Use of amyloid PET/CT with 18F-Florbetaben in the management of patients with Alzheimer's disease.

INTRODUCTION AND AIM: Amyloid PET/CT is an "in vivo" imaging that may radically change management of Alzheimer's disease (AD) thanks to its ability to identify AD at the earliest stage. A diagnosis of dementia is currently made in terms of probability and is based on clinical evaluation (neuropsycological tests) as well as on the results of morphological imaging investigations (MRI) that can be supported by biohumoral (CSF analysis), and functional imaging only in the case of uncertain diagnosis of disease. The present study aimed to evaluate the role of amyloid PET/CT in the management of patients with suspicion of AD, through comparison with instrumental and clinical evaluation. METHODS: 38 consecutive patients with suspicion of AD (23 female, 15 male; median age 63 years old, range 46-72), who performed 18F-florbetaben PET/CT, were retrospectively reviewed. All of them performed a previous instrumental evaluation. A subgroup of patients (24/38) were evaluated with Mini Mental State Examination (MMSE). Cohen's K test was used as a measure of agreement between previous instrumental examinations/clinical evaluation and beta-amyloid PET results. RESULTS: Twenty-five/38 (65.8%) amyloid PET/CT scans resulted positive for amyloid deposition. Among the four target regions, precuneus was the most frequently involved. Previous instrumental evaluation was: MRI in 26/38 patients (24/26 positive for atrophy), CT in 9/38 (8/9 positive for atrophy), perfusion SPECT in 12/38 (8/12 areas of hypo-perfusion), 18F-FDG PET/CT in 2/38 (1/2 hypometabolism in frontal cortex). The agreement between previous instrumental examinations and beta-amyloid PET results was low (K= 0.084). In the subgroup of 24/38 patients, MMSE was scored positive (MMSE<24) in 14/24 (58.4%) and negative (MMSE>24) in 10/24 (41.6%). The agreement between clinical evaluation (MMSE) and beta-amyloid PET results was fair (K= 0.217). CONCLUSION: The low agreement between amyloid PET/CT and previous clinical and instrumental assessments that we found in our study suggests that the amyloid PET/CT provides additional and early information. To perform an early and differential diagnosis of AD could have a great impact on the patient's management and cost of care in order to perform the correct therapeutic interventions and to allow family members to manage adequately the patient's demanding care.

Impact of pre-treatment variables on the completion of 223radium-dichloride therapy in mCRPC patients with bone metastases.

INTRODUCTION: Radium-223 dichloride (223Ra) is an alpha-particle-emitter radiopharmaceutical, approved for metastatic castration-resistant prostate cancer (mCRPC) patients with symptomatic bone metastases and no visceral involvement. Its administration is based on a schedule of intravenous injection (55kBq/kg) every four weeks for up to six cycles. Because the biological effectiveness of 223Ra-therapy is dose-dependent, the main goal is to complete the entire treatment to achieve a better patient outcome. This study aims to identify potential pre-treatment variables that could impact on 223Ra-treatment completion and then be used to improve the clinical and supportive management of mCRPC patients. MATERIALS AND METHODS: 30 consecutive mCRPC patients (mean age 77 years old), who were admitted for Ra223-therapy at our Department from February 2016 to October 2018, were enrolled for the analysis. The population was grouped as patients who completed 223Ra-therapy (group Ra223-C) and patients who do not (group 223Ra-U). For each group, we analyzed the effects of potential pre-treatment variables (age, Gleason Score, tumor burden, "Time From Diagnosis To 223Ra therapy", type and number of previous treatments, hemoglobin level, Alkaline Phosphatase, Prostate Specific Antigen and pain) on the Ra223-therapy completion. Statistical analysis was performed to evaluate the association between the completion of 223Ra therapy and the variables examined. RESULTS: 16/30 (53%) patients were 223Ra-C, conversely 14/30 (47%) patients were 223Ra-U because of an early interrupted treatment. A statistically significant association was found only with tumor burden: 68.7% of patients who completed 223-therapy had less than 20 bone metastases (χ2=4.821, p=0.028). CONCLUSION: Our preliminary analysis demonstrates that the high tumor burden represents the most important pre-treatment factor that could affect treatment completion and that needs to be considered before starting 223Ra-therapy to achieve a better outcome in mCRPC patients.

