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In recent years, the frequency of infections due to saprophytic fungi has increased. Cryptococcus laurentii, recently classified as Papiliotrema laurentii, is responsible for fungemia, meningitis, and superficial infections. Here, we report the first case of cutaneous Papiliotrema (Cryptococcus) laurentii infection in a 23-year-old Caucasian woman affected by an autoimmune thyroiditis with hypothyroidism. Impairments of the immune system are often associated with unusual fungal infections, which cannot be neglected. The isolate strain was susceptible to Amphotericin B while resistant to fluconazole, itraconazole, voriconazole, and terbinafine. The patient was successfully treated with Amphotericin B.

Summary We are reporting the case of a nine-month-old Pakistani female with complaint of growth retardation who presented multiple intestinal parasitic infections. Probably because of contamination with fecal matter, the initial microscopic examination of the urinary sample revealed the presence of eggs of Enterobius vermicularis, cysts of Entamoeba coli, and an organism similar to mites. Stool samples were obtained after two weeks and microscopic investigation confirmed the presence of Enterobius vermicularis eggs, cysts of Entamoeba coli, and hookworm eggs. The patient was immediately subjected to mebendazole therapy associated with trimethoprim-sulfamethoxazole, to which she responded well. Follow-up stool re-examinations performed 15 and 30 days after the treatment tested negative for all parasitic ova and cysts. This study reflects the importance of considering multiple parasitic infestations in low socio-economic populations and highlights the need of improving poor hygienic conditions to prevent such infections, in particular in children.

The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.

Clostridiodes difficile infection (CDI) is the most important cause of healthcare-associated diarrhea. The decreasing trend of CDI from 15 % to 4 % observed at the Italian Hospital of Desio over a 10-year period is due to prevention strategies. Our data highlight the importance of surveillance studies to control CDI.

The correlation of clinical, radiological and laboratory findings of patients at admission in the Emergency Department (ED) with clinical severity and risk of mortality was investigated. Adult coronavirus disease 2019 (COVID-19) patients hospitalized in March 2020 in Desio Hospital, Lombardy, were retrospectively included in the study, and categorized in terms of disease severity and adverse outcome. Out of the 175 patients enrolled, 79% presented one or more comorbidities, with cardiovascular disease being the most frequent (62%). More than half of the patients showed lymphocytopenia and 20% thrombocytopenia. The patients in the severe group presented higher absolute neutrophil count (ANC), C-reactive protein (CRP), AST, LDH, procalcitonin (PCT) and BUN values compared to the non-severe group (p 106.08 µmol/L (OR = 2.87; 95% CI 1.04–7.92) and creatine kinase > 2.90 µkat/L (OR = 3.80; 95% CI 1.31–10.9) were observed on admission (p

Highlights • We conducted a retrospective study on 61 patients admitted to the ICU • We investigated the relationship among SARS-CoV-2 and bacterial/fungal colonization • Among positive bronchial aspirates, 80 % were colonized by fungi or P. aeruginosa • No multidrug-resistant bacteria or fungi were isolated • The failure in the regulation of defenses against pathogens other than SARS-CoV-2

The infrequency of urinary tract and blood stream infections caused by Aerococcus urinae is most probably due to the difficulties in the identification of this bacterium using standard microbiological methods. With the introduction of more sensitive and accurate techniques in clinical microbiology, such as genetic approaches and Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry (MS), the incidence of infections due to A. urinae increased. Herein, we described a case of urinary tract and bloodstream infection caused by A. urinae, which occurred in an 86-year-old Caucasian man with a previous history of prostate cancer. The identification of A. urinae was performed by MALDI-TOF MS, since this microorganism cannot be identified by biochemical reactions. In this report, we highlight the need to consider MALDI-TOF MS as technique of choice for A. urinae identification in the presence of subjects with predisposing factors, such as old age, male gender, and genitourinary tract pathologies.

