Nuclear reaction measurements on tissue-equivalent materials and GEANT4 Monte Carlo simulations for hadrontherapy.

When a carbon beam interacts with human tissues, many secondary fragments are produced into the tumor region and the surrounding healthy tissues. Therefore, in hadrontherapy precise dose calculations require Monte Carlo tools equipped with complex nuclear reaction models. To get realistic predictions, however, simulation codes must be validated against experimental results; the wider the dataset is, the more the models are finely tuned.Since no fragmentation data for tissue-equivalent materials at Fermi energies are available in literature, we measured secondary fragments produced by the interaction of a 55.6 MeV u(-1) (12)C beam with thick muscle and cortical bone targets. Three reaction models used by the Geant4 Monte Carlo code, the Binary Light Ions Cascade, the Quantum Molecular Dynamic and the Liege Intranuclear Cascade, have been benchmarked against the collected data. In this work we present the experimental results and we discuss the predictive power of the above mentioned models.

Is a single focus of low-grade prostate cancer diagnosed on saturation biopsy predictive of clinically insignificant cancer?

OBJECTIVES: To evaluate the incidence of indolent prostate cancer (PCa; <0.5 ml cancer and Gleason score, GS,

Posttransplant proteinuria associated with everolimus.

Anti-mTOR may induce proteinuria when utilized after renal transplantation. Little is known about the pathogenesis and composition of proteinuria. To clarify this unresolved aspect, we analyzed urinary protein composition utilizing an integrated proteomics approach, including quantitative assays, 2-dimensional electrophoresis, MALDI-TOF, and Western blots among 48 renal transplant recipients treated with everolimus (EVL; n = 31) or enteric-coated mycophenolic acid (EC-MPA; n = 17). High (>3 g/d) or intermediate levels of proteinuria (1-3 g) developed in 12 EVL patients (39%) compared with 4 subjects (23%) in the EC-MPA group. Proteinuria, which started during the first 2 days after EVL, tended to reduce during the follow-up. Quantitative proteomics showed an increase in low molecular proteins beta2 microglobulin (P < .001) and alpha1 microglobulin (P < .025). Qualitative proteomics showed a marked increase among all urinary components in EVL and EC-MPA patients. Major changes involved typical components of glomerular damage: albumin, Zn-alpha1 glycoprotein, alpha2HS glycoprotein, and leucine-rich alpha2 glycoprotein. In addition, we observed specific biomarkers for EVL: clusters of alpha1-antitrypsin fragments and monoclonal lambda chains. In conclusion, EVL induced proteinuria of a mixed glomerular and tubular origin that correlated with the start of treatment and reached nephrotic ranges in few cases. The specific urinary markers may reflect renal alterations related to the transplant or specific alterations associated with the drug.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

Circulating anti-actin and anti-ATP synthase antibodies identify a sub-set of patients with idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome (iNS) with resistance or dependence to steroids is a common disease in children but in spite of an increasing clinical impact its pathogenesis is unknown. We screened for the presence of circulating antibodies against glomerular (podocytes, mesangium) and tubular cells (tubular epithelia) a cohort of 60 children with iNS including 8 patients with a familial trait of iNS or with proven mutation of NPHS1-NPHS2 and 12 with good sensitivity to steroids. Positive sera were found in 8 cases, all belonging to the category without familial trait/molecular defects. The targets of antibodies were characterized with Western blot and MALDI-Mass utilizing beta-hexyl cell extracts separated with two-dimensional electrophoresis. In all cases antibodies of the IgM class were directed against ATP synthase beta chain alone (4 cases) or in combination with actin (3 cases); one child presented IgG against aldose reductase. The clinical picture was nephrotic syndrome with steroid resistance or dependence and variable cyclosporin sensitivity; 3 patients developed end stage renal failure. The basic pathology picture was focal segmental glomerulosclerosis (FSGS) in 4 cases and mesangial proliferative glomerulonephrites with deposition of IgM in 2. Overall, patients with circulating auto-antibodies could not be readely differentiated on clinical grounds with the exception of 3 children who developed positivity for antinuclear antibodies during the follow-up. Affinity-purified IgM from one patient who underwent plasmapheresis for therapeutical pourposes (but not from a normal pool) induced proteinuria in Sprague-Dawley rats and concomitant human IgM deposition within glomeruli. This is the first report of circulating anti-actin/ATP synthase beta chain antibodies in a subset of patients with iNS. Both pathological significance and clinical impact given by the presence of these antibodies and the relationship with other conditions such as lupus-erythematosus, characterized by their presence, must be defined.