The paleoradiology importance in the study of relics: the unique densitometric analysis of a bone relic of Saint Nicholas.

INTRODUCTION: DXA have greatly contributed to the development of paleoradiology, a branch of diagnostic imaging that allows to obtain information about human remains in contexts of archaeological and/or forensic interest. In this manuscript we report the unique experience of DXA performed on the relic of a Saint; in particular we analyzed a skeletal fragment of St. Nicholas, kept in the Basilica of Bari (Italy) since 1087. MATERIALS AND METHODS: The bone to be examined consisted of the posterior arch of the ninth left rib that was 12cm long, 1.2cm maximum width and 1.7cm thick at the body. The data acquired from the densitometric study were performed using the anthropometric measures reported in historical records of St. Nicholas' life: sex (male), age (75 years), weight (70kg), height (167cm), and ethnicity (Caucasian). In addition to the examination of the relic, a comparison assessment was made with the rib of a healthy 60 years old man (height of 170cm, without known skeletal pathologies). This sample had a length of 19cm, maximum width at the head 1cm, and 0.7cm thick at the body. The analysis of bone fragments is different from the analysis of bones in the context of the human body (where soft tissues are placed around the skeleton); for this reason, one of the most critical issues was to create a support that would allow the analysis of bone fragments. We simulated conditions similar to those occurring in patients: a density scale was established, using a specific plexiglass phantom on which the bone fragments to be examined were placed. From the analysis it was calculated the parameter bone mineral density (BMD), express in g/cm2, that indicates the relation between mass of bone mineral content and area of examined bone segment. BMD data was compared to a range normalized by age, sex and ethnicity (BMD-N). RESULTS: The results of the scannnig of St. Nicholas' rib showed a BMD of 0.97g/cm2 with a BMD-N between 0.77 and 1.08g/cm2. Simultaneous measurements of the relic compared with a reference rib showed highlighted BMD of 0.84g/cm2 for the relic and 0.50g/cm2 for the reference rib. The St. Nicholas data are 168% higher than reference bone. All our measurements of the relic indicated a high bone mineral density, most likely due to the presence of a high concentration of calcium salts. A relatively higher mineral density of the relic was seen compared to the healthy subject's rib. From the history of St Nicholas' life, we know of the long imprisonment at the age of 51 in damp and unhealthy environment. The results of this study suggest that a good bone mineral density was maintained by the Saint even in old age. An additional element that can influence bone mineral density is diet, certainly different during the time of St. Nicholas. The good bone densitometry indicates that the Saint maintained a proper diet, with a generally fair state of health. CONCLUSION: For this first DXA analysis of the rib relic of Saint Nicholas was necessary a long and complex experimental work to modify standard technique procedure to particular and unusual sample and Create specific supports and complementary instruments. Perform DXA analysis on relics permit to obtain additional information to living conditions, economical situation, behaviours, diet, diseaes, conservations conditions of remains, change of life style in different age. Our experimental work, the first of its kind, creates the way to analyze precious relics that often include only few bone fragments and data obtained by our work can be useful for a better management and movement of fragile relics. We ourselves are working on a new challenge for the analysis of bone finds from shipwrecks found at the bottom of the sea.

Cetuximab continuation after first progression in metastatic colorectal cancer (CAPRI-GOIM): a randomized phase II trial of FOLFOX plus cetuximab versus FOLFOX.