Bacterial vaginosis (BV) is one of the most frequent causes of vaginal discharge in women during reproductive age worldwide. This disease is characterized by the replacement of the normal vaginal flora with an overgrowth of anaerobic bacteria most of them producing sialidase enzyme. BV is associated with an increased risk of adverse outcomes in pregnancy and susceptibility to several sexually transmitted diseases. In the present study, we evaluated the detection of sialidase activity by OSOM BVBlue test in association with routine microbial cultures and Nugent’s score, considered as the gold standard, for the diagnosis of bacterial vaginosis. Three vaginal swabs were collected from 352 women older than 12 years in age. A swab collected into Amies transport medium was employed for standard microbial cultures, a FLOQSwab for Gram stain, and a second FLOQSwab for the BVBlue test. According to Nugent’s score, BV frequency was 16.5 % (58 samples). The sensitivity of microbial culture and BVBlue test, when compared with Nugent’s score, was 69.8 % and 39.6 %, respectively. However, BVBlue test detected five cases with no bacterial growth in culture, whereas 14 samples with bacterial cultures positive for Gardnerella vaginalis showed a BVBlue test negative. The combination of microbial culture and BVBlue test increased the sensitivity to 75 % compared with Nugent’s score. In conclusion, BVBlue test alone appears not to be an efficient screening test, but, when associated with microbial cultures, can improve the diagnosis of BV

Dientamoebiasis is globally distributed and detected in a large number of subjects with diarrhea, abdominal discomfort, flatulence, fatigue and loss of appetite. The life cycle and transmission of Dientamoeba fragilis are poorly understood. Microscopic examination of permanent stained smears is traditionally employed to diagnose the infection. However, this approach is time-consuming and the success in detecting D. fragilis depends on the microscopist’s experience. Hence, only a few laboratories routinely carry out tests for D. fragilis. Consequently, the prevalence of D. fragilis infection is probably underestimated. Although novel, rapid and more sensitive diagnostic tests are becoming available for detecting intestinal parasites, they also possess some limitations. The aim of this study was to emphasize the importance of performing microscopic examination of permanent stained smears from at least one fresh stool specimen after sample arrival at the laboratory, as a mandatory practice for the diagnosis of dientamoebiasis, particulary where it is not possible to perform molecular assays.

Monoclonal gammopathy of uncertain significance (MGUS) identifies a clinically asymptomatic and laboratory-based situation characterized by a modest monoclonal component (MC). In a limited percentage of cases, on a probabilistic basis, the asymptomatic genepremalignant stage could lead to multiple myeloma (MM). Materials and Methods. Based on literature data and available Guidelines on the subject, the diagnostic criteria and a methodological path are here suggested to the Occupational Physician to formulate a judgment of suitability for the task with exposure risk to RI or pesticides. Results. Some studies have evaluated the prevalence of MGUS in subjects exposed professionally to pesticides. Numerous other studies conducted on the survivors of the atomic bombing of Hiroshima and Nagasaki have highlighted a possible association with exposure to ionizing radiation (IR). The guidelines relating to the diagnosis and management of MGUS cases (with respect to the potential evolution in MM allow) to draw important operational indications for the competent/authorized physician. Conclusions. The routinely use of laboratory tests for subjects exposed to the studied risk factors is generally indicated starting from the worker's 50 years of age. The finding of a MGUS in the absence of further laboratory alterations represents the situation most frequently and does not require further measures, other than those of foreseeing even blood controls at least every two years. In this situation, there are no justified restrictions on work activities with exposure risks to IR or pesticides. If alterations suggestive for an increased risk of evolution in a neoplastic way could be identified, a close periodicity - every 3-6 months - of haematological checks is recommended. In these cases, it appears justified an abstention from activities involving exposure to ionizing radiation for a period of time that will be evaluated based on the evolution of the framework and by the progress of laboratory tests in the monitored period.Introduzione. La gammopatia monoclonale di incerto significato (MGUS) identifica un quadro clinicamente asintomatico e laboratoristicamente caratterizzato da una modesta componente monoclonale (CM). In una limitata percentuale di casi può essere considerata, su base probabilistica, lo stadio premaligno asintomatico che precede il mieloma multiplo (MM). Scopo. Sulla base dei dati di letteratura e delle Linee Guida sull’argomento, vengono individuati i criteri e un percorso metodologico per il Medico del Lavoro quando viene chiamato a formulare un giudizio di idoneità alla mansione con rischio espositivo a RI o pesticidi. Materiali e Metodi. Viene analizzata la letteratura scientifica sull’argomento. Alcuni studi hanno valutato la prevalenza della MGUS in soggetti esposti professionalmente a pesticidi. Altri numerosi studi condotti sui sopravvissuti ai bombardamenti atomici di Hiroshima e Nagasaki hanno evidenziato una possibile associazione con l’esposizione a radiazioni ionizzanti (RI). Le linee Guida relative alla diagnosi e gestione dei casi di MGUS rispetto alla potenziale evoluzione in MM consentono di trarre importanti indicazioni operative per il medico competente/autorizzato. Conclusioni. Il ricorso routinario a test di laboratorio per soggetti esposti ai fattori di rischio studiati appare in linea generale indicato a partire dai 50 anni di età del lavoratore. Il riscontro di una MGUS in assenza di ulteriori alterazioni laboratoristiche rappresenta la situazione di più frequente riscontro e non necessita di ulteriori provvedimenti, se non quelli di prevedere controlli anche ematochimici con periodicità almeno biennale. In questa situazione non appaiono giustificate restrizioni all’attività lavorativa con rischio espositivo a RI o pesticidi. In caso di concomitante presenza di alterazioni che possono essere indicative o suggestive di un aumentato rischio di evoluzione in senso neoplastico, è raccomandata una periodicità ravvicinata - ogni 3-6 mesi - dei controlli ematologici. In questi casi appare giustificato un allontanamento da attività comportanti esposizione a radiazioni ionizzanti per un periodo di tempo che andrà valutato sulla base dell’evoluzione del quadro e dell’andamento degli esami di laboratorio nel periodo di monitoraggio.

