Acute oral toxicity in mice of a new palytoxin analog: 42-hydroxy-palytoxin.

The acute oral toxicity of a new palytoxin congener, 42-hydroxy-palytoxin (42-OH-PLTX), was investigated in female CD-1 mice. The toxin (300-1697 µg/kg), administered by gavage, induced scratching, jumping, respiratory distress, cyanosis, paralysis and death of mice, with an LD50 of 651 µg/kg (95% confidence limits: 384-1018 µg/kg) within 24 h. Hematoclinical analyses showed increased plasma levels of lactate dehydrogenase and aspartate-aminotransferase at doses of 600 µg/kg and above, as well as of alanine-aminotransferase, creatine phosphokinase and potassium ions at ≥ 848 µg/kg. Histology revealed inflammatory lesions in the non-glandular area of the stomach of mice that survived up to 24 h after gavage (424-1200 µg/kg). Although no histological alterations were seen in skeletal and cardiac muscles, changes in some plasma biomarkers (creatine phosphokinase, lactate dehydrogenase) suggested involvement of these tissues in 42-OH-PLTX oral toxicity, in agreement with epidemiological data on seafood poisonings ascribed to palytoxins. Complete recovery of the tissue and hematological changes was observed two weeks post-exposure. Furthermore, 42-OH-PLTX induced in vitro delayed erythrocyte hemolysis at concentrations similar to those of PLTX (EC50 = 7.6 and 13.2 x 10⁻¹² M, respectively). This hemolysis could be completely neutralized by a monoclonal anti-PLTX antibody. The in vivo data, together with the in vitro data recorded for 42-OH-PLTX, seem to indicate Na+/K+-ATPase as one of the key cellular targets of this toxin.

In search of adult renal stem cells.

The therapeutic potential of adult stem cells in the treatment of chronic degenerative diseases has becoming increasingly evident over the last few years. Significant attention is currently being paid to the development of novel treatments for acute and chronic kidney diseases too. To date, promising sources of stem cells for renal therapies include adult bone marrow stem cells and the kidney precursors present in the early embryo. Both cells have clearly demonstrated their ability to differentiate into the kidney's specialized structures. Adult renal stem cells have yet to be identified, but the papilla is where the stem cell niche is probably located. Now we need to isolate and characterize the fraction of papillary cells that constitute the putative renal stem cells. Our growing understanding of the cellular and molecular mechanisms behind kidney regeneration and repair processes - together with a knowledge of the embryonic origin of renal cells - should induce us, however, to bear in mind that in the kidney, as in other mesenchymal tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells. Thus, by displaying their plasticity during kidney maintenance and repair, terminally differentiated cells may well function as multipotent stem cells despite being at a later stage of maturation than adult stem cells. One of the major tasks of Regenerative Medicine will be to disclose the molecular mechanisms underlying renal tubular plasticity and to exploit its biological and therapeutic potential.

[In search of renal stem cells]. / Alla ricerca della cellula staminale renale.

The therapeutic potential of stem cell research is very promising. Although arising ethical questions, especially in the field of embryonic stem cells (ES), it is astonishing how, in the last few years, the potential application of stem cells for treating proliferative as well as degenerative diseases, is becoming increasingly evident. It was recently demonstrated that somatic stem cells showed unexpected plasticity similar to ES. In fact, if somatic stem cells are exposed to proper stimuli they can differentiate into a multitude of cell types that may be different from those of the tissue they belong to. In addition, it was recently demonstrated that circulating blood stem cells, probably of bone marrow origin, were recruited at the sites of injury to regenerate or repair damaged tissues. Very little is known about renal stem cells. Although the great capacity of the kidney to regenerate injured nephrons is well established, renal somatic stem cells have yet to be identified. The question we are now faced with is whether renal stem cells exist and, if they do exist, where do they reside. In the attempt to answer this question, the present review will focus on the achievements both in the fields of somatic stem cells and renal embryogenesis and in the field of renal repair and regeneration mechanisms.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis.

Structural elucidation of a new cytotoxin isolated from mussels of the Adriatic sea.