BACKGROUND: Cetuximab plus chemotherapy is a first-line treatment option in metastatic KRAS and NRAS wild-type colorectal cancer (CRC) patients. No data are currently available on continuing anti-epidermal growth factor receptor (EGFR) therapy beyond progression. PATIENTS AND METHODS: We did this open-label, 1:1 randomized phase II trial at 25 hospitals in Italy to evaluate the efficacy of cetuximab plus 5-fluorouracil, folinic acid and oxaliplatin (FOLFOX) as second-line treatment of KRAS exon 2 wild-type metastatic CRC patients treated in first line with 5-fluorouracil, folinic acid and irinotecan (FOLFIRI) plus cetuximab. Patients received FOLFOX plus cetuximab (arm A) or FOLFOX (arm B). Primary end point was progression-free survival (PFS). Tumour tissues were assessed by next-generation sequencing (NGS). This report is the final analysis. RESULTS: Between 1 February 2010 and 28 September 2014, 153 patients were randomized (74 in arm A and 79 in arm B). Median PFS was 6.4 [95% confidence interval (CI) 4.7-8.0] versus 4.5 months (95% CI 3.3-5.7); [hazard ratio (HR), 0.81; 95% CI 0.58-1.12; P = 0.19], respectively. NGS was performed in 117/153 (76.5%) cases; 66/117 patients (34 in arm A and 32 in arm B) had KRAS, NRAS, BRAF and PIK3CA wild-type tumours. For these patients, PFS was longer in the FOLFOX plus cetuximab arm [median 6.9 (95% CI 5.5-8.2) versus 5.3 months (95% CI 3.7-6.9); HR, 0.56 (95% CI 0.33-0.94); P = 0.025]. There was a trend in better overall survival: median 23.7 [(95% CI 19.4-28.0) versus 19.8 months (95% CI 14.9-24.7); HR, 0.57 (95% CI 0.32-1.02); P = 0.056]. CONCLUSIONS: Continuing cetuximab treatment in combination with chemotherapy is of potential therapeutic efficacy in molecularly selected patients and should be validated in randomized phase III trials.

Regorafenib monotherapy as second-line treatment of patients with RAS-mutant advanced colorectal cancer (STREAM): an academic, multicenter, single-arm, two-stage, phase II study.

BACKGROUND: Maintaining angiogenesis inhibition and switching the chemotherapy backbone represent the current second-line therapy in patients with RAS-mutant metastatic colorectal cancer (mCRC). Regorafenib, an oral multikinase inhibitor, prolonged overall survival (OS) in the chemorefractory setting. MATERIALS AND METHODS: STREAM was an academic, multicenter, single-arm phase II trial, evaluating the activity of regorafenib in RAS-mutant mCRC, in terms of the rate of patients who were progression-free after 6 months from study entry (6mo-PF). Patients were pretreated with fluoropyrimidine, oxaliplatin, and bevacizumab. According to Simon's two-stage design, ≥18 patients 6mo-PF were needed in the overall population (N = 46). Secondary endpoints were safety, objective response rate (ORR), progression-free survival (PFS), and OS. Early metabolic response by [18F]2-fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography ([18F]-FDG PET/CT) scan was an exploratory endpoint. EudraCT Number: 2015-001105-13. RESULTS: The number of patients 6mo-PF was 8/22 at the first stage and 14/46 in the overall population. The ORR was 10.9%, disease control rate was 54.6%, median (m)PFS was 3.6 months [95% confidence interval (CI) 1.9-6.7 months], mOS was 18.9 months (95% CI 10.3-35.3 months), and mPFS2 (from study entry to subsequent-line progression) was 13.3 months (95% CI 8.4-19.7 months). Long benefiter patients (>6mo-PF) significantly more often had a single metastatic site and lung-limited disease. No unexpected toxicity was reported. Grade ≥3 events occurred in 39.1% of patients, with hand-foot syndrome (13%), fatigue, and hyperbilirubinemia (6.5%) occurring mostly. Baseline metabolic assessment was associated with OS in the multivariate analysis, while early metabolic response was not associated with clinical outcomes. CONCLUSIONS: The study did not meet its primary endpoint. However, regorafenib was well tolerated and did not preclude subsequent treatments. Patients with good prognostic features (single metastatic site and lung-limited disease) reported clinical benefit with regorafenib. The exploratory metabolic analysis suggests that baseline [18F]-FDG PET/CT might be useful to select patients with a favorable outcome. A chemotherapy-free interval with regorafenib was associated with durable disease control in a selected group of patients with favorable clinical characteristics.