Nuclear translocation of a clusterin isoform is associated with induction of anoikis in SV40-immortalized human prostate epithelial cells.

Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.

Inhibition of renal permeability towards albumin: a new function of apolipoproteins with possible pathogenetic relevance in focal glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a degenerative renal disease characterized by the accumulation of extracellular matrix and lipids within the glomerular tuft. It has been proposed that an abnormal renal permeabilization towards proteins induced by a putative plasma factor is, in some way, involved in the pathogenesis of the disease. In this paper, we measured the plasma permeability activity (Palb) in several sera of patients with FSGS and found a mean activity of 0.82+/-0.03 which means a marked increase compared to a mean Palb of 0.16+/-0.03 in normal controls. Coincubation of FSGS and normal serum reduced the permeability activity within the normal range; normal serum added to the incubation medium after the glomeruli had already been exposed to the FSGS serum had no effect, suggesting the presence of inhibitory substances with a direct effect on a circulating substrate. Finally, the antipermeability activity was retained when heated to 60 degrees C but not to 100 degrees C. By serial fractionations of normal serum and reported activity measurements at each step, five natural occurring inhibitors of albumin permeabilization were purified and characterized by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), as components of apolipoproteins (apo) (apo E2 and E4, apo L, the high Mr apo J and a 28 kDa fragment of apo A-IV). Coincubation of each apolipoprotein with FSGS serum inhibited permeability, but only apo J and apo E2 and E4 were found to be crucial for the process. In conclusion, we have purified from normal serum five inhibitors of permeability induced by FSGS serum, all corresponding to apolipoproteins. An imbalance between permeability factors and apolipoproteins may play a pathogenetic role in FSGS.

New high-performance liquid chromatographic method for the detection of picolinic acid in biological fluids.

A HPLC method is described to quantify picolinic acid in milk, blood serum and tissue culture supernatant. The method requires very little sample preparation because acid precipitation allows total recovery of picolinic acid. High specificity and sensitivity were obtained using ion-pair chromatography on a C18 reversed-phase column with tetrabutylammonium hydrogen sulfate as ion pairing reagent. We describe the conditions for the automated testing of multiple samples and for the detection of L-tryptophan and L-kynurenine together with picolinic acid. This system will be utilized to elucidate the relationship between picolinic acid production and human disease. Furthermore, we provide the first evidence of picolinic acid in human blood serum.

Humoral permeability factors in the nephrotic syndrome: a compendium and prospectus.

The concept that increased glomerular albumin permeability in steroid-resistant nephrotic syndrome is induced by circulating humoral factors is not new. Zimmermann (1) was among the first to demonstrate that serum from a renal transplant patient with recurrent focal segmental glomerulosclerosis (FSGS) could provoke increased albumin excretion when infused in the aorta of intact rats. Unfortunately, the experiment was not easily reproducible, and the possibility that human serum could induce serum sickness in rats was a serious limitation of the original experiment. We now know that inhibitors of permeability activity are present in both normal human and rat serum (see below), which explains the difficulty in replicating the disease in intact animals. In 1974 Shalhoub (2) theorized that a disordered clone of T lymphocytes, present in both minimal change disease and FSGS, secreted a circulating lymphokine "toxic" to the glomerular barrier. In support of this hypothesis, Koyama et al (3) formed hybridomas from T cells from four patients with minimal change disease and three control subjects. The hybridomas of the patients produced a substance that induced proteinuria when injected intravenously into normal rats. However, the study utilized stimulated and not quiescent T cells, and therefore the relevance to the pathogenesis of FSGS is unknown. Hoyer and colleagues first described recurrence of idiopathic nephrotic syndrome after renal transplantation in 1972 (4). Numerous subsequent reports have established the rate of recurrence as being about 30%. Timely plasmapheresis associated with aggressive immunosuppression resolves the proteinuria and disease progression in a large proportion of cases (5). FSGS not only recurs after renal transplantation, but the diseased kidney can also recover when kept protected from the pathological milieu. Rea et al (6) demonstrated that kidneys from a donor with FSGS transplanted into two uremic recipients were free from proteinuria, and that renal function was normal after one year. Ethical and legal considerations aside, recurrence of FSGS after transplantation is strong evidence supporting the role of a humoral factor in the pathogenesis of the disease.

Analytical titration curves of glycosyl hydrolase Cel45 by combined isoelectric focusing-electrophoresis.