Trichophyton rubrum and Trichophyton mentagrophytes spp. are two of the most frequently isolated dermatophytes causing dermatophytosis worldwide. Since the incidence of resistance to antifungal agents is increasing, antifungal susceptibility tests are needed to successfully treat dermatophytoses. Most of the methods currently available are complicated, time-consuming and lack of reference procedures. The aim of this work was to establish a simple protocol to test the susceptibility of dermatophytes isolated from clinical samples against five antifungal drugs using E-test and disk diffusion methods. We used the E-test on non-supplemented Mueller–Hinton agar plates to determine the minimum inhibitory concentrations (MICs) of fluconazole, itraconazole, voriconazole and amphotericin B, and disk diffusion method to determine the interpretive MIC of terbinafine. Fifty dermatophytes—10 T. rubrum and 40 T. mentagrophytes spp.—were assessed after only 96 h of colony growth. Terbinafine was the most active antifungal agent with an inhibition diameter greater than 70 mm (sensitivity > 20 mm), followed by voriconazole, itraconazole and amphotericin B with MICs ranging from 0.032 to 0.38 µg/mL, from 0.006 to 0.125 µg/mL and from 0.5 to 1.5 µg/mL, respectively. All isolates were resistant to fluconazole. Collectively, the less laborious E-test and disk diffusion method were shown to be suitable and reliable to determine antifungal sensitivity of dermatophytes. This simple standard protocol could be employed in the routine of clinical laboratories.

OBJECTIVE: The analysis of the protein pattern of Klebsiella pneumoniae carbapenemase (KPC)-producing strains by Bruker Matrix-Assisted Laser Desorption Ionization (MALDI) Biotyper system has revealed the presence, in the majority of cases, of an 11.109 m/z peak. The peak corresponds to the gene product named p019 of the bla(KPC)-bearing plasmids and has been suggested as a candidate for a biomarker that is able to distinguish KPC-producers from non-KPC-producers. The aim of this study was to evaluate the rapid detection of the 11.109 m/z peak of KPC-producer strains in the clinical laboratory routine by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) technique, using the Vitek(®) Research-User-Only (RUO) Mass Spectrometry (MS) system without changing the instrument parameters. MATERIALS AND METHODS: Globally, 373 K. pneumoniae isolates were investigated and identified by MALDI-TOF MS analysis. KPC-producers were distinguished from non-KPC-producers by Antimicrobial Susceptibility Testing (AST) and phenotypic carbapenemase resistance assays. RESULTS: The MALDI-TOF Vitek MS RUO detected the 11.109 m/z peak in 95.7% of KPC-producers with 100% specificity before traditional test results became available. CONCLUSION: Our approach is appropriate as a first screening step for the rapid identification of KPC isolates, which will help to improve infection control in clinical practice and prevent the outbreak and dissemination of resistant bacteria.