Prevalence and clinical impact of incidental findings on the unenhanced CT images of PET/CT scan in patients with multiple myeloma: the value of radiological reporting in the multimodal hybrid imaging.

OBJECTIVE: In the hybrid Positron Emission Tomography/Computed Tomography (PET/CT) method, the functional evaluation is integrated with the morphological information provided by co-registered CT, still performed for attenuation correction and lesion localization. However, co-registered CT images could provide additional diagnostic information that PET alone could underestimate. To optimize the diagnostic potential of this hybrid examination, we evaluated the prevalence and the clinical significance of incidental findings detected on co-registered CT images in a cohort of multiple myeloma (MM) patients. PATIENTS AND METHODS: We evaluated 112 MM patients (mean age 65.8 y), who underwent [18F]FDG-PET/CT during their regular workup. All co-registered CT images were retrospectively reviewed by two expert radiologists and each non-myelomatous incidental finding (nM-IF) was collected and clinically graded according to a nM-IF Reporting and Data System (nM-RADS). In addition, nM-IFs were classified according to anatomic localization (skull, lung, mediastinum, abdomen, breast, gastrointestinal, genitourinary and cardiovascular system and muscle/soft tissue). RESULTS: 163 nM-IFs were detected in 94/112 patients (83.9%) (mean value: 1.5 IFs per patient). The most interested anatomic districts were the lung (n=33; 20.2%), genitourinary (n=33; 20.2%) and gastrointestinal (n=30; 18.4%) systems. Focusing on the clinically significant findings (nM3+nM4), 92/163 (56.4%) IFs could have been required further investigations, of which 38/163 (23.3%) were potentially important and detected in 33/112 (29.5%) patients. CONCLUSIONS: The high percentage of potentially clinically significant IFs detected in MM patients emphasizes that co-registered CT images hold precious information often missed. Giving more relevance to co-registered CT with tailored acquisition and reconstruction protocols and dedicated reporting could optimize the potentiality of this multimodality imaging method with impact on clinical management.

The clinical impact of a cardiologic follow-up in breast cancer survivors: an observational study.

Anthracycline-containing chemotherapy (A-CHT) can induce late cardiotoxicity adding a considerable burden to cardiovascular risk. Irradiation of left breast cancer has also been associated to an increased risk of cardiovascular disease. The aim of this observational study is to prove the usefulness of an accurate cardiovascular evaluation in left breast cancer survivors treated with radiotherapy (RT) and A-CHT. Patients with left breast cancer, on follow-up after treatment with A-CHT plus RT in an adjuvant setting, were eligible for this observational study. Patients underwent cardiovascular assessment with myocardial perfusion imaging. Thirty patients were enrolled in the study: mean age at diagnosis 55.8 years; stage: I/III; Er and/or pgR status: positive in 24/30 pts; 3 patients in pre-menopausal status. Twenty-two patients (73.3 percent) had normal perfusion imaging, 1 patient (3.3 percent) had a fixed myocardial perfusion defect, 7 patients (23.3 percent) had reversible myocardial perfusion defects; 1 patient (3 percent) with normal perfusion scan showed depressed rest and stress LVEF. Only 1 patient had a large defect and underwent coronary angiography and percutaneous coronary intervention. Five patients with small defect showed normal coronary arteries at Multislice Computed Tomography. Cardiovascular followup may reveal signs of A-CHT or RT-induced cardiotoxicity. A stress test combined with MPI- and GATED-derived data of ventricular systolic performance after stress can give information on the coronary reserve and the contractile reserve and allow early appropriate treatment.

Cell surface changes associated with cellular immune reactions in Drosophila.

In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts.

