A Phospholipid Profile at 4 Months Predicts the Onset of Celiac Disease in at-Risk Infants.

Celiac disease (CeD) is a multifactorial disease influenced by both genetic and environmental risk factors. CeD genetic components are mainly due to HLA class II genes, which account for approximately 40% of the disease heritability. The environmental factor is linked to gliadin ingestion. Despite genetic and epigenetic studies, the pathological molecular mechanism remains unclarified. The strong genetic component does not explain more than half of the hereditability; we identified several epigenetic features that contribute to the understanding of the missing hereditability. The lipid profile of infants has been proposed as a potential biomarker of CeD metabolism that can be measured before they exhibit developmental disorders and clinical symptoms. We suggest that the state of the host is a main factor for the abnormal immune response to gluten. Long before any exposure to the offending agent or any production of specific antibodies, several molecular mechanisms are differentially expressed in infants who will develop CeD compared to their peers matched for the same genetic profile. The present study explored the serum phospholipid profile of a group of infants at risk for celiac disease, followed up to 8 years to monitor the onset of CeD. We compared 30 patients who developed the disease with 20 age- and sex-matched peers with similar genetic profiles who did not develop the disease within 8 years. Serum phospholipids were analysed at 4 months, before exposure to gluten, and at 12 months of age, when none showed any marker of disease. In the 30 CeD patients, we also analysed the serum at the time of diagnosis (>24 months). The serum phospholipid profile was fairly constant across 4 and 12 months of age and, in CeD, up to 24-36 months. The phospholipid signature was dramatically different in infants who developed CeD when compared to that of control NY-CeD (Not Yet developing Celiac Disease) peers. We identified a specific serum phospholipid signature that predicts the onset of celiac disease in HLA at-risk infants years before the appearance of antibodies specific for CeD in the serum and before any clinical symptoms, even before gluten introduction into the diet at 4 months. Specifically, lysophosphatidylcholine, phosphatidylcholine, alkylacyl-phosphatidylcholine, phosphoethanolamines, phosphatidylserines, phosphatidylglycerol and phosphatidylinositol were found to be differentially represented in CeD versus NY-CeD. A set constituted by a limited number of alkylacyl-phosphatidylcholine and lyso-phosphatidylcholine, together with the duration of breast-feeding, allows the discrimination of infants who develop celiac disease before 8 years of age from those at a similar genetic risk who do not develop the disease. In addition to recent discovery, our paper unveiled a specifc phopholipid profile, able to discriminate infants who eventually develop celiac disease years before antibodies or clinical symptoms ensue.

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.

Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor.

Nerve growth factor (NGF) is a member of the neurotrophin family. These growth factors support neuronal survival and differentiation. Neurotrophins are synthesized as pre-pro-proteins. Whereas the pre-sequences mediate secretion, the function of the pro-peptides is largely unknown. To test the role of the pro-sequence as a folding enhancer, recombinant human pro-NGF (rh-pro-NGF) was produced in Escherichia coli. The oxidative refolding of rh-pro-NGF and rh-NGF was studied using electrospray mass spectrometry (ESIMS) time-course analysis. This analysis permitted both the identification and quantification of intermediates present during the process. The disulfide bonds formed at different times of the refolding processes were characterized by proteolytic digestion followed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) analysis. Folding yields and kinetics of rh-pro-NGF were significantly enhanced when compared to the in vitro refolding of mature rh-NGF. These results suggest that the pro-sequence of NGF promotes folding of the mature part.

Early intermediates in the PDI-assisted folding of ribonuclease A.

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.

Comparison of the kinetics of S-S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme.

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.

Effect of deamidation on folding of ribonuclease A.

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.

Analysis of RNase A refolding intermediates by electrospray/mass spectrometry.

Electrospray/mass spectrometry (ES/MS) was extensively used to obtain information on disulphide-containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular S-S are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides. The results indicate that ES/MS is a suitable technique to detect and characterize refolding intermediates.

Lithotripsy in the treatment of urinary lithiasis.

