Non-collagen genes role in digenic Alport syndrome.

BACKGROUND: Alport syndrome is a clinically heterogeneous nephropathy characterized by severe symptomatology at kidney level due to ultrastructural lesions of the glomerular basement membrane (GBM) as consequence of mutations in COL4 genes. The disease has been linked to COL4A3/COL4A4/COL4A5 mutations, which impair GBM functionality and can be inherited in a dominant, recessive or X-linked transmission. Although a targeted Next Generation Sequencing approach has allowed identifying families with pathogenic mutations in more than one COL4 α3-α4-α5 heterotrimer encoding genes, leading to conclude for a digenic pattern of inheritance, the role of non-collagen genes in digenic Alport syndrome has not yet been established. METHODS: We employed a whole-exome sequencing approach on three families in whom a digenic pattern of transmission could be suspected because of a likely biparental contribution or an unexplained phenotype in the proband. RESULTS: We identified in the three probands hypomorphic LAMA5 mutations co-inherited with pathogenic COL4 α4-α5 chains mutations. Segregation analysis revealed that the combination of LAMA5/COL4 variants co-segregate with a fully penetrant phenotype in line with a digenic inheritance. In one of the three probands an hypomorphic variant in NPHS2 was also found, suggesting that role of other kidney disease related-genes as modifiers. CONCLUSION: These findings validate the impact of LAMA5 mutations in digenic ATS and highlight the role of extracellular matrix's genes, basement membrane, slit diaphragm and podocyte cytoskeleton in ATS. This underline the need for a more extensive panel approach in the presence of a digenic ATS, in order to better define clinical severity and recurrence risk for family members.

Microbiologic investigation on patients with cystic fibrosis subjected to bilateral lung transplantation.

BACKGROUND: In cystic fibrosis (CF) patients, lung transplantation is the only way to improve both quality and length of life. Data in the literature show that, in 80% of the cases, mortality after lung transplantation in CF patients is due to infections. METHODS: We microbiologically monitored 34 patients subjected to bilateral lung transplantation in during 1996 to 1999 to ascertain whether a change in the bacterial species isolated from the lower respiratory tract took place that might have influenced the clinical conditions of the patients. RESULTS: Our results show that the percentage of nonfermenting Gram-negative bacteria isolated from the lower respiratory tract remains high even in the posttransplantation phase. Nevertheless, the general clinical conditions of most of the patients were good and the three patients who died did not do as a consequence of an infection. CONCLUSIONS: Lung transplantation constitutes a valid therapeutic choice for CF patients because the microorganisms that we isolated from the lungs of the patients in our study behave mostly as contaminants rather than as colonizers. However, the transplanted patients remain at risk and thus require constant microbiological surveillance.

Mechanism of action of indomethacin in tubular defects.

Indomethacin, a potent prostaglandin synthesis inhibitor, has been proven to be effective in a number of tubular defects characterized by enhanced prostaglandin (namely, prostaglandin E2 (PGE2) production, but its mechanism of action is poorly understood. To elucidate further the mechanism(s) by which indomethacin reverses the abnormal tubular functions, five children with different tubular defects (nephrogenic diabetes insipidus, three cases; Fanconi syndrome, one case; and pseudohypoaldosteronism, one case) were treated with indomethacin. Indomethacin, 1 mg/kg every eight hours, was given for 1 week to all children and then was given chronically to four of the children who responded to the drug. Its use was suspended in a 10 year-old-boy with nephrogenic diabetes insipidus because it proved ineffective. To assess the site along the nephron where indomethacin affects the solute and water excretion, an acute water load study was performed in three responsive children before and during the treatment. Indomethacin did not significantly alter the glomerular filtration rate but was effective in reducing diuresis and levels of urinary sodium and potassium excretion. In the child with Fanconi syndrome, indomethacin was also effective in controlling the urinary loss of phosphate, urate, glucose, and bicarbonate. Results of the water load studies show that indomethacin decreases the delivery of solute from the proximal tubule, reduces the fractional free water clearance, and increases the urine-plasma osmolar ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term follow-up of minoxidil therapy in refractory hypertension. A prospective trial in patients with various degrees of renal insufficiency.