Safety of new DAAs for chronic HCV infection in a real life experience: role of a surveillance network based on clinician and hospital pharmacist.

BACKGROUND: Direct Antiviral Agents (DAAs) for HCV therapy represents a step ahead in the cure of chronic hepatitis C. Notwithstanding the promising results in several clinical trials, few data are available on adverse effects in real life settings. METHODS: We have evaluated 170 patients with persistent infection and on those eligible to treatment we have followed up them through a network managed by clinician and hospital pharmacist. RESULTS: According to our data we have found that 41% (32 out of 78) of enrolled patients experienced adverse reactions, of these 40% were in those under 65 years while 60% was in patients older than 65 years, SVR was achieved in 88% of the patients (including drop-out). We had 4 drop-out treatment due to major adverse reaction (heart and lung related). CONCLUSION: Even if new antiviral drugs seem to be promising, according to SVR, they require careful follow-up, possibly managed by clinician and hospital pharmacist, to avoid unrecognized side effects which may affect adherence and the real impact of these drugs on chronically infected subjects.

Therapeutic Strategies in HCC: Radiation Modalities.

Patients with hepatocellular carcinoma (HCC) comply with an advanced disease and are not eligible for radical therapy. In this distressed scenario new treatment options hold great promise; among them transarterial chemoembolization (TACE) and transarterial metabolic radiotherapy (TAMR) have shown efficacy in terms of both tumor shrinking and survival. External radiation therapy (RTx) by using novel three-dimensional conformal radiotherapy has also been used for HCC patients with encouraging results while its role had been limited in the past for the low tolerance of surrounding healthy liver. The rationale of TAMR derives from the idea of delivering exceptional radiation dose locally to the tumor, with cell killing intent, while preserving normal liver from undue exposition and minimizing systemic irradiation. Since the therapeutic efficacy of TACE is being continuously disputed, the TAMR with (131)I Lipiodol or (90)Y microspheres has gained consideration providing adequate therapeutic responses regardless of few toxicities. The implementation of novel radioisotopes and technological innovations in the field of RTx constitutes an intriguing field of research with important translational aspects. Moreover, the combination of different therapeutic approaches including chemotherapy offers captivating perspectives. We present the role of the radiation-based therapies in hepatocellular carcinoma patients who are not entitled for radical treatment.

The effects of glutathione and ascorbic acid on the oxidations of 6-hydroxydopa and 6-hydroxydopamine.

The interactions of ascorbic acid (AA) and reduced glutathione (GSH) in the oxidations of the catecholaminergic neurotoxins 6-hydroxydopa (TOPA) and 6-hydroxydopamine (6-OHDA) were investigated by both high performance liquid chromatography with electrochemical detection (HPLC-ED) and spectrometric methods. These comparative studies showed TOPA and 6-OHDA to be extremely unstable, with 100% of the trihydroxyphenyls oxidized within 0.5 min at physiological pH in potassium phosphate buffer. Neither AA nor GSH was found capable of significantly impeding the oxidations of these trihydroxyphenyls, or of regenerating these substances by reducing back their oxidation products, even though such a redox exchange mechanism was demonstrated for AA and the dihydroxyphenyl dopamine. Although ineffective in keeping TOPA and 6-OHDA as reduced molecules, GSH may nevertheless influence the neurotoxicity of trihydroxyphenyls by interacting with their oxidation products forming glutathionyl conjugates, thereby switching the reaction pathway away from potentially toxic eumelanin precursors and toward the production of pheomelanin. Electrochemical analyses established the formation of two oxidation products derived from each trihydroxyphenyl, one detected at -100 mV and the other at +700 mV. AA had no effect on either oxidation product, whereas GSH significantly decreased the levels of both oxidation products. The component detected at +700 mV is the cyclized, reduced leukochrome. The identity of the component detected at -100 mV was not established, but it is considered to be either the p-quinone or the cyclized, oxidized aminochrome.

Comparative studies of enhanced iron-mediated production of hydroxyl radical by glutathione, cysteine, ascorbic acid, and selected catechols.