Based on an extensive review of the literature and on our own clinical experience, this article attempts to present clear guidelines for the management of various kidney stones, particularly regarding the extracorporeal shock waves lithotripsy (ESWL) treatment nowadays. Few technical developments have changed medicine more within a short period of time than ESWL. Fifteen years after the first clinical application, ESWL has gained world-wide acceptance as first choice therapy for most forms of urolithiasis. Ninety-eight per cent of stones can be successfully fragmented by the application of shock-waves, but the ability of the kidney and ureter to clear the resulting fragments is far more important in terms of successful treatment outcome. Increasing experience with new ultrasound-guided lithotriptors has shown that there are some advantages: cost reduction, permanent monitoring and lack of exposure to ionising radiations. ESWL is a safe procedure for the treatment of urolithiasis; nevertheless some problems remain. In ureteric stones, ureteroscopy (rigid or flexible device) allows a rate of stone-free patients better than ESWL. For treatment of large staghorn calculi combined approach of PCNL and ESWL is preferred. For stones located at lower calyx, the stone-free rate in patients treated by ESWL fell to 50%, when unfavourable anatomy is present. The potential long-term renal damage, associated with ESWL in children, have delayed the acceptance of shock-waves into paediatric practice. Recent reports suggest that the renal damage, including the potential risk of hypertension induced by ESWL, is mild and transient. A subgroup of patients (e.g. solitary kidney, impaired renal function, children) required further attention. The fate of residual fragments is unclear. In some cases residual lithiasis tend to result in regrowth and further progression, although ESWL itself does not increase the recurrence rate of urolithiasis. Nevertheless follow-up of stone patients after ESWL is mandatory and the ultimate goal of treating stones by whatever means is to get the patient stone-free and prevent recurrence.

[Urologic laparoscopy: preliminary experience]. / Laparoscopia urologica: esperienza preliminare.

Laparoscopy is a safe and reliable procedure for treatment of cryptorchid testicle, varix ligation, staging of lymph nodes in prostatic and in bladder cancer. Recently this procedure is adopted in management of renal cysts. From 2/92 to 7/92, we performed laparoscopic assessment in 31 patients: in 15 cases for varicocelectomy, in 9 for renal cysts, in 2 for lymph nodes dissection, in 3 for preoperatory evaluation, in 1 for unpalpable testicle and in 1 case for ileal perforation in orthotopic neobladder. In 1 case the procedure was unsuccessful because of damage of the spleen. All the patients were mobilized within 1 day postoperative treatment and discharged within 3 days. There are many present and potential application of laparoscopic surgery in urologic pathology, but we think that this technique must be carefully applied in order to avoid complications.

[Benign prostatic hypertrophy. Optimized diagnosis]. / Ipertrofia prostatica benigna. Diagnostica ottimizzata.

The optimal diagnostics of B.P.H. must be efficient, cheap and of spare invasiveness either physical than psychological. The optimization of diagnosis is the obtainment of high quality in the services provided, matched to a correct and shrewd utilisation of resources. To optimise a diagnosis efficiency is necessary, which means the largest degree of standardization of diagnostical procedures, with responsible management of economical factors. A course of standardization, cause his excessive simplification, cannot shared to a lot of Urologists, because in their opinion there is a risk to lose informations in every single patient, but is essential to use a universal language that make easy the comparison with patients and with results whether in order to clinics or trails. The standardisation of diagnostics consists to obtain the maximum of informations from present methodology and that is possible to realize in two way: improving the technique of execution and including the diagnostic test in the appropriate point of algorithm.

[External devices: for which patients?]. / Dispositivi esterni: a quali pazienti proporli?

Vacuum therapy is a reversible, non-invasive form of treatment for partial impotence, with great success in USA. The story of vacuum therapy begins about 1960, when Osbon developed a vacuum tumescence device which he personally used for more than 20 years. The device was made commercially available many years ago and has been marketed under several names. The newer systems have incorporated a negative pressure pump to achieve vacuum. Osbon's system was patented in 1983, sale is permitted by the U.S. Food and Drug Administration, and it is available by prescription only. More than 10,000 units have been sold. There are four different types of vacuum therapy: 1) loading cone + constriction band; 2) external splint + negative pressure; 3) Negative pressure + constriction band; 4) Negative pressure + intracavernous injections, without the use of constriction band. Each basic system will be described. The authors make a comparison between the use of negative pressure devices plus tension band and the use of negative pression devices without tension band (plus C.I.D. with vaso-active agents). In the second case there's a sort of synergistic action between vaso-active agents and vacuum therapy, representing a sort of "vaso-active exercise" of the erectile tissue. There are no absolute contraindications to use of external penile devices and potential contraindications are few. The external penile devices described represent a reversible therapeutic modality that can augment an inadequate erection and they should prove useful in any man who needs erectile enhancement. These devices appear to be particularly effective in men with partial impotence in whom only erectile enhancement is needed.

[Orthotopic ileal neobladder of the Emikock type: technical points and functional results]. / La neovescica ileale ortotopica tipo Emikock: attualità tecniche e risultati funzionali.

Ileal orthotopic neobladder represents, nowadays, the best urinary diversion after cystectomy. Emikock procedure was performed, in our institution, in 26 patients with bladder cancer T2-T4. At 6-60 months of follow-up 3 pts were died with local or at distance neoplastic recurrence, 2 were alive with neoplasms and 21 were NED. Nocturnal continence was good in 22 cases (88%) and only 3 patients were obstructed because of pseudodyssynergia in 2 and stricture in 1. Emikock neobladder even if needs a longer surgical time than other procedure and a long ileal tract is almost free from severe metabolic disorders. This technique offers a good protection of high urinary tract because of antireflux nipple and avoid the uretero-intestinal stricture. It not feasible, now, to know the functional trend of this reservoir on the long term. Adequate postoperative training is recommended to avoid the pseudodyssynergia and functional obstruction of reservoir.

