance. Results suggest that noninvasive cerebral impedance measurements do reflect intracranial events, and are able to detect cerebral edema following hypoxia-ischaemia in the neonate. (C) 2002 Elsevier Science B.V. All rights reserved.

A new technique for the quantification of peripheral edema with application in both unilateral and bilateral cases

Current noninvasive techniques for the routine and frequent quantification of peripheral lymphedema in patients are total limb volume measurement (by water immersion or by circumferential measurements) and bioelectrical impedance analysis (BIA). However both of these techniques require standardizing the measurement using a contralateral measurement from the unaffected limb, Hence these techniques are essentially restricted to unilateral lymphedema. This paper describes the results from a preliminary study to investigate an alternative approach to the analysis of the data from multiple frequency BIA to produce an index of lymphedema without the need for normalization to another body segment. Twenty patients receiving surgical treatment for breast cancer were monitored prior to surgery and again after diagnosis with unilateral lymphedema. The data recorded were total limb volume, by circumferential measurements; and BIA measurements of both limbs. From these measurements total limb volumes and extracellular fluid volumes were calculated and expressed as ratios of the affected limb to that of the unaffected limb. An index of the ratio of the extracellular fluid volume to the intracellular fluid volume was determined. This ECW/ICW index was calculated for both the affected and unaffected limbs at both measurement times. Results confirmed that the established techniques of total limb volume and extracellular fluid volume normalized to the unaffected contralateral limb were accurate in the detection of lymphedema (p < 10(-6)). Comparison of the ECW/ICW index from the affected limb after diagnosis with that from the pre-surgery measurement revealed a significant (p< 10(-6)) and considerable (75%) increase. The results of this pilot study suggest that by using multiple frequency bioelectrical impedance analysis, an index of the ECW/ICW ratio can be obtained and this index appears to have an equal, or better, sensitivity, than the other techniques in detecting lymphedema. More importantly, this index does not require normalization to another body segment and can be used to detect all types of peripheral edema including both unilateral and bilateral lymphedema.

Four cytotoxic N4-substituted thiosemicarbazones derived from 2-hydroxynaphthalene-1-carboxaldehyde

The X-ray crystal structures are reported of four novel and potentially O,N,S-tridentate donor ligands that demonstrate antitumour activity. These ligands are 1-[(4-methylthiosemicarbazono)methyl]-2-naphthol, C13H13N3OS, (III), 1-[(4-ethylthiosemicarbazono)methyl]-2-naphthol, C14H15N3OS, (IV), 1-[(4-phenylthiosemicarbazono)methyl]-2-naphthol, C18H15N3OS, (V), and 1-[(4,4-dimethylthiosemicarbazono)methyl]-2-naphthol dimethyl sulfoxide solvate, C14H15N3OS.C2H6OS, (VI). These chelators are N4-substituted thiosemicarbazones, each based on the same parent aldehyde, namely 2-zhydroxynaphthalene-1-carboxaldehyde isonicotinoylhydrazone. Conformational variations within this series are discussed in relation to the optimum conformation for metal-ion binding.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Diferenciação histopatológica e imunoistoquímica das alterações epiteliais no nódulo vocal em relação aos pólipos e ao edema de laringe

