The autopsy at the time of SARS-CoV-2: Protocol and lessons.

A new viral disease named COVID-19 has recently turned into a pandemic. Compared to a common viral pneumonia it may evolve in an atypical way, causing the rapid death of the patient. For over two centuries, autopsy has been recognized as a fundamental diagnostic technique, particularly for new or little-known diseases. To date, it is often considered obsolete giving the inadequacy to provide samples of a quality appropriate to the sophisticated diagnostic techniques available today. This is probably one of the reasons why during this pandemic autopsies were often requested only in few cases, late and discouraged, if not prohibited, by more than one nation. This is in contrast with our firm conviction: to understand the unknown we must look at it directly and with our own eyes. This has led us to implement an autopsy procedure that allows the beginning of the autopsy shortly after death (within 1-2 h) and its rapid execution, also including sampling for ultrastructural and molecular investigations. In our experience, the tissue sample collected for diagnosis and research were of quality similar to biopsy or surgical resections. This procedure was performed ensuring staff and environmental safety. We want to propose our experience, our main qualitative results and a few general considerations, hoping that they can be an incentive to use autopsy with a new procedure adjusted to match the diagnostic challenges of the third millennium.

Prognostic Potential of the Panfungal Marker (1 → 3)-ß-D-Glucan in Invasive Mycoses Patients.

We analyze the prognostic potential of (1 → 3)-ß-D-glucan (BG) levels in predicting clinical outcomes in patients with invasive fungal infections, on a population undergoing 253 episodes (177 with positive and 76 with negative outcome). Using linear regression analysis, we assessed the prognostic potential of kinetically evaluated BG levels and we found an overall sensitivity and specificity of 68 and 82%, respectively. Moreover, using an interpretative algorithm based on two distinct cutoff values, we were able to predict the outcome in 84% of the studied population with a diagnostic accuracy of 82%.

Clinical performance of a commercial real-time PCR assay for Aspergillus DNA detection in serum samples from high-risk patients: comparison with a galactomannan enzyme immunoassay.

We investigated the clinical performance of a polymerase chain reaction (PCR)-based commercial platform, the Myconostica MycAssay™ Aspergillus (MAP), for fungal DNA detection in the serum of patients at risk of invasive aspergillosis (IA). Sixty-four hospitalized patients were prospectively enrolled and a total of 71 different episodes were investigated (30 episodes were clinically/microbiologically classified as IA and 41 as control episodes). When MAP was compared to the galactomannan (GM) assay, no significant differences were found in terms of sensitivity (46.7% vs. 50.0%), specificity (97.6% vs. 95.1%), positive predictive value (PPV) (93.3% vs. 88.2%), and negative predictive value (NPV) (71.4% vs. 72.2%). The corresponding areas under the curve (AUC) of the receiver operating characteristic (ROC) curves were also superimposable. Overall, because of the good agreement between the two assays and considering the high specificity and PPV of the MAP, we suggest the use of this PCR-based platform as a second-level examination for the evaluation of clinically undefined cases where culture or GM have provided positive results.

Routine use of a protease zymogen-based colorimetric assay for the detection of Beta-glucan and its role in clinical practice.

The detection of Aspergillus antigen (galactomannan) is considered a reliable marker for the diagnosis of invasive aspergillosis (IA), yet the sensibility and specificity of the assays commonly employed in routine are not optimal. The aim of the present study was to investigate whether the detection of another panfungal antigen, the (1,3)-b-D-glucan could have an auxiliary role in the identification of patients with IA. The study was carried out on 63 sera belonging to patients who had been screened for galactomannan, according to the clinical suspect of IA. Our data show that the positive galactomannan results were not confirmed by positive (1,3)-b-D-glucan results in patients receiving therapy with beta-lactam antibiotics associated with tazobactam, whereas in all the other cases, with the exception of four, the results of the (1,3)-b-D-glucan test were confirmatory of the galactomannan results.

An antibody reactivity-based assay for diagnosis of invasive candidiasis using protein array.

The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicans proteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.

Inhibitory effects of different lactobacilli on Candida albicans hyphal formation and biofilm development.