[Iatrogenic obstructive azoospermia]. / Azoospermie ostruttive iatrogene.

Obstructive azoospermia is a common cause of sterility in men. In the past infection played an important role in the aetiology of obstructive azoospermia. Recently, however, the aetiology of obstructive azoospermia appears to be changing. So iatrogenic obstructive azoospermia has reached an important role in the field of obstructive azoospermia. In this work we show international literature about iatrogenic obstructive azoospermia. Unfortunately it is poor, in spite of an interesting item. We divided iatrogenic obstructive azoospermia into six groups, considering the possible anatomical site of obstruction. So we show the possible damages at the different levels: testis, epididymis, vas deferens, seminal vesicles, prostate and ejaculatory ducts.

[TURP: what and when. Indications, results, complications: our experience]. / TURP: quale e quando. Indicazioni--risultati--complicanze: nostra esperienza.

Transurethral resection of the prostate (T.U.R.P.) has indeed replaced open surgery in the great majority of cases, particularly when the gland is estimated to weight 50-60 g or less. After prostatectomy the patient resumes a normal voiding pattern and obstructive symptoms quickly disappear, although irritative symptoms tend to persist for some time.

[Radical prostatectomy: our experience]. / Prostatectomia radicale: la nostra esperienza.

Between July 1989 and June 1994 32 men (48-73 years old) underwent radical retropubic prostatectomy for prostate cancer. 26 patients (70%) presented with symptoms of bladder outflow obstruction. The primary tumour was understaged preoperatively in 5 patients (15.5%). At follow-up (6-66 months, average 24) significant urinary incontinence not occurred in our patients; sixty-two per cent reported a substantial problem with reduced or absent erection. A total of 23 patients was free of clinical or biochemical progression, observed in 28% of cases as distant or local progression. Radical prostatectomy is being performed with increasing frequency: trends in morbidity have been identified.

[Penile nuclear magnetic resonance (NMR)]. / La risonanza magnetica nucleare (RMN) peniena.

Nuclear Magnetic Resonance (NMR) is a new diagnostic technique with great opportunities of application in the field of the penile pathologies. A new interest for this diagnostic technique was born when the use of vasoactive agents, like papaverine or PGE1, and the use of para-magnetic contrast agents, like gadolinium, were introduced. The introduction of dynamic NMR in andrology allowed a better definition of anatomical details and a better knowledge of penile micro-circulation. N.M.R. is showing a great diffusion, because of a little invasiveness (X-rays are not used in this technique). The Authors show a wide spread of possible applications of NMR in penile pathologies, helping in the interpretation of the images. In conclusion the authors describe NMR as a diagnostic technique with great possibilities of improvement, even if the high costs don't allow a better diffusion until now.

[pT1G3 bladder carcinoma: parameters of a correct therapy]. / Carcinoma vescicale pT1G3: parametri per una corretta terapia.

Between 13.8% and 27% of all superficial bladder cancers are represented by pT1G3 neoplasm. In the Department of Urology of Policlinico S. Marco-Zingonia, between February 1988 and June 1994, we treated 22 patients suffering for pT1G3 bladder tumor. TUR-B has demonstrated to be a good approach for treatment of superficial bladder cancer, with low morbility; on the opposite side, we have to underline the high rate of recurrence and of progression of the urothelium disease. Now a day our best approach for the treatment of pT1G3 bladder tumor is represented by radical cystectomy supplied by chemotherapy.

[RIRS through semi-rigid ureteroscope and holmium laser in the treatment of ureteral stones retropulsion]. / La RIRS di necessità con ureteroscopio semirigido e laser ad olmio nel trattamento della calcolosi ureterale retropulsa.