Thirty patients with various degrees of renal insufficiency (4 on dialysis treatment) and severe hypertension refractory to conventional agents were treated with minoxidil and followed for up to 182 weeks. BP averaging 203 +/- 8.8/125 +/- 4.1 mm Hg supine and 194 +/- 7.8/125 +/- 3.9 upright before treatment, fell to 155 +/- 6.2/92 +/- 3.4 and 146 +/- 6.3/92 +/- 3.2 respectively within 1 week with 5 to 30 mg daily of minoxidil and remained stabilized around these normal values for three years or more. However, increasing doses of the drug became necessary after 52 weeks of therapy. Serum creatinine increased slightly during long-term treatment in most patients suggesting that the fall in renal function with time should be attributed to the natural progression of normotensive chronic renal failure. Adequate dialysis (in the 4 dialyzed patients) and high-ceiling diuretics prevented fluid imbalance during minoxidil therapy, while beta-blocking agents allowed complete control of reflex tachycardia.

Biological significance of the C3 nephritic factor in membranoproliferative glomerulonephritis.

Serum levels of the C3 nephritic factor (C3NeF), an IgG autoantibody directed against the C3bBb convertase of the alternative complement pathway, and of eight complement components (C1q, C4, C3, C3d, C5, C9, fB and properdin) were measured in 109 serum samples from 27 patients with idiopathic membranoproliferative glomerulonephritis (MPGN) (type I, 20 cases, and type II, 7 cases) and 14 patients with secondary MPGN. Correlations between the concentrations of C3NeF, serum complement levels and progression of the renal damage were studied during the course of the disease in 14 patients with C3NeF activity. The results showed that (1) C3NeF activity was more frequent in patients with type II MPGN than in patients with type I disease; nevertheless there was a high incidence of this splitting activity in patients with secondary MPGN, (2) high levels of the complement components were present in patients with MPGN, (3) low levels of C3 occurred frequently in type II disease and in secondary MPGN, (4) there was no correlation between C3, fB and C3NeF levels, (5) the presence of C3NeF was associated with a more rapid deterioration of renal function. Longitudinal studies showed that serum levels of C3NeF were not satisfactory for monitoring the clinical course of the illness and, in this respect, are similar to the levels of other autoantibodies in patients with autoimmune disease. As MPGN is a clinical syndrome with various pathogeneses, we suggest that the autoantibody, C3NeF, should be considered only as a marker of some forms of MPGN.

[Vesico-ureteral reflux and vesico-sphincteral dysfunction in children]. / Reflusso vescico-ureterale e disfunzioni vescico-sfinteriche in età pediatrica.

The neurologically normal children affected by v.u. reflux frequently have miction disorders. The bladder-sphincter dysfunction can determine high intravesical pressures causing distortion of the vesico-ureteral junction in the same way of anastomotic obstruction. The urodynamic study allows the diagnosis of bladder-sphincter dysfunction and the careful therapeutic management. The medical treatment of the dysfunctional voiding may improve the evaluation of the reflux and reduce postoperatively complications and urinary tract infections. The Authors analyse their experience in the medical treatment of 40 children with vesico-ureteral reflux associated with miction disorders. The reflux resolved after medical treatment in 25% of cases and miction disorders in 50% of cases. The Authors stress the importance of careful diagnosis and treatment to improve results in this group of patients.

Advanced ossification of the posterior longitudinal ligament in a mildly symptomatic patient. A case report and literature review.

We describe the imaging findings of a man who developed neurologic symptoms due to ossification of the posterior longitudinal ligament with narrowing of the spinal canal and compression of the spinal cord. CT study allowed a detailed evaluation of the stenosis and the extension of the ossification while MRI gave an excellent visualization of the spinal lesions caused by spinal cord compression by the mass. The neurological status of patients with ossification of the posterior longitudinal ligament depends on many factors such as the degree of spinal canal stenosis, life style, accidental mechanical stress and trauma.

Effects of the remote C fibres stimulation induced by capsaicin on the blink reflex in chronic migraine.

The aim of this study was to test the function of the diffuse noxious inhibitory control system (DNIC) in chronic and episodic migraine, exploring the blink reflex (BR) modifications induced by topical application of capsaicin on the hand. We evaluated 11 migraine without aura (MA) and nine chronic migraine (CM) patients during the not symptomatic phase; they were compared with 14 non-headache subjects (N). The BR was elicited by weak electrical stimuli delivered to the right supraorbital nerve; it was obtained 10 min and 20 min after the application of 1 ml of 3% capsaicin in a cream base (Teofarma) on the skin of the dorsum of the right hand, and 60 min after capsaicin removal. The subjective pain sensation induced by capsaicin was significantly increased in CM with respect to both MA patients and normal subjects; the R2 area was increased in CM patients during capsaicin application, with respect to controls and MA patients, who did not exhibit any reflex alterations. These results may suggest a failure of DNIC and a disturbed control of the trigeminal reflex at the central level, linked with migraine frequency.