A sensitive electrochemical detection system was employed together with a specific salicylate hydroxylation assay to comparatively assess the effects of various substances on the iron-mediated generation of the hydroxyl radical (.OH). Hydroxyl radical production was found to be enhanced significantly by reduced glutathione, cysteine, ascorbic acid, and selected catechols, but not by mannitol, melatonin or tyramine. The data showed that over the range of concentrations examined, the augmented effects were linearly proportional to the amount of added reductant for a given amount of iron in the system. The pro-oxidant activity of thiols and ascorbate reduced and recycled iron providing both hydrogen peroxide (H2O2) and catalytic ferrous ions for augmented .OH production by the Fenton reaction. The enhanced production of .OH by catechols resulted from their oxidation either by molecular oxygen or ferric ions, with the accompanying formation of semiquinones, superoxide anion and H2O2. These data caution against therapeutic applications of thiols and ascorbate for ameliorating oxy-radical-induced tissue damage in environments where free redox-active metal ions may be present to function both as foci for site-specific peroxidative activity, and as catalysts to promote the pro-oxidant properties of certain endogenous reductants, thereby elevating rather than diminishing .OH levels.

Hydroxyl radical formation via iron-mediated Fenton chemistry is inhibited by methylated catechols.

The differing effects of O-methylated catecholamines and their dihydroxyphenyl precursors on the production of *OH were quantified using a previously established specific salicylate hydroxylation assay in conjunction with a sensitive electrochemical detection system. The production of *OH by the Fenton reaction was diminished significantly by O-methylated catecholamines (O-methyldopa, O-methyldopamine, O-methyltyrosine, and N-acetyl-O-methyldopamine), whereas radical production was augmented by dihydroxyphenyls (DOPA, dopamine, and N-acetyldopamine), including those with methylated side chains (N-methyldopamine and alpha-methyldopa). Monohydroxyphenyls such as octopamine, tyramine, tyrosine, and alpha-methyltyrosine had little or no effect on radical production. These data show that a methyl group positioned on the side chain of a catecholamine does not alter its pro-oxidant behavior, while a methyl group positioned on the aromatic ring renders the catecholamine sterically or kinetically unfavorable for coordination with transition metals, thus preventing the promotion of Fenton chemistry. These results highlight the importance of O-methylation in forming catechols that are less reactive than their dihydroxyphenyl precursors. Thus, factors regulating the methylation of brain catecholamines may play a crucial role in mediating neuronal integrity during aging and in the pathogenesis of certain neurodegenerative disorders. Competitive side-chain methylation reactions may sustain or perpetuate some dihydroxyphenyls, creating an oxidatively less favorable environment for cells than would result from compounds formed by O-methylation.

Hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide.

The highly reactive and cytotoxic hydroxyl radical (OH) was found by electrochemical detection to be produced in reactions involving hydrogen peroxide (H2O2) and the nitric oxide (NO) donor diethylamine- NO complex. Using aromatic hydroxylation of salicylate as a specific indicator of OH, three salicylate hydroxylation products were identified; catechol, 2,3- and 2,5-dihydroxybenzoic acid. Four additional compounds were detected but not identified. The interactions of H2O2 and NO represent a biologically feasible reaction mechanism that can account for OH-induced damage in cellular environments where transition metal ions are unavailable for participation in the superoxide-mediated Fenton reaction. The ability of the NO/H2O2 complex to generate OH independently of iron or other transition metals provides a new focus for studies concerned with the origin of tissue-specific damage caused by oxygen-derived species.

Contrasting effects of catecholic and O-methylated tetrahydroisoquinolines on hydroxyl radical production.