A detailed analysis of the toxic composition in the hepatopancreas of mussels from northern Adriatic sea has been performed. Along with some polyether toxins of DSP (diarrhetic shellfish poisoning) type, such as yessotoxin and its analogues, which are responsible for a variety of human seafood poisonings throughout the world, we have now isolated a new type of toxin, the chlorosulfolipid 1, which is completely different in structure from the polyether DSP-toxins isolated so far. The structural determination of the new toxin, including its absolute stereochemistry, has been performed by extensive NMR analysis and molecular mechanics and dynamics calculations.

42,43,44,45,46,47,55-Heptanor-41-oxohomoyessotoxin, a new biotoxin from mussels of the northern Adriatic sea.

The diarrhetic shellfish toxin composition in the digestive glands of mussels from the northern Adriatic sea was investigated. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, 42,43,44,45,46,47,55-heptanor-41-oxohomoyessotoxin, 1. Its structure was determined by (1)H NMR spectroscopy and mass spectrometry.

Results and complications of facial reanimation following cerebellopontine angle surgery.

The present study was undertaken to evaluate the results of a group of patients following treatment for cerebellopontine angle lesions who developed postoperative facial palsy and underwent facial nerve repair in order to reanimate the muscles of facial expression. A retrospective study was performed on 23 patients treated between 1988 and 1997 at the 2nd and 4th ENT chairs of University "La Sapienza" of Rome for facial palsy following cerebellopontine angle surgery. Tumors included acoustic neuromas (n = 3). Seventeen patients underwent hypoglossal-facial anastomoses [10 with end-to-end anastomoses, 4 with May's interposition "jump-nerve" grafts and 3 with partial (30%) use of the hypoglossal nerve plus a facial cross-over]. The remaining patients were operated on using a cable graft with the sural nerve (n = 2) and the great auricular nerve (n = 4). Postoperative facial function was determined by the House-Brackmann 6-scale classification The hypoglossal-facial anastomoses resulted in long-term grade III or IV findings. Cable grafts improved facial function from grade VI to grade III. None of the patients operated on with the modfied VII-XII anastomosis developed swallowing disturbances. The ten patients having traditional hypoglossal-facial anastomoses showed different degrees of tongue disability and retention of residue in the oral cavity. Surgical recovery of postoperative facial palsy can be obtained with various techniques according to the availability of the proximal facial nerve stump at the brain stem. Since a traditional hypoglossal-facial anastomosis procedure can be a source of a separate disability for the patient, techniques are preferred that leave the hypoglossal nerve mostly intact and uncompromised.

Reconstruction of the posterior auditory canal with hydroxyapatite-coated titanium.

There are a variety of techniques for treating chronically discharging radical mastoid cavities. The purpose of this article is to report the preliminary results of an original technique for reconstruction of the posterior auditory canal using a titanium net combined with porous hydroxyapatite coating. Titanium is fixed with two screws to the mastoid tip and zygomatic root to prevent the risk of implant dislocation. Eight patients with chronically discharging radical mastoid cavities that failed medical management underwent reconstruction of the mastoid cavity using this technique. After surgery, all cases had rapid healing and good aeration of the middle ear and mastoid. One tympanic membrane reperforated, and no extrusion of the prostheses were detected clinically or on computed tomography scanning. The minimum postoperative follow-up period has been 12 months (range 12-48 months). To date, there has been no evidence of cholesteatoma recurrence. The preliminary results remain encouraging. Larger series and longer follow-up, however, are advisable to prove real validity.

Structure determination of carboxyhomoyessotoxin, a new yessotoxin analogue isolated from adriatic mussels.

The contamination of shellfish with marine biotoxins derived from microalgae represents a serious problem for shellfish industries and public health. This study investigated the composition of diarrhetic shellfish toxins in the digestive glands of mussels from the Northern Adriatic Sea. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, carboxyhomoyessotoxin, whose structure was determined by mass spectrometry and (1)H NMR spectroscopy.

Saxitoxin and neosaxitoxin as toxic principles of Alexandrium andersoni (Dinophyceae) from the Gulf of Naples, Italy.

A clonal culture of Alexandrium andersoni, obtained from germination of a resting cyst, collected from the Gulf of Naples, was found positive for PSP toxicity by mouse bioassay. The toxicity profile of this dinoflagellate consists mainly of toxins belonging to the saxitoxin class, in particular of Saxitoxin (STX) and Neosaxitoxin (NEO), as determined by a wide MS and (1)H NMR analysis. This represents the first report of the presence of A. andersoni in the Mediterranean Sea, as well as of its toxicity.