The role of cyanide in the removal of type 2 copper from laccase.

We have improved a method for the removal of the type 2 copper from tree laccase under anaerobic reducing conditions and developed a mechanistic model. We identify two key steps in the reaction: (i) dissociation of copper(I) catalyzed by trace levels of cyanide in a weakly acidic medium and (ii) sequestration of the released metal by an appropriate chelator such as 2,9-dimethyl-1,10-phenanthroline. We maintain a steady-state concentration of cyanide in a pH 5.5 acetate buffer under a constantly exchanging nitrogen atmosphere by introducing a cyanometalate ion as a cofactor or by continuously injecting the ion into the protein solution. The type 2-depleted product is identical to previous preparations as regards its spectral properties, activity level and ability to recombine with copper(I). The mechanistic insights appear to be quite general and should form the basis for the development of methods for removing the type 2 copper from other related systems.

Renal tubular dysgenesis without pulmonary hypoplasia.

Only a few cases of renal tubular dysgenesis (RTD) have been diagnosed. RTD is associated with a very early death, always due to lethal pulmonary hypoplasia. We report an additional patient, interesting for two reasons: (1) the rarity of RTD, (2) the absence of microscopic and macroscopic anatomical alterations of the lungs. The infant had only a mild, transient pulmonary maladaptation; after a few days of life the pulmonary function returned to normal. Although renal function dramatically improved after institution of peritoneal dialysis at 6 days of life, the patient died after 2 weeks with an overwhelming Candida sepsis. We suggest that the lack of pulmonary hypoplasia might be due to a short exposure to oligohydramnios.

Variable clinical presentation of carbonic anhydrase deficiency: evidence for heterogeneity?

The clinical and biochemical findings in an Italian sibship affected by carbonic anhydrase II deficiency are described. Evidence of clinical heterogeneity and an increased frequency of the disease in the Mediterranean area and the Middle East are discussed.

Mechanism of inhibition of electron transfer by amino acid replacement K362M in a proton channel of Rhodobacter sphaeroides cytochrome c oxidase.

The three-dimensional structure of cytochrome coxidase (COX) reveals two potential input proton channels connecting the redox core of the enzyme with the negatively charged (N-) aqueous phase. These are denoted as the K-channel (for the highly conserved lysine residue, K362 in Rhodobacter sphaeroides COX) and the D-channel (for the highly conserved aspartate gating the channel at the N-side, D132 in R. sphaeroides). In this paper, it is shown that the K362M mutant form of COX from R. sphaeroides, although unable to turnover with dioxygen as electron acceptor, can utilize hydrogen peroxide as an electron acceptor, with either cytochrome c or ferrocyanide as electron donors, with turnover that is close to that of the wild-type enzyme. The peroxidase activity is similar to that of the wild-type oxidase and is coupled to the generation of a membrane potential and to proton pumping. In contrast, no peroxidase activity is revealed in the D-channel mutants of COX, D132N, and E286Q. Reduction by dithionite of heme a3 in the fully oxidized oxidase is severely inhibited in the K362M mutant, but not in the D132N mutant. Apparently, mutations in the D-channel arrest COX turnover by inhibiting proton uptake associated with the proton-pumping peroxidase phase of the COX catalytic cycle. In contrast, the K-channel appears to be dispensable for the peroxidase phase of the catalytic cycle, but is required for the initial reduction of the heme-copper binuclear center in the first half of the catalytic cycle.

Role of the K-channel in the pH-dependence of the reaction of cytochrome c oxidase with hydrogen peroxide.