Fungal peritonitis is a rare but serious complication of peritoneal dialysis. This infection has been reported to be mostly caused by Candida species, and less frequently by a variety of other yeasts and moulds, such as Aspergillus, Penicillium, and Fusarium spp. are commonly isolated from soil, plants and environmental surfaces, and rarely from non-immunosuppressed subjects. In this report, author describe a case of infection caused by Fusarium solani in a 59-year-old man undergoing continuous ambulatory peritoneal dialysis. The fungus was recovered from cultures of peritoneal dialysate and the pathogen identification was carried out by mass spectrometry. The patient's outcome was favorable without complications after liposomal amphotericin B treatment along with peritoneal dialysis catheter removal.

Clostridium like species, particularly Clostridium perfrigens, are the second most common causes of human anaerobic infections, including myonecrosis and bacteremia. Clostridium paraputrificum is an infrequent isolate, which has been identified in only 1% of reported cases of clostridial infections. We herein report a rare case of C. paraputrificum bacteremia in a 78-year-old Caucasian man diagnosed with an intestinal carcinoma and liver neoplastic lesions. The isolate was susceptible to chloramphenicol, meropenem, metronidazole, vancomycin, and resistant to clindamycin and penicillin, and the patient was successfully treated with metronidazole. Malignancy and inflammatory bowel diseases are often associated with clostridial bacteremia, which cannot be neglected.

Background New Hevylite® assay quantifies the immunoglobulin classes, including IgM bound to light chains, allowing distinguishing immunoglobulins involved and uninvolved in plasma cell disorders. Objective To compare data obtained by IgM Hevylite® (IgM-HLC) assay with conventional methods used in routine laboratory practice for monitoring IgM plasma cell disorders. Methods Serum samples (n = 122) from 50 patients with IgM monoclonal protein (MP) identified by Immunofixation (IFE) before the beginning of the study were collected during monitoring from December 2012 to September 2014 (2 Waldestrom's macroglobulinemia, 4 NH-lymphoma, 44 MGUS) and were assessed using IgM Hevylite® (HLC) assay, Capillary Electrophoresis (CE), Immunofixation (IFE), serum Free Light Chain (FLC) assay and total IgM measurements. Results IgM MP was detected by IFE in 85/122 samples (71 IgMk, 10 IgMl, 4 IgMk/IgMl), while in 37/122 was undetectable although CE measured small MP, probably as a consequence of disease stimulating inflammatory immuno-response. Among the 85 positive samples, the HLC ratio but not the FLC ratio was altered in 36 samples while in 4 sera only FLC was altered. Out of 37 IFE negative samples 24 had normal HLC and FLC ratios. Conclusions Since the partial overlap of abnormalities identified by HLC and FLC assays, IgM Hevylite assay can provide valuable information on the evolution of IgM monoclonal disease and may support the recognition of a transitory monoclonality leading to an improvement in routine laboratory practice.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is becoming a popular technology in clinical microbiology. It is a fast and highly specific method for the routine identification of micro-organisms. In this study, we evaluated the suitability of dermatophyte identification after only 2 days of colony growth using MALDI-TOF MS. Two protein extraction protocols were also evaluated consisting of either formic acid alone or of ethanol-formic acid-acetonitrile to achieve a complete protein extraction. Morphology-based techniques were used as the diagnostic standard methods and MALDI-TOF MS results were obtained using the manufacturer's spectral library. Using the formic acid protein extraction protocol after 2 days of colony growth, 70 and 46% of dermatophytes were properly identified at the genus and species-level respectively. The addition of ethanol-formic acid-acetonitrile extraction protocol increased the identification to 90 and 62%. Based on our observations, we propose a two-step workflow for the fast and reliable identification of dermatophytes after only 2 days of colony growth. This flow chart consists of a first direct deposition procedure with the addition of formic acid, followed by a complete protein extraction when dermatophyte identification is not successful. Significance and impact of the study In this study, a two-step workflow for the identification of clinical dermatophytes using MALDI-TOF analysis and commercially available spectral library was developed. The workflow consists of an initial direct deposition of the sample on the MALDI plate and formic acid protein extraction at 2 days of growth culture; if dermatophyte identification is not successful, a complete protein extraction using ethanol-formic acid-acetonitrile is subsequently performed. Using this workflow, the correct isolate identifications increase up to 90%; of these, 27% are identified at the genus-level, providing sufficient information to start an antifungal treatment. The method here proposed represents a fast and useful approach to differentiate dermatophytes grown in culture.