Direct effect of chronic cyclosporine treatment on collagen III mRNA expression and deposition in rat kidneys.

BACKGROUND: It is hypothesized that in acute and chronic CsA nephrotoxicity, in vivo models CsA side-effects are mediated by Renin-Angiotensin II (RAS)-TGF-beta-1 pathway. However, to induce chronic nephrotoxicity, CsA administration has to be combined with a low salt diet, which causes hemodynamic changes and RAS up-regulation. MATERIALS AND METHODS: In order to define any direct correlation between CsA and nephrotoxicity, we studied in normal sodium fed rats, the chronic effects of CsA administration (group-1 treated with 12.5 mg/Kg/day of CsA subcutaneously; group 2 received daily placebo; group 3 interrupted CsA injection after 60 days), on renal TGF-beta-1 and collagen III expression, and on TGF-beta-1, collagen III and IV deposition. Sacrifices were performed after 2, 4, 8 and 12 weeks (wks) and kidneys were harvested for immunohistological studies and RT/PCR analysis. RESULTS: No difference of TGF-beta-1 expression and deposition was found among groups. Starting from the 2nd week of treatment, an increased collagen III deposition was evident in vessels and in outer medulla with subsequent extension at the 4th week to medullary rays and to cortex interstitium. The deposition paralleled the renal collagen III mRNA up-regulation: it was significantly higher in group 1 than in group 2 (p < 0.009 at 2nd wk; p < 0.016 at 4th wk). Collagen IV deposition did not differ between groups at any point. CONCLUSIONS: Our results suggest that chronic CsA administration can induce, in normal fed rats, the process of interstitial fibrogenesis through TGF-beta non-related mechanisms.

Acoustic neuroma: postoperative quality of life.

OBJECTIVE: Evaluating patients who have had surgical management of acoustic neuroma has relied heavily on the surgeon's viewpoint for determining success. However, the perspective of the surgeon may be different from that of the patient. Thus, a recent increased interest in terms of quality of life has been documented by the literature on this specific topic essentially through the use of a questionnaire. The objective of this paper was to review this topic in our series of patients operated on for acoustic neuroma to ascertain the personal and social impact that surgery has had on their lifestyle. DESIGN: This retrospective study was devoted to increasing statistics to provide more detailed and valid information during the counselling phase. METHODS: This study was carried out on 82 patients who underwent surgery for acoustic neuroma between 1988 and 1997. Each patient was recalled and assessed for his/her postoperative quality of life. Detailed information was requested on the initial postoperative facial, vestibular, and hearing functions; their evolution; and their social consequence. Finally, at the end of the interview, each patient was invited to give a final comment on his/her opinion regarding the outcomes of surgery and preoperative information. RESULTS: Facial function showed a grade I-III in 85.4% of cases, with postoperative neurovegetative dysfunction (taste and lacrimation) in 43%. Audiologic abnormalities (worsening hearing and tinnitus) were complained of in 90% and 57% of the cases, respectively. Twenty-three percent of the patients had various degrees of gait instability; 6% reported postoperative headache at 1-year follow-up. Social consequence (reduced work ability, vocational change, new education, state pension, etc.) was not influenced by surgery in 80%. CONCLUSIONS: Our experience is in general agreement with previously reported statistics. It is interesting to note that our patients exhibited more disturbances linked to the sensory component of facial nerve. In contrast, dysequilibrium had a less negative influence. These outcomes suggest the importance of thorough preoperative counselling in candidates for surgery for acoustic neuroma in order to motivate them and, at the same time, to reduce their psychological discomfort.

Mesangial cell proliferation in long-term streptozotocin-induced diabetes mellitus in the rat and the renoprotective activity of heparin.