The reaction of cytochrome c oxidase (COX) from Rhodobacter sphaeroides with hydrogen peroxide has been studied at alkaline (pH 8.5) and acidic (pH 6.5) conditions with the aid of a stopped-flow apparatus. Absorption changes in the entire 350-800 nm spectral range were monitored and analyzed by a global fitting procedure. The reaction can be described by the sequential formation of two intermediates analogous to compounds I and II of peroxidases: oxidized COX + H2O2 --> intermediate I --> intermediate II. At pH as high as 8.5, intermediate I appears to be a mixture of at least two species characterized by absorption bands at approximately 607 nm (P607) and approximately 580 nm (F-I580) that rise synchronously. At acidic pH (6.5), intermediate I is represented mainly by a component with an alpha-peak around 575 nm (F-I575) that is probably equivalent to the so-called F* species observed with the bovine COX. The data are consistent with a pH-dependent reaction branching at the step of intermediate I formation. To get further insight into the mechanism of the pH-dependence, the peroxide reaction was studied using two mutants of the R. sphaeroides oxidase, K362M and D132N, that block, respectively, the proton-conducting K- and D-channels. The D132N mutation does not affect significantly the Ox --> intermediate I step of the peroxide reaction. In contrast, K362M replacement exerts a dramatic effect, eliminating the pH-dependence of intermediate I formation. The data obtained allow us to propose that formation of the acidic form of intermediate I (F-I575, F*) requires protonation of some group at/near the binuclear site that follows or is concerted with peroxide binding. The protonation involves specifically the K-channel. Presumably, a proton vacancy can be generated in the site as a consequence of the proton-assisted heterolytic scission of the O-O bond of the bound peroxide. The results are consistent with a proposal [Vygodina, T. V., Pecoraro, C., Mitchell, D., Gennis, R., and Konstantinov, A. A. (1998) Biochemistry 37, 3053-3061] that the K-channel may be involved in the delivery of the first four protons in the catalytic cycle (starting from reduction of the oxidized form) including proton uptake coupled to reduction of the binuclear site and transfer of protons driven by cleavage of the dioxygen O-O bond in the binculear site. Once peroxide intermediate I has been formed, generation of a strong oxene ligand at the heme a3 iron triggers a transition of the enzyme to the "peroxidase conformation" in which the K-channel is closed and the binuclear site becomes protonically disconnected from the bulk aqueous phase.

[Preoperative assessment of Blount disease]. / Valutazione pre-chirurgica del morbo di Blount.

Osteochondrosis of the medial proximal tibial epiphyseal plate is known as Blount disease. To date, two types have been described: infantile and juvenile disease. Six patients (5 men and 1 woman, mean age: 7.2 years) affected with infantile Blount disease diagnosed with plain radiography were submitted to Magnetic Resonance Imaging (MRI) for preoperative lesion staging. The lesions were bilateral in 5 patients. According to Langenskiöld classification, 2 patients were in stage II-III, 3 in stage III-IV and 1 in stage V-VI. Each patient underwent MRI of the more affected knee and 1 patient, who exhibited a more prominent metaphyseal beak, underwent CT of both knees. MRI depicted proximal tibia varus deformity and the degree of its angulation, which helped plan osteotomy; impaired growth of the medial portion of the proximal tibia involving physeal cartilage, metaphysis and epiphysis; alterations of menisci and ligaments (hypertrophic medial meniscus, medial collateral ligament laxity), the presence of bone bridges; the presence and integrity of unossified epiphysis and of chondral growth plate which was quite completely ossified in one case. To conclude, MRI is a completely atraumatic and noninvasive technique yielding many pieces of information necessary for the preoperative assessment of Blount disease.

The post-translational modification in cytochrome c oxidase is required to establish a functional environment of the catalytic site.

Mutation of tyrosine-288 to a phenylalanine in cytochrome c oxidase from Rhodobacter sphaeroides drastically alters its properties. Tyr-288 lies in the CuB-cytochrome a3 binuclear catalytic site and forms a hydrogen bond with the hydroxy group on the farnesyl side chain of the heme. In addition, through a post-translational modification, Y288 is covalently linked to one of the histidine ligands that is coordinated to CuB. In the Y288F mutant enzyme, the "as-isolated" preparation is a mixture of reduced cytochrome a and oxidized cytochrome a3. The cytochrome a3 heme, which is largely six-coordinate low-spin in both oxidation states of the mutant, cannot be reduced by cytochrome c, but only by dithionite, possibly due to a large decrease in its reduction potential. It is postulated that the Y288F mutation prevents the post-translational modification from occurring. As a consequence, the catalytic site becomes disrupted. Thus, one role of the post-translational modification is to stabilize the functional catalytic site by maintaining the correct ligands on CuB, thereby preventing nonfunctional ligands from coordinating to the heme.

[Computed tomography and magnetic resonance in the follow-up to interventions for the prosthetic replacement of the carpal bones]. / Tomografia Computerizzata e Risonanza Magnetica nel follow-up degli interventi di sostituzione protesica delle ossa del carpo.