Bacillus pumilus is commonly isolated from a wide variety of soils, plants and environmental surfaces, but rarely from human specimens. In this report, we describe a case of infection caused by B. pumilus in a healthy 10-years-old child. The microorganism was recovered from a severe wound of the left knee after three days from trauma. Pathogen identification was carried out by mass spectrometry. The patient's outcome was positive following an ampicillin/sulbactam treatment without complications.

Background Environmental toxicants are allegedly involved in decreasing semen quality in recent decades; however, definitive proof is not yet available. In 1976 an accident exposed residents in Seveso, Italy, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Objective The purpose of this study was to investigate reproductive hormones and sperm quality in exposed males. Methods We studied 135 males exposed to TCDD at three age groups, infancy/prepuberty (1–9 years), puberty (10–17 years), and adulthood (18–26 years), and 184 healthy male comparisons using 1976 serum TCDD levels and semen quality and reproductive hormones from samples collected 22 years later. Results Relative to comparisons, 71 men (mean age at exposure, 6.2 years; median serum TCDD, 210 ppt) at 22–31 years of age showed reductions in sperm concentration (53.6 vs. 72.5 million/mL; p = 0.025); percent progressive motility (33.2% vs. 40.8%; p < 0.001); total motile sperm count (44.2 vs. 77.5 × 106; p = 0.018); estradiol (76.2 vs. 95.9 pmol/L; p = 0.001); and an increase in follicle-stimulating hormone (FSH; 3.58 vs. 2.98 IU/L; p = 0.055). Forty-four men (mean age at exposure, 13.2 years; median serum TCDD, 164 ppt) at 32–39 years of age showed increased total sperm count (272 vs. 191.9 × 106; p = 0.042), total motile sperm count (105 vs. 64.9 ×106; p = 0.036), FSH (4.1 vs. 3.2 UI/L; p = 0.038), and reduced estradiol (74.4 vs. 92.9 pmol/L; p < 0.001). No effects were observed in 20 men, 40–47 years of age, who were exposed to TCDD (median, 123 ppt) as adults (mean age at exposure, 21.5 years). Conclusions Exposure to TCDD in infancy reduces sperm concentration and motility, and an opposite effect is seen with exposure during puberty. Exposure in either period leads to permanent reduction of estradiol and increased FSH. These effects are permanent and occur at TCDD concentrations < 68 ppt, which is within one order of magnitude of those in the industrialized world in the 1970s and 1980s and may be responsible at least in part for the reported decrease in sperm quality, especially in younger men.

Aquaporins (AQPs) are an ubiquitous family of proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs. At present, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules (AQPO, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), or also glycerol and other small solutes (AQP3, AQP7, AQP9, AQP10, AQP12). The genomic, structural, and functional aspects of this family are briefly described. In particular, proteomic approaches to identify and characterize the most studied AQPs, mainly through SDS-PAGE followed by MS analysis, are discussed. Moreover, the clinical importance of the best studied aquaporin (AQP1) in human diseases is also provided. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

The heat shock proteins (hsp) are ubiquitous molecules induced in cells exposed to sublethal heat shock, present in all living cells, and highly conserved during evolution. Their function is to protect cells from environmental stress damage by binding to partially denatured proteins, dissociating protein aggregates, to regulate the correct folding, and to cooperate in transporting newly synthesized polypeptides to the target organelles. The molecular chaperones are involved in numerous diseases, including cancer, revealing changes of expression. In this review, we mainly describe the relationship of hsp expression with human cancer, and discuss what is known about their post-translational modifications according to malignancies.

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.