Tetrahydroisoquinolines (TIQs) are intraneuronal, catecholamine-derived alkaloids that have been implicated in the etiology of Parkinson's disease and in alcohol related disorders. The in vitro production of the cytotoxic hydroxyl radical (*OH) was recorded during the autoxidation of salsolinol (SAL) and salsolinol-1-carboxylic acid (SAL-1C), but not when these two catecholic TIQs were oxidized by tyrosinase. Significantly higher levels of the radical were produced when these catecholic TIQs were incubated with *OH generating complexes, or with chelated iron. In contrast, mono-O-methylated TIQs such as salsoline (SLN) and salsoline-1-carboxylic acid (SLN-1C) did not generate *OH during autoxidation or when incubated with chelated iron or tyrosinase. Radical production by *OH-generating complexes was reduced in the presence of O-methylated TIQs. The neurotoxicity of TIQs may result from their propensity to autoxidize and generate reactive quinoids and ensuing oxygen radicals. The functional significance of the replacement of a hydroxyl group attached to C-7 of SAL or SAL-1C with a methoxyl group remains to be determined. This single structural modification may prevent mono-O-methylated TIQs from participating in catalytic redox cycling reactions that would otherwise augment *OH production. If true, then O-methylation and other cellular mechanisms that circumvent the autoxidation of catecholamine-derived TIQs may reduce the likelihood of these substances forming cytotoxic quinoids and influencing endogenous *OH-generating reactions.

5-S-cysteinyldopa, a diffusible product of melanocyte activity, is an efficient inhibitor of hydroxylation/oxidation reactions induced by the Fenton system.

Interest in 5-S-cysteinyldopa (5-S-CD), a major excretion product of normal and malignant melanocytes, has traditionally concentrated on its significance as a biosynthetic precursor of pheomelanins, the characteristic pigments of red hair, and as a specific biochemical marker for monitoring melanoma progression. The present study shows that 5-S-CD is a potent inhibitor of hydroxylation/oxidation reactions mediated by hydrogen peroxide and the Fe2+/EDTA complex under both aerobic and anaerobic conditions. The inhibitory effect of 5-S-CD, as determined by the deoxyribose and salicylic acid assays in phosphate buffer (pH 7.4), is much stronger than that of dopa, acetylsalicylic acid and mannitol, increases with increasing ligand-to-metal ratio, and is inversely proportional to the concentration of EDTA present in the Fenton system. Spectrophotometric evidence and competition experiments indicate that 5-S-CD forms a chelate complex with ferric ions (lambda max = 500 nm at pH 7.4), which may account for both an altered production of hydroxyl radicals by the Fenton reagent and a site-specific localization of oxidative damage on the chelate complex itself.

Genetics of anti-parasite resistance in invertebrates.

This review summarizes and compares available data on genetic and molecular aspects of resistance in four well-described invertebrate host-parasite systems: snail-schistosome, mosquito-malaria, mosquito-filarial worm, and Drosophila-wasp associations. It underlies that the major components of the immune reaction, such as hemocyte proliferation and/or activation, and production of cytotoxic radicals are common to invertebrate hosts. Identifying genes responsible for naturally occurring resistance will then be helpful to understand the mechanisms of invertebrate immune defenses and to determine how virulence factors are used by parasites to overcome host resistance. Based on these four well-studied models, invertebrate resistance appears as generally determined by one major locus or a few loci, displaying at least partial dominance. Interestingly, specificity of resistance is highly variable and would involve processes other than simple recognition mechanisms. Finally, resistance was shown to be generally costly but is nevertheless observed at high frequencies in many natural populations, suggesting a high potential for host parasite coevolution.

Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity.