The electrical charge of endocellulase Cel45-core has been determined by combined isoelectric focusing-electrophoresis in the range of pH 3-9. In order to transform electrophoretic mobility to absolute electrical charge value, several corrections were applied: the frictional coefficient theoretically calculated from the molecular dimensions depends on porous gel structure and on the ionic strength of the solution. By comparing the curve calculated according to the Linderstrom-Lang equation, the number of charged electrical groups exposed to the solvent and their apparent ionization constants, pK(o)i, can be determined. Furthermore, the macromolecule structure can be assumed not to change in this pH range. This finding is necessary to understand the structure and the electrical properties of the entire Cel45 molecule.

Identification of HSP-60 as the specific antigen of IgM produced by BRG-lymphoma cells.

In previous studies we described a patient with Burkitt's lymphoma and AIDS, whose cells recognized a molecule expressed by normal and malignant breast cells. In the present study, we identified this antigen by two-dimensional (2-D) electrophoresis and Western blotting using the antibody produced by lymphoma cells. The antigen so identified consisted of two clusters of spots with a molecular mass (Mr) of 60 and 50 kDa, respectively. Preparative immobilized pH gradient (IPG) was subsequently used to isolate the clusters of spots of higher molecular masses, from which peptide fragments of approximately 10 aa were separated on reverse-phase chromatography and sequenced. This procedure enabled the identification of the antigen recognized by the lymphoma cells as HSP-60. By means of serological analyses it was possible to identify the lower molecular mass cluster of spots as a molecule related to HSP-60. It is hypothesized that this molecule is a membrane form of HSP-60 that differs from HSP-60 in a COOH terminal portion.

Rapid detection of gammaT cell receptor gene rearrangements in acute lymphoblastic leukemia by electrophoresis and silver staining: implications for detection of minimal residual disease.

Minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) was studied using polymerase chain reaction (PCR). GammaT cell receptor (TCRG) genes are ideal targets for PCR-based detection of MRD due to their molecular characteristics. Polyacrylamide gel electrophoresis (PAGE) analysis of PCR products followed by silver staining was performed for 72 children with ALL at the onset of disease. Silver staining is an effective technique to detect gene rearrangements without the use of ethidium bromide. Moreover, this method may show heteroduplex bands of a clonal nature when both TCRG alleles are rearranged. PCR products subjected to a rapid staining protocol were recovered from the gel, reamplified by a second PCR and directly sequenced. After sequencing, we identified the junctional region and obtained patient-specific probes. In more than half of the patients we detected TCRG rearrangements that were used as molecular markers for residual disease.

Resolution of fibronectin and other uncharacterized proteins by two-dimensional polyacrylamide electrophoresis with thiourea.

Several proteins, which are recognized components of serum, are not resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) under standard conditions. One major example is fibronectin, which is detected in fairly high concentration (milligram range) by immunoassays, while undetectable in 2D-PAGE gels. Following several experiments with a combination of zwitterionic and chaotropic substances we obtained a good resolution of the protein in gels containing 0.5 M thiourea plus 8 M urea. By this technique, fibronectin was, for the first time, found to be microheterogeneous between pl values of 5.3 and 5.6. Besides fibronectin we detected three other families of uncharacterized proteins with Mr of 130000, 110000 and 34000 respectively, whose identity and function are currently under investigation.

alpha 1-Antitrypsin (AAT) deficiency and ANCA-positive systemic vasculitis: genetic and clinical implications.

A high incidence of alpha 1-antitrypsin (AAT) deficiency has been reported in patients with C-ANCA systemic vasculitis in association with antibodies against proteinase-3 (PR3). To clarify the role of AAT deficiency in the acute vasculitic process as well as in progression of the disease, we studied 84 patients with either C-ANCA or P-ANCA vasculitis with special reference to: (a) the AAT gene, (b) the phenotypic (Pi) variants and (c) the serum levels during both acute illness and remission. The PiZ gene was found in six patients (8% vs. 1.5% controls) irrespective of the type of autoantibodies (C-ANCA vs. P-ANCA). All PiZ patients displayed the ability to raise their AAT serum levels up to the normal range during acute illness. In contrast, 24 patients with the PiM phenotype presented low AAT serum levels during acute illness. In all these patients, the AAT levels returned to normal values during the remission. Low AAT levels were associated with low levels of C-reactive protein (PCR) (P < 0.001), with a less severe renal involvement or a minor risk of death, and, in one tested patient, with a novel point mutation (TCGA-->TCAA) at the enhancer-promoter region of the AAT gene. Low AAT serum levels did not correlate with either type/titre of autoantibody or distribution/severity of the vasculitis process. In the case-control study, high AAT levels emerged as a major determinant of progression towards end-stage renal failure [odds ratio 3 (95% CI 1.1-8.4)]. These results indicate: (a) a high incidence of the PiZ gene of AAT in systemic vasculitis irrespective of the type of autoantibodies; (b) a novel form of AAT deficiency associated with the normal PiM phenotype becoming manifest only during acute illness; (c) dysregulation of the acute-phase response affecting selectively AAT or both AAT and PCR; (d) correlation between low plasma levels of AAT and less severe renal involvement or risk of death.