At present, it is not clear whether mesangial proliferation underlies mesangial expansion in diabetic nephropathy. To address this issue and the relationship between heparin's renoprotective and antimitogenic activities, we studied three streptozotocin-induced diabetic rat groups 5 and 12 months after diabetes induction: two groups were administered a modified heparin, each with a different protocol, and two healthy rat groups, one of which was treated with the same heparin, served as controls. Untreated diabetic animals developed clear evidence of nephropathy, namely expansion of the glomerular extracellular matrix, as expressed by glomerular basement membrane thickening, and increased mesangial deposition of type IV collagen. These alterations were prevented/cured by heparin treatment. Kidney sections were processed immunohistochemically for proliferating cell nuclear antigen and smooth muscle alpha-actin which is expressed only by proliferating mesangial cells. The number of proliferating cell nuclear antigen positive nuclei and alpha-actin-positive cells per glomerulus did not differ between groups at both 5 and 12 months. In conclusion, there is no evidence that mesangial proliferation is increased in late experimental diabetic nephropathy, and heparin seems to be renoprotective through mechanisms other than antiproliferation.

Isolation of 45-hydroxyyessotoxin from mussels of the Adriatic Sea.

The diarrhetic shellfish toxin composition in the hepatopancreas of mussels from the northern Adriatic sea was investigated. The major toxins were shown to be yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxyyessotoxin (45-OHYTX), identified by comparison of their chromatographic and spectral properties with those reported in the literature.

Molecular biology of diabetic glomerulosclerosis.

Diabetic nephropathy is one of the leading causes of renal failure in Western countries, where diabetic patients account for nearly half of all patients on haemodialysis. Progressive expansion of the mesangial matrix, and thickening of the glomerular and tubular basement membranes without signs of major cell proliferation are hallmarks of human and experimental diabetic nephropathy. These lesions eventually lead to glomerular fibrosis, a central pathological feature in many human acute and chronic kidney diseases, which progressively destroys the renal filtration unit, and may finally cause renal failure. Indeed, structure function relationship studies have shown that mesangial matrix expansion is strongly related to the clinical manifestation of diabetic nephropathy.

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Von Willebrand factor abnormalities in IgA nephropathy.

BACKGROUND: Plasma concentration of von Willebrand factor (vWF) has been used as an index of endothelial dysfunction. Increased release of vWF from endothelial cells has been reported in several conditions, and there is also evidence that dysfunctioning endothelial cells synthesize defective molecules. In fact, unusually large vWF multimers have been described and related to the pathogenesis of some microangiopathic diseases. Abnormal levels of vWF have been reported in primary glomerulitis, but this was no referred to histological diagnosis. Furthermore, no qualitative vWF analysis was performed in these glomerulopathies. Therefore the aim of our study was to analyse quantitatively and qualitatively vWF in patients with IgA (IgAN) and non-IgA mesangial proliferative glomerulonephritis (PGN). METHODS: Fourteen IgAN patients, eight PGN patients, seven subjects with different glomerulonephritides, and 10 healthy controls formed the basis of this study. On peripheral venous blood collected in the presence of protease inhibitors, vWF parameters were investigated. vWF antigenic activity (vWF:Ag) was measured by electroimmunodiffusion. vWF subunits mobility was studied by crossed immunoelectrophoresis (CIE) and in some patients vWF multimeric analysis was performed by SDS-agarose gel electrophoresis. RESULTS: Mean vWF:Ag was significantly higher in PGN patients as compared to controls, while there was no significant difference between PGN and IgAN patients and between IgAN and controls. CIE revealed a pre-peak in 12 of 14 IgAN patients and a migration index which did not differ between controls, IgAN, and PGN subjects. No pre-peak was observed in PGN and in other glomerulonephritides. Analysis of plasma vWF multimeric pattern by SDS-agarose gel electrophoresis disclosed in four IgAN patients abnormally large vWF multimers that were not documented in PGN subjects. CONCLUSIONS: This study, by showing the presence of a pre-peak and of large vWF multimers in IgAN patients, suggests an altered postsecretory handling of the vWF in IgAN and possibly a different role of the vWF in IgAN in respect to PGN.

Yessotoxin in mussels of the northern Adriatic Sea.

This study investigated the composition of diarrhoetic shellfish toxins in the hepatopancreas of mussels from the northern Adriatic Sea. The major toxins were shown to be yessotoxin, identified by its chromatographic properties and spectral data, and okadaic acid, detected both by fluorometric high-performance liquid chromatography and by comparison of its spectral properties with those of an authentic sample.