Retrograde displacement of ureteral stones into the renal cavities during ureteroscopic lithotripsy represents a frequent and adverse event that leads to additional procedures (ESWL, PCNL, Retrograde Intra-renal lithotripsy with flexible instruments, DJ stent placement and subsequent EWSL) to obtain full clearence of calculi. All these procedures require a further time of treatment. Between 1/2008 and 3/2009, a total of 48 patients harbouring proximal (21 cases) and distal (27 cases) ureteral stones underwent Holmium Laser lithotripsy. In 3 patients previous percutaneous nephrostomy was performed to drain the excretory way. In 12 cases (25%) stone retropulsion occurred; in 3 patients in the upper calix and in 5 in the renal pelvis. Only in 4 cases the stone migrated in the lower or medium calix. In 8 cases we attempted the immediate treatment of intrarenal displaced stones by advancing the semi-rigid instrument into the renal cavities. In 2 cases the treatment aborted because of the shortness of ureteroscope. The instillation of lubricating lidocaine jelly prevented in 3 cases furher displacement of stone. Washing with saline solution through nephrostomic catheter allowed an effective mobilization of stone and an easy lasertripsy. RIRS was successful in 4 cases. When flexible devices or immediate ESWL are not available, rigid or semi-rigid retrograde lithotripsy with holmium laser immediately performed after ureteral stone displacement represents a safe and effective method to treat displaced stones. Several tricks are required to obtain a good stone-free rate.

[Neuroendocrine small-cell bladder cancer: our experience]. / Carcinoma neuroendocrino a piccole cellule della vescica; nostra esperienza.

INTRODUCTION: Neuroendocrine bladder cancer is extremely rare, with an estimated incidence of 0.35-0.70% of all bladder tumors. The small-cell carcinoma represents the most frequent histologic variant described. Small-cell carcinoma is an epithelial tumor associated with a more aggressive behavior and poorer prognosis than transitional cell bladder carcinoma. The overall survival rate at 5 years does not exceed 8%. At the time of presentation 59% of patients have clinical stage >T2 and 56% show metastatic disease. In 50% of the patients, fatal progression occurs within 6 months. Local recurrence after radical surgery occurred in 50-70% of cases. PATIENTS AND METHODS: We report three cases of pure neuroendocrine small-cell bladder cancer. Hematuria was the most common presenting symptom. Local advanced disease was present in all the cases with stage >T2, metastatic disease in 1 case, lymph node involvement and ureteral bilateral obstruction in 2. Two patients were treated by radical cystectomy, bilateral pelvic limph node resections and urinary derivation. Platinum-based adjuvant chemotherapy was proposed but only two patients received the treatment. One patient with liver metastasis was managed only by extensive TUR and support regimen. RESULTS: In 2 patients residual or relapsed cancer reappered within 2 months after surgery. All of the three patients died of metastatic disease at 5, 7, and 13 months. Median overall survival was 7 months. The most common site of relapse and spread of disease was the peritoneum and intestinal tract, and the reason of death was uncontrolled acute hemorrhage from gastro-intestinal district. CONCLUSIONS: In the absence of a prospective study, and because of the rarity of the disease, the best treatment for small-cell bladder cancer remains uncertain. Neoadjuvant chemotherapy with platinum regimen plus aggressive surgical approach will be the treatment of choice. The association of chemotherapy and radiotherapy should also be considered.

[Urethral recurrence of invasive carcinoma following BCG treatment for bladder Ca in situ]. / Recidiva uretrale di Ca infiltrante dopo trattamento con BCG per Ca in situ vescicale.

CIS is a flat, high-grade, non-invasive microscopic urothelial carcinoma. It is considered a precursor of invasive bladder cancer. CIS is classified as primary, secondary or concurrent, when occurred as isolated CIS without cuncurrent papillary tumors, or detected during the follow-up of patients with a previous papillary tumor, or finally in the presence of bladder neoplasm. BCG is widely established as the treatment of choice for CIS with a success rate of approximately 70%. BCG reduces the risk of progression of CIS into invasive carcinoma in 30 to 50% of cases. Direct and prolonged contact between the urothelium and BCG is a prerequisite for successful therapy. Discovery of CIS in the prostatic or membranous urethra represents an ominous sign. CIS may be present only in the epithelial lining of the prostatic urethra or in the ducts, or in the worst case it may be found in the prostatic tissue stroma. Urethral involvement by CIS is at high risk of tumor progression and development of metastases due to reduced thickness of lamina propria and absence of muscolaris mucosa. 83 patients, enrolled from 1/1996 to 12/2005 at our urological department with CIS: primary (focal and multifocal) in 25, secondary in 7 and cuncurrent in 51 (associated with T1bG3 cancer in 37 cases), and urethral CIS in 5 and conservatively treated by TUR and intravescical instillations of BCG, 4 developed afterwords only invasive cancer of the urethra in the absence of bladder involvement. In 2 cases cancer arised from the prostatic fossa after TURP, in 1 from membranous urethra and in the last from prostatic ducts. Among the 4 patients, 3 were treated by cystoprostatourethrectomy and Platinum-based chemotherapy, 1 refused surgical treatment. Two patients died for disseminated disease. 1 patient is alive at 60-month's follow-up. In the last patient cancer relapsed at 36-month's follow-up. We conclude that prostatic/urethral involvement during follow-up after successful intravesical treatment with BCG in CIS represents a high risk of developing invasive and incontrolled cancer. A careful watch is recommended in these patients.