The aim of this study was to investigate the effects of different species of Lactobacilli on hyphal formation and biofilm development by the opportunistic fungal pathogen Candida albicans. We employed 4 different Lactobacillus species, namely L. rhamnosus, L. acidophilus, L. plantarum and L. reuteri, and 2 C. albicans strains, the reference DAY286 and its isogenic hwp1/hwp1 mutant, the FJS24 strain. As assessed by morphological analysis and quantitative colorimetric assays, Lactobacillus crude filtrate supernatant fluids (CFSF) affected Candida, impairing both hyphal formation and biofilm production. The CFSF-mediated phenomenon occurred in a dilution- and time-dependent manner and was consistently observed, irrespective of the C. albicans HWP1 genotype.

DOG1 expression in neuroendocrine neoplasms: Potential applications and diagnostic pitfalls.

Neuroendocrine neoplasms represent a heterogeneous group of rare tumors, more frequently arising from gastroenteropancreatic tract and lungs. At the time of diagnosis, 20% of cases are metastatic, and 10% of cases are considered as cancer of unknown primary origin. Several immunohistochemical markers are routinely used to confirm the neuroendocrine differentiation, first among all Synaptophysin and Chromogranin-A; on the other hand, different immunohistochemical markers are used to establish primary anatomical site, as TTF1, CDX2, Islet-1 and Calcitonin, but no marker is available in order to distinguish among different sites of the digestive tract. DOG1 (discovered on GIST-1) is a gene normally expressed in interstitial cells of Cajal and, in routine practice, DOG1 immunostaining is used in diagnosis of GIST (gastrointestinal stromal tumor). DOG1 expression has been described in several neoplasms other than GIST, both in mesenchymal and epithelial neoplasms. In the present study, DOG1 immunostaining has been performed in a large cohort of neuroendocrine neoplasms, including neuroendocrine tumors and neuroendocrine carcinomas, in order to evaluate frequency, intensity and pattern of expression in different anatomical site and in different tumor grade. DOG1 expression was detected in a large percentage of neuroendocrine tumors, with statistically significant association between DOG1 expression and gastrointestinal tract neuroendocrine tumors. As a consequence, DOG1 could be included in marker panel for the identification of primary site in neuroendocrine metastases of unknown primary origin; moreover, these results recommend careful evaluation of DOG1 expression in gastrointestinal neoplasms, in particular in differential diagnosis between epithelioid GIST and neuroendocrine tumors.

Protein microarrays on midtrimester amniotic fluids: a novel approach for the diagnosis of early intrauterine inflammation related to preterm delivery.

Novel technologies that allow simultaneous assessment of multiple biomarkers provide new and promising diagnostic/prognostic approaches. By protein microarrays, here we analyzed amniotic fluids (AF) from 50 women with preterm delivery (PTD) and 50 control women, who delivered at term. In detail, cytokines, chemokines, matrix metalloproteinases and antigen-specific antibodies were assessed. The AF analysis showed significant differences between women with preterm and term delivery in the levels of IL-1alpha, IL-1beta, IL-4, IL-6, IL-8, MCP-1, IFN-gamma and anti-HSV2 IgG. No significant differences were observed in the levels of TNF-alpha, MMP-2, MMP-9 and specific IgG for seven vertically transmitted pathogens. In conclusion, we demonstrated the feasibility of protein microarrays in the diagnosis of early intrauterine inflammation. The significant association between the increased levels of certain cytokines and preterm delivery argues on their relevance as early pathogenetic markers for identification of risk patients.

A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections.

The detection of specific serum antibodies is mainly achieved by enzyme-linked immunosorbent assay (ELISA). Here, we describe the setting up of a microarray-based serological assay to screen for IgG and IgM against vertically transmitted pathogens (Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus types 1 and 2, varicella zoster virus, Chlamydia trachomatis). The test, accommodated onto a restricted area of a microscope slide, consists of: (a) the immobilization of antigens and human IgG and IgM antibody dilution curves, laid down in an orderly manner; (b) addition of serum samples; (c) detection of antigen-serum antibodies complexes by indirect immunofluorescence. The IgG and IgM curves provide an internal calibration system for the interpolation of the signals from the single antigens. The test was optimized in terms of spotting conditions and processing protocol. The detection limit was 400 fg for the IgG assay and 40 fg for the IgM assay; the analytical specificity was >98%. The clinical sensitivity returned an average value of 78%, the clinical specificity was >96%, the predictive values were >73%, and the efficiency was >88%. The results obtained make this test a promising tool, suitable for introduction in the clinical diagnostic routine of vertically transmitted infections, in parallel (and in future as an alternative) to ELISA.

The morphology and clinical importance of the axillary arch.