Renal cell carcinoma (RCC), a human kidney cancer from the proximal tubular epithelium, accounts for about 3% of adult malignancies. Molecular and cytogenetic analysis have highlighted deletions, translocations, or loss of heterozygosity in the 3p21–p26, a putative RCC locus, as well as in 6q, 8p, 9pq, and 14pq. Studies on phenotypic expression of human kidney tissue and on post-translational modifications in RCC have not yet provided a marker for early renal cell carcinoma diagnosis. Current dignostic methods do not help to detect the tumor before advanced stages. We therefore used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to study normal and tumor kidney tissues in ten patients suffering from RCC. A human kidney protein map in the SWISS-2DPAGE database accessible through the ExPASy WWW Molecular Biology Server was established. Of 2789 separated polypeptides, 43 were identified by gel comparison, amino acid analysis, N-terminal sequencing, and/or immunodetection. The comparison between normal and tumor kidney tissues showed four polypeptides to be absent in RCC. One of them was identified as ubiquinol cytochrome c reductase (UQCR), whose locus has elsewhere been tentatively assigned to chromosome 19p12 or chromosome 22. A second polypeptide was identified as mitochondrial NADH-ubiquinone oxido reductase complex I whose locus is located on chromosome 18p 11.2 and chromosome 19q 13.3. These result suggest that the lack of UQCR and of mitochondrial NADH-ubiquinone oxidoreductase complex I expression in RCC may be caused by unknown deletions, or by changes in gene transcription or translation. It might indicate that mitochondrial disfunction plays a major role in RCC genesis or evolution.

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.

The quality of samples and of pre-analytical steps are crucial in all biological tests, this is dramatically true in proteomics analysis. In renal cell carcinoma preparation for two-dimensional gel electrophoresis the time elapsed between sample collection and treatment, and the heterogeneity of tissues are considered in order to obtain high quality and reproducibility of spots. The mechanical dissection and cell separation by magnetic beads coated with anti-Ber and EP4 antibodies to minimize the contamination of nonepithelial cells are described.

Background: In recent decades, young men in some industrialized areas have reportedly experienced a decrease in semen quality. Objective: We examined effects of perinatal dioxin exposure on sperm quality and reproductive hormones. Methods: We investigated sperm quality and hormone concentrations in 39 sons (mean age, 22.5 years) born between 1977 and 1984 to mothers exposed to dioxin after the accident in Seveso, Italy (1976), and 58 comparisons (mean age, 24.6 years) born to mothers exposed only to background dioxin. Maternal dioxin levels at conception were extrapolated from the concentrations measured in 1976 serum samples. R esults: The 21 breast-fed sons whose exposed mothers had a median serum dioxin concentration as low as 19 ppt at conception had lower sperm concentration (36.3 vs. 86.3 million/mL; p = 0.002), total count (116.9 vs. 231.1; p = 0.02), progressive motility (35.8 vs. 44.2%; p = 0.03), and total motile count (38.7 vs. 98 million; p = 0.01) than did the 36 breast-fed comparisons. The 18 formula-fed exposed and the 22 formula-fed and 36 breast-fed comparisons (maternal dioxin background 10 ppt at conception) had no sperm-related differences. Follicle stimulating hormone was higher in the breast-fed exposed group than in the breast-fed comparisons (4.1 vs. 2.63 IU/L; p = 0.03) or the formula-fed exposed (4.1 vs. 2.6 IU/L; p = 0.04), and inhibin B was lower (breast-fed exposed group, 70.2; breast-fed comparisons, 101.8 pg/mL, p = 0.01; formula-fed exposed, 99.9 pg/mL, p = 0.02). C onclusions: In utero and lactational exposure of children to relatively low dioxin doses can permanently reduce sperm quality

Caveolin-1 and flotillin-1 belong to plasma membrane microdomains. They are characterized by peculiar lipid and protein composition and are involved in fundamental cellular events such as: signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 30 human renal cell carcinoma (RCC) and adjacent normal kidney (ANK) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 over-expression was found in RCC tissues compared to ANK, and was confirmed by immunohistochemistry. Caveolin-1 and flotillin-1 protein levels were found by 1-D, 2-DE, and MS to be increased also in RCC microdomain-enriched subcellular fractions purified from paired RCC and ANK samples.

Several efforts are today focused on studying the most wide form of tumor affecting human kidney, renal cell carcinoma (RCC), because of our inability to diagnose and treat this very aggressive neoplasia. Different complementary approaches based on genomic and proteomic tools are used to highlight its altered molecular processes, and new developed methods and techniques are implemented in the search of possible biomarkers. However, notwithstanding the great work done by several groups and the enormous amount of information present in literature, knowledge about its pathogenesis is still incomplete, and several markers of RCC are proposed but not yet validated.

Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Renal cell carcinoma (RCC) tissue is composed of a mixture of neoplastic and normal cells, which complicate proteome analysis. The aim of our study was to investigate whether it is feasible to establish primary cell cultures of RCC and of renal cortex maintaining the tissue phenotype along with a more homogeneous and enriched cytological material. Fourteen (82.3%) primary cultures from 17 surgical cases were established and characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, Western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry. Cultures showed >90% cytokeratine-positive epithelial cells. In primary tumor cultures, the molecular phenotype of manganese superoxide dismutase and heat shock protein 27 was the same as that found in tumor tissues with overexpression and increased number of isoforms. Moreover, 27 out 28 specific proteins and their isoforms, present in spots excised from 2-DE gel of cortex or RCC cultures, corresponded to those identified on the 2-DE tissue cortex reference map, suggesting that these primary cultures retain the proteomic profile of the corresponding tissues.

Proteomics methodologies hold great promise in basic renal research and clinical nephrology. The classical approach for proteomic analysis couples two-dimensional gel electrophoresis (2-DE) with protein identification by mass spectrometry, to produce more global information regarding normal protein expression and alterations in different physiological and pathological states. In this report we have expanded the identification of proteins in the renal cortex, improving the previously published map to facilitate the study of different diseases affecting the human kidney. About 250 spots were analyzed by peptide mass fingerprinting, 89 proteins and 74 isoforms for some of them were identified and implemented in the normal human renal cortex 2-DE reference map. This more comprehensive view of the proteome of the human renal cortex could be of invaluable help to the differential proteomic display of urological diseases.

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.

The phosphorylation of heat shock protein 27 (HSP27) occurs differently in human renal cell carcinoma (RCC) compared to homologous normal kidney tissue. Two-dimensional electrophoresis was used to separate and visualize HSP27, via immunostaining with anti-HSP27 antibody, in tumor and normal renal samples, obtained after surgery resection from patients with RCC. The mean number of protein species was 21 in RCC and 15 in normal tissues. Selected spots were in-gel digested with trypsin, extracted and analyzed by microcapillary liquid chromatography (LC) electrospray ionization tandem mass spectrometry to confirm HSP27 protein identity and reveal phosphorylation sites. Loss of phosphopeptides due to extensive plumbing and/or metal components in automated LC-systems was limited by manual loading of samples directly onto the LC system using a homemade pressure vessel. Mass spectrometry (MS) analysis revealed that in three of the HSP27 protein species phosphorylation occurred at Serine 15 and in five at Serine 82 in a different pattern. The phosphorylation of Serine 15 and 82 was also investigated by immunohistochemistry on tissue sections. The data obtained using anti-HSP27Serine82phos-antibody are consistent with MS results, while the variance between results achieved by anti-HSP27Serine15phos-antibody and by MS is probably due to the low specificity of the antibody. Knowledge of the diversity and modulation of HSP27 phosphorylation protein species might represent useful markers involved in the differentiation of RCC. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 pI range and 18-29 kDa M(r) range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.

Renal cell carcinoma (RCC) originates in the renal cortex. It accounts for 2 —3 percent of all cancers occurring in adults and it is characterised by lack of early clinical manifestations, un- predictable outcome, and absence of effective treatment modalities except early surgery. RCC com- prises a heterogeneous group of tumours with various molecular and cytogenetic abnormalities and different histological features as cell types and tumour architecture. Molecular genetic and proteo- mic tools led to the discovery of potential diagnostic prognostic and therapeutic biomarkers of RCC. In this review we discuss recent developments in understanding genotype-phenotype rela- tionships, with attention to manganese superoxide dismutase, a mitochondrial enzyme related to the redox cycle which affects various regulatory functions of cells. The expression of this protein has been evaluated in numerous human tumour types including RCC, and post-translational modifica- tions are being investigated. Keywords. Two-dimensional electrophoresis / Renal cell carcinoma / Manganese superoxide dis- mutase / Post-transitional modifications / Review Abbreviations. IFN, interferon; IL, interleukin; Mn-SOD, manganese superoxide dismutase; RCC, renal cell carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.