OBJETIVO: Avaliar, por meio de técnicas histológicas e imunoistoquímicas, o epitélio nos nódulos vocais em relação aos pólipos, ao edema de laringe e às pregas vocais sem lesões macroscópicas. FORMA DE ESTUDO: Clínico retrospectivo. MATERIAL E MÉTODO: Por meio de levantamento de prontuário, foram identificados vinte e seis pacientes com lesões laríngeas inflamatórias (nódulos, pólipos e edema de laringe), que haviam sido submetidos à cirurgia. Pregas vocais sem alterações macroscópicas foram obtidas a partir de autópsia de cadáveres. Para análise do epitélio, foram realizadas colorações pela técnica da hematoxilina-eosina e do ácido periódico de Schiff e técnicas de imunoistoquímica com anticorpos dirigidos contra laminina e colágeno IV. A leitura das lâminas foi realizada por meio de microscopia óptica. RESULTADOS: Houve maior imunoexpressão de laminina e colágeno IV nos nódulos quando comparados aos pólipos (p=0,034 e p=0, 036, respectivamente), assim como quando comparados às pregas vocais sem lesões macroscópicas (p=0,019 e p=0, 021, respectivamente). Nódulos tendem a apresentar maior espessura da membrana basal, quando utilizamos coloração pela técnica do PAS, em relação aos pólipos (p=0,102). O edema de laringe não se diferenciou das demais nas técnicas utilizadas (p> 0,10). CONCLUSÕES: O Nódulo Vocal diferencia-se dos pólipos, nas três técnicas utilizadas para detecção da membrana basal (PAS, laminina, colágeno IV), e das pregas vocais sem lesões macroscópicas em duas das técnicas utilizadas (laminina e colágeno IV). Edema de laringe não se diferencia das demais lesões, nem de pregas vocais sem lesões macroscópicas, quando utilizadas as técnicas anteriormente descritas, para estudo da membrana basal.

Perimetria de hiperacuidade preferencial no estudo do edema macular diabético

Introdução – A diabetes é uma das maiores epidemias do último século. Mais de 250 milhões de pessoas, em todo o mundo, sofrem de diabetes. Das complicações derivadas da diabetes são as principais causas de cegueira, de insuficiência renal e de amputação de membros inferiores, derivando estes, predominantemente, da disfunção vascular. Quando surge perda de pericitos na parede vascular ocorrem uma série de alterações da microcirculação que levam ao aparecimento de microaneurismas e outras alterações vasculares que possibilitam a passagem de componentes sanguíneos para o tecido retiniano adjacente que, em situação de normalidade, não ocorreriam, sendo esta uma das causas do edema macular exsudativo diabético. A perimetria de hiperacuidade preferencial (PHP) é um teste psicofísico que pretende detetar metamorfopsias na Degenerescência Macular ligada à Idade (DMI). Uma vez que o edema macular diabético (EMD) se destaca como uma das principais causas de deficiência visual e baixa visão, pretende-se verificar a eficácia do PHP no estudo do edema macular diabético, respondendo à seguinte questão: “Qual a capacidade do perímetro de hiperacuidade preferencial em detetar metamorfopsias em pacientes com edema macular diabético?“ Metodologia – Estudo quantitativo, do tipo descritivo e correlacional. Selecionou-se uma amostra de 33 pacientes, onde se analisou um total de 60 olhos. Resultados – A sensibilidade do PHP na deteção de metamorfopsias associadas ao EMD na tomografia de coerência ótica (OCT) foi de 70,6%, a especificidade foi de 11,5% e a eficiência global do teste de 45%. Comparando os resultados encontrados no PHP e no OCT, constatou-se a existência de uma correlação inversa fraca (Phi = -0,215). Conclusões – Este novo método de diagnóstico revela-se sensível, contudo pouco específico e eficaz na deteção de metamorfopsias consequentes da existência de EMD. - ABSTRACT - Introduction – Preferential hyperacuity perimeter (PHP) is a new psychophysical test, which principle is based on the detection of metamorphopsia in age-related macular degeneration (AMD). It is intended to verify its effectiveness in the study of diabetic macular edema (DME). When there is loss of pericytes in the vascular wall occur a number of microcirculatory changes that lead to the appearance of microaneurysms and other vascular changes that allow the passage of blood components to the surrounding retinal tissue than in normal situation does not occur, this being one of the causes exudative diabetic macular edema. Methodology – It was performed a quantitative study, using descriptive and correlational analysis. A sample of 33 patients was selected, and 60 eyes were analyzed. Results – The sensitivity of PHP on the detection of metamorphopsia associated to EMD was 70.6%, the specificity was 11.5% and the global efficiency of the test was 45%. It was found a weak negative correlation (Phi= -0.215) between the PHP and optical coherence tomography (OCT). Conclusions – This new method of diagnosis was sensitive, but not very specific and effective on the detection of metamorphopsia, due to the DME.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

A new natural spiro heterocyclic compound and the cytotoxic activity of the secondary metabolites from Juniperus brevifolia leaves

Copyright © 2010 Elsevier B.V. All rights reserved.