The axillary arch is the main variation of the axillary muscle. It was first described by Ramsay in 1795. In its classical form, it arises from the latissimus dorsi muscle and extends from this towards the pectoralis major, crossing the base of the axilla and creating a close relationship with the elements of the axillary neurovascular bundle. We describe the finding of 9 axillary arches, including one case of a bilateral arrangement. We develop a searching and finding technique for the axillary arch, essential for the safe and successful development of surgical procedures in the axillary region. Knowledge of this muscle variation and the possibility of finding it during axillary procedures is crucial for lymph node staging and lymphadenectomy and is also important for differential diagnosis in compressive pathologies of the axillary vessels and brachial plexus.

Heterogeneity of hematopoietic cells immortalized by v-myc/v-raf recombinant retrovirus infection of bone marrow or fetal liver.

The J2 recombinant retrovirus expressing v-myc/v-raf (also known as MYC/RAF1) immortalized macrophages from the bone marrow of lipopolysaccharide-responsive mouse strains, producing the ANA-1 cell line from C57BL/6 mice and the INF-3A cell line from C3H/HeN mice. In contrast, J2 recombinant retrovirus infection of the fetal liver from C57BL/6-Ly-5a mice immortalized a cell line (GGD) that did not exhibit the characteristics of mature macrophages. The GGD cell line was classified as leukocytic on the basis of its expression of the Ly-6B.2, Fc gamma R, and Ly-5.2 antigens. Our results indicate that the J2 recombinant retrovirus selectively immortalizes macrophages from the bone marrow of C57BL/6 and C3H/HeN mice but immortalizes cells without definitive macrophage characteristics from murine fetal liver under the same culture conditions.

Erythroid differentiation and modulation of c-myc expression induced by antineoplastic drugs in the human leukemic cell line K562.

The human leukemia cell line K562 expresses constitutively high levels of c-myc mRNA and can be induced to differentiate along the erythroid lineage. Treatment of K562 cells with the antineoplastic drugs 1-beta-D-arabinofuranosylcytosine and daunomycin causes differentiation into hemoglobin-producing cells. The differentiation process is associated with an early block of cellular proliferation occurring during the first 24 h of treatment. RNA synthesis is progressively reduced to 20 to 30% of the control levels after 3 days of exposure to the drugs. Dot and Northern blot analyses were performed to evaluate the levels of c-myc or globin mRNA during the differentiation of K562. Daunomycin and 1-beta-D-arabinofuranosylcytosine, despite their distinct chemical nature, induced similar modulation of mRNA levels. Globin mRNA did not change during the first 24 h of culture and began to increase after 48 h of treatment with drugs, reflecting the kinetic of appearance of hemoglobin-producing cells. In contrast, a transient decrease of c-myc mRNA was observed after the first 24 h of drug treatment, followed by a return to normal levels of c-myc mRNA after 48 h of treatment. Thus, the expression of c-myc mRNA in K562 did not reflect their proliferative activity nor their stage of differentiation. We speculate that the transient down-regulation of c-myc mRNA may be an initial event in the erythroid differentiation of K562.

Potent activation of mouse macrophages by recombinant interferon-gamma.

The ability of recombinant interferon-gamma (IFN-gamma) to activate mouse macrophages was investigated. The use of recombinant IFN-gamma has the advantage of being devoid of contaminating lymphokines. Two preparations of IFN-gamma were utilized, one which was not glycosylated and which was highly purified from Escherichia coli another which was glycosylated and which was from transfected COS-7 monkey cells. Both preparations of recombinant IFN-gamma activated murine macrophages to kill lymphoma and melanoma tumor targets, suggesting that glycosylation of the protein or the presence of other mammalian proteins is not essential for activation. Significant levels of cytolytic activity were induced from IFN-gamma (1 to 10 units/ml). This activity was undiminished by treatment of the IFN-gamma preparations with polymixin B at doses which neutralized endotoxin (50 micrograms/ml). Similarly, IFN-gamma, at low concentrations, induced an inhibition of migration by macrophages. Based on antiviral activity, IFN-gamma was shown to be 100 to 1000 times more potent than was IFN-beta as a macrophage-activating agent. Taken together, these results demonstrate that murine IFN-gamma is a macrophage-activating factor which is effective at physiological concentrations. Of particular interest is the observation that the nonglycosylated E. coli-derived IFN-gamma is active and therefore may be of value for therapeutic studies, since it can be easily produced in large amounts.