Insects transmit the causative agents for such debilitating diseases as malaria, lymphatic filariases, sleeping sickness, Chagas' disease, leishmaniasis, river blindness, Dengue, and yellow fever. The persistence of these diseases provides testimony to the genetic capacity of parasites to evolve strategies that ensure their successful development in two genetically diverse host species: insects and mammals. Current efforts to address the problems posed by insect-borne diseases benefit from a growing understanding of insect and mammalian immunity. Of considerable interest are recent genomic investigations that show several similarities in the innate immune effector responses and associated regulatory mechanisms manifested by insects and mammals. One notable exception, however, is the nearly universal presence of a brown-black pigment accompanying cellular innate immunity in insects. This response, which is unique to arthropods and certain other invertebrates, has focused attention on the elements involved in pigment synthesis as causing or contributing to the death of the parasite, and has even prompted speculation that the enzyme cascade mediating melanogenesis constitutes an ill-defined recognition mechanism. Experimental evidence defining the role of melanin and its precursors in insect innate immunity is severely lacking. A great deal of what is known about melanogenesis comes from studies of the process occurring in mammalian systems, where the pigment is synthesized by such diverse cells as those comprising portions of the skin, hair, inner ear, brain, and retinal epithelium. Fortunately, many of the components in the metabolic pathways leading to the formation of melanin have been found to be common to both insects and mammals. This review examines some of the factors that influence enzyme-mediated melanogenic responses, and how these responses likely contribute to blood cell-mediated, target-specific cytotoxicity in immune challenged insects.

Drosophila serpin 27A is a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response.

Avirulent strains of the endoparasitoid Leptopilina boulardi succumb to a blood cell-mediated melanotic encapsulation response in host larvae of Drosophila melanogaster. Virulent wasp strains effectively abrogate the cellular response with substances introduced into the host that specifically target and effectively suppress one or more immune signaling pathways, including elements that control phenoloxidase-mediated melanotic encapsulation. The present study implicates involvement of the Drosophila Toll pathway in cellular innate immunity by regulating the serine protease inhibitor Serpin 27A (Spn27A), which normally functions as a negative regulator of phenoloxidase. The introduction of Spn27A into normally highly immune competent D. melanogaster larvae significantly reduced their ability to form melanotic capsules around eggs of L. boulardi. This study confirms the role of Spn27A in the melanization cascade and establishes that this pathway and associated blood cell responses can be activated by parasitization. The activation of phenoloxidase and the site-specific localization of the ensuing melanotic response are such critical components of the blood cell response that Spn27A and the signaling elements mediating its activity are likely to represent prime targets for immune suppression by L. boulardi.

Superoxide anion generation in Drosophila during melanotic encapsulation of parasites.

Quinoid precursors of melanin and/or reactive oxygen species (ROS) generated during melanogenesis have been implicated as cytotoxic molecules in the immune responses of insects against their internal metazoan parasites. No study has yet identified the killing components produced in conjunction with melanotic encapsulation responses, or explained how cytotoxic molecules generated in the open circulatory system of an insect can selectively destroy foreign tissues. Strains of Drosophila melanogaster with differing immune capabilities against the wasp parasitoid Leptopilina boulardi were examined for superoxide anion (O2-.) formation during parasitization. Elevated levels of O2-. were produced by immune reactive (R-strain) hosts during melanotic encapsulation of the parasitoid, but not by susceptible (S-strain) hosts in which the parasitoid developed unmolested. Both a superoxide dismutase (SOD)-deficient strain (cSODn108, red/TM3/Sb Ser) and a catalase (CAT)-deficient strain (Catn1) also produced melanotic capsules and elevated levels of O2-. when infected, but these reactions were unsuccessful and the parasitoids survived, indicating that neither the quinoid precursors of melanin nor O2-. per se were cytotoxic. Immune incompetence in SOD-deficient and CAT-deficient hosts is attributed in part to defects in hydrogen peroxide (H2O2) metabolism, and/or the inability of these metalloenzyme-deficient strains to initiate the metal-mediated reductive cleavage of H2O2 required for the production of the cytotoxic hydroxyl radical (.OH). The role proposed for O2-. in Drosophila cellular immunity is one of potentiating the formation of .OH. Melanin, which contains both oxidizing and reducing components, may serve a dual role in producing O2-. and sequestering redox-active metal ions, thereby confining the production of ROS. Host-parasite susceptibility in the Drosophila-Leptopilina system may be determined by the ability of the parasitoid to modulate hemocyte activity and prevent both effective melanotic encapsulation and the generation of cytotoxic levels of ROS.