Negative staining of proteins in polyacrylamide gels with methyl trichloroacetate.

This paper describes a new, sensitive (in the nanogram range), and rapid (two-step) technique for the negative staining of proteins in polyacrylamide gels in the presence or absence of sodium dodecyl sulfate. After separation, gels are incubated with 8% methyl trichloroacetate ester in 38% isopropanol and then washed in water to produce a negative image of colorless proteins against an opaque background. The technique allows unmodified proteins to be recovered for biological studies or transblot for amino acid sequence. Finally, owing to the reversibility of the process, gels can be restained after rapid visualization. For these reasons, negative staining with methyl trichloroacetate should become the method of choice for rapid and sensitive staining of proteins prior to further processing, including stable staining with silver ions.

Purification of alpha-1-antitrypsin monomer by preparative electrophoresis.

Alfa-1-antitrypsin (alpha 1AT) was purified by pseudoligand chromatography and preparative electrophoresis from the serum of a patient with alpha 1AT deficiency. The combination of the two techniques yielded a high grade batch of alpha 1AT monomer and this was successfully used to purify the protein from the serum of PiMIM1, PiMIM2, and PiZZ phenotype subjects. This procedure should facilitate structural studies of alpha 1AT variants susceptible to intracellular accumulation.

Intact renal albumin downregulates the extracellular matrix expression by mesangial cells and renal fibroblasts in vitro.

Chronic Adriamycin (ADR) nephropathy is invariably associated with glomerulosclerosis and tubulointerstitial fibrosis. To investigate the hypothesis that severe albuminuria plays a role in the pathogenesis of both processes, we purified the protein from conditioned media of rats with advanced ADR nephropathy and tested the fibrogenic effect on renal fibroblasts and mesangial cells in vitro. Albumin was purified by pseudoligand chromatography and was identified on the basis of the NH2 amino terminus. Furthermore, it was differentiated from the urinary homologue, being more anionic and more fatted while maintaining a conserved peptide composition. The exposure of renal cells to renal albumin induced a dose-dependent reduction in collagen synthesis with a half-maximal decrease with 0.2 microgram/ml of albumin. With renal albumin levels of 0.4 microgram/ml the collagen incorporation of 3H-proline by mesangial cells and renal fibroblasts (primary cultures and cell lines) was reduced by 76, 81 and 45% respectively. A qualitative analysis by SDS-polyacrylamide electrophoresis and immunoprecipitation of radiolabelled collagens demonstrated a drastic and unselective decrease in all major collagens synthesized by mesangial cells and fibroblasts, including type I, III and V. Previous immunoprecipitation of the protein with anti-rat albumin antibodies completely reversed this phenomenon. Finally, albumin purified from urines of rats with ADR nephropathy downregulated the synthesis by renal cells of the same collagens but this effect was less evident compared to renal albumin. These findings demonstrate that renal albumin drastically reduces the synthesis of collagens by mesangial cells and renal fibroblasts, this effect being most evident on those components which constitute the extracellular matrix in glomerulosclerosis and interstitial fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the human COL5A1 gene to chromosome 9q34.3.

A 353-bp region encoding for the NH2 terminus of the noncollagenic part of the alpha 1(V) chain was amplified by the polymerase chain reaction (PCR), subcloned and sequenced. The subcloned PCR product (pGC1) presented the same nucleotide sequence as the original fragment from the published sequence of COL5A1. In situ hybridization, using pGC1 as a probe, mapped the COL5A1 gene to chromosome 9q34.3. This assignment shows that COL5A1 is not synthetic with COL5A2, which is localized together with other collagen genes on chromosome 2.

Extracellular matrix formation by epithelial cells from human polycystic kidney cysts in culture.

Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [3H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [alpha (III)]3 and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M(r) of alpha 1(I) and alpha 2(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize alpha 1(I) and alpha 2(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cytogenetic mechanisms in vivo.