The strain of mouse and assay conditions influence whether MuIFN-gamma primes or activates macrophages for tumor cell killing.

Investigators from two different laboratories have compared several variables in the short-term macrophage-mediated cytotoxicity assays used by each group to study the role of MuIFN-gamma in macrophage activation. The findings suggest that the capacity of MuIFN-gamma to activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.

Comparison of five short-term assays that measure nonspecific cytotoxicity mediated to tumor cells by activated macrophages.

Five different short term assays (less than 48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.

Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins.

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity.

A web service-based Grid portal for Edgebreaker compression.

BACKGROUND: In health applications, and elsewhere, 3D data sets are increasingly accessed through the Internet. To reduce the transfer time while maintaining an unaltered 3D model, adequate compression and decompression techniques are needed. Recently, Grid technologies have been integrated with Web Services technologies to provide a framework for interoperable application-to-application interaction. OBJECTIVES: The paper describes an implementation of the Edgebreaker compression technique exploiting web services technology and presents a novel approach for using such services in a Grid Portal. The Grid portal, developed at the CACT/ISUFI of the University of Lecce, allows the processing and delivery of biomedical images (CT--computerized tomography--and MRI--magnetic resonance images) in a distributed environment, using the power and security of computational Grids. METHODS: The Edgebreaker Compression Web Service has been deployed on a Grid portal and allows compressing and decompressing 3D data sets using the Globus toolkit GSI (Globus Security Infrastructure) protocol. Moreover, the classical algorithm has been modified extending the compression to files containing more than one object. RESULTS AND CONCLUSIONS: An implementation of the Edgebreaker compression technique and related experimental results are presented. A novel approach for using the compression web service in a Grid portal allowing storing and preprocessing of huge 3D data sets, and subsequent efficient transmission of results for remote visualization is also described.

A radiolabel release microassay for phagocytic killing of Candida albicans.

The chromium release technique for quantifying intracellular killing of radiolabelled Candida albicans particles was exploited in a microassay in which murine and human phagocytes acted as effectors under peculiarly simple conditions. At appropriate effector: target ratios and with a 4 h incubation, up to 50% specific chromium release could be detected in the supernatant with no need for opsonization or lysis of phagocytes. This simple microassay permits easy-to-perform, simultaneous testing of a variety of different phagocytes even if only available in limited amounts, and provides an objective measurement of intracellular killing of Candida albicans.

A rapid objective immunofluorescence microassay. Application for detection of surface and intracellular antigens.

An indirect immunofluorescence microassay, which permits automated reading, has been employed for simple, rapid and objective detection of surface and intracellular antigens. Initially, the cells, spun in microplates, are fixed with glutaraldehyde (0.25% v/v in PBS). Following fixation, the cells can be stored at 4 degrees C for up to 2 weeks before being used in the immunofluorescence microassay. The fixed cells are then stained according to standard procedures using appropriate first and fluorescein-conjugated second antibodies. An automated and quantitative evaluation of the fluorescence intensity of the cell samples was achieved using the Titertek Fluoroskan II automatic reader. This microassay was shown to be suitable for the detection of the surface MAC1 antigen and intracellular v-myc protein in the GG2EE macrophage cell line.

Microglial cell-mediated anti-Candida activity: temperature, ions, protein kinase C as crucial elements.

An in vitro established microglial cell line, BV-2, constitutively exhibits high levels of anti-Candida activity. To elucidate the cascade of events leading to the accomplishment of such activity, we studied its dependence on temperature and ion availability. The role of protein kinases has also been studied by the specific inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA 1004). We found that (a) the BV-2 cell/Candida conjugate formation is a discrete step, temperature-, ion- and protein kinase-independent; (b) the phagocytic event, which is protein kinase-independent, is significantly impaired by temperature decrease and ion deprivation; (c) the fulfillment of anti-Candida effects is strictly dependent upon temperature, ion availability and functional protein kinase. Functional protein kinase C, but not other kinases, is required for the accomplishment of anti-Candida activity, which, in fact, is selectively abrogated by H7 but not HA. Furthermore, protein kinase C activators, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl glycerol (OAG), consistently potentiate BV-2 cell-mediated anti-Candida activity, the phenomena being dose-dependent. These results indicate that the multistep events leading a microglial cell to express anti-Candida activity can be dissected and differentiated for biochemical and biological demands, the latest along the cascade being the most demanding steps.