The authors examined with CT and MRI 12 patients submitted to prosthetic replacement of necrotic carpal bones--7 of them because of scaphoid proximal pole posttraumatic osteonecrosis and 5 because of lunate Kienböck's disease. The prosthetic implants were autologous in all patients: they were taken from palmaris gracilis tendon and modified to give them a rounded shape, to adapt them to the new anatomic site. All patients exhibited postoperative limitation in flexion-extension movements; 5 of them reported associated wrist pain. The authors investigated the anatomic reasons of the postoperative symptoms and tried to assess CT and MR diagnostic capabilities in depicting these conditions. CT was performed with thin sections (1.5 mm) and multiplanar reconstructions, with a Philips Tomoscan LX unit. MRI was performed with a GE MR Max Plus unit at 0.5 T and a Medical Advances transmit-receiving extremity coil, on the axial, coronal and sagittal planes, with T1- and T2-weighted sequences. All patients had been submitted to conventional radiography of the wrist. In 6 patients CT and MRI showed severe synovial reaction in the surgical site, with new ligament absorption in 5 of them. In 5 of these patients CT identified some nodular calcifications, while MRI better depicted the fibrotic portion, if present. MRI demonstrated the carpal dorsal intercalated instability which was present in all the patients submitted to scaphoid proximal pole resection; in the patients operated on for Kienböck's disease, volar tilt of the scaphoid was increased. Both kinds of carpal instability were correlated with scaphoid-lunate surgical dissociation. These instabilities were greatly correlated with the postoperative symptoms. Currently, MRI is the gold standard in studying carpal instability and it is therefore fundamental in investigating the complex anatomic and biomechanical features of these patients postoperatively.

Lysinuric protein intolerance characterized by bone marrow abnormalities and severe clinical course.

STUDY OBJECTIVE: To evaluate phenotypic variability of lysinuric protein intolerance in a cohort of nine Italian patients. DESIGN: Retrospective analysis of patient records. SUBJECTS: Nine Italian patients (seven independent families), all originating from southern Italy, observed during the last 14 years. RESULTS: Some of the patients had unique clinical features, including bone marrow abnormalities featuring erythroblastophagocytosis (five patients) and clinical course and the outcome of the disease, have also been observed: respiratory involvement was present in five cases, with a lethal picture of "alveolar proteinosis" in one. Severe kidney involvement, with both glomerular and tubular damage and rapidly progressing to chronic renal failure, has been observed in one case. CONCLUSION: Lysinuric protein intolerance may cause severe multisystem involvement, which requires early and careful monitoring. Some peculiar clinical findings observed in Italian patients point to a genetic heterogeneity of lysinuric protein intolerance.

[Computerized tomography assessment of replacement of the cruciate ligaments of the knee]. / Valutazione con Tomografia Computerizzata degli interventi di sostituzione dei legamenti crociati del ginocchio.

January, 1993, to December, 1995, we examined with CT 44 patients submitted to surgical replacement of knee cruciate ligaments, namely 18 anterior cruciate ligaments (ACL) and 6 posterior cruciate ligaments (PCL). ACL was replaced with a patellar tendon graft (Eriksson technique) in 23 cases and with a semitendinous graft (Lindemann-Bousquet technique) in 7 cases. The autologous new ligament appeared as a laminar structure of intermediate density. The tendon had been covered with a synthetic lining (Leeds-Kejo) in 10 of the above cases, while the new ACL graft was completely synthetic in 8 cases, which synthetic allografts appeared hyperdense relative to autologous new ligaments. PCL replacement had been performed with Augustine technique (patellar tendon graft in a tibial tunnel) in 3 cases, with Hughston technique (medial gastrocnemius graft in a medial condylar tunnel) in 2 cases and with a synthetic graft (Gore-tex) in 1 case. Twelve of 38 new ACLs were injured (7 Eriksson, 3 Lindemann-Bousquet and 2 synthetic grafts): the injured new ligament was enlarged and hypodense, or involved in atrophic absorption. The new ligament was partially calcified in one of the 3 patients with PCL replacement with a patellar tendon graft (Augustine technique). The bone bract was dislocated in the intercondylar notch in a patient submitted to Hughston surgery because of proximal disinsertion of the new ligament. The Gore-tex new PCL was sinuous in the proximal tract, with intra-articular calcified loose bodies. CT adequately depicted surgical results and diagnosed the new ligament injuries and other articular abnormalities. MRI is currently the gold standard in the multiplanar studies of the postoperative knee, but CT can be confidently used to identify ligament injuries and intra-articular calcified loose bodies.