Ureterocystoplasty (bladder augmentation) in a 16 year-old boy with Goldenhar syndrome.

The use of the dilated ureter for bladder augmentation is universally accepted for its lower rate of complications compared to the use of gastrointestinal segments. We report the case of a 16 yearold boy affected by Goldenhar syndrome who presented with neurogenic bladder with small-capacity, 5° grade vescico-ureteral reflux (VUR) with megaureter and bilateral hydronephrosis. Bladder augmentation using the distal dilated ureter, transuretero-ureterostomy left to right and Mitrofanoff's appendicovescicostomy were performed. Six months after surgery voiding cystourethrogram (VCUG) revealed a compliant bladder with a functional capacity of 400 ml. Ureterocystoplasty is a safe and effective method of augmenting small capacity urinary bladder. We suggest using the ureter, when available, instead of using gastrointestinal segments.

Laparoscopic transposition of lower pole crossing vessels in children with extrinsic pelvi-ureteric junction obstruction: a worthy alternative to dismembered pyeloplasty.

Pelviureteric junction obstruction (PUJO) due to intrinsic or extrinsic causes is a common problem in childhood. Extrinsic compression by a lower pole-crossing blood vessel can present symptomatically in older children. In these cases, laparoscopies Vascular Hitch can represent a valid alternative to pyeloplasty dismembered. We analyzed the data of 4 children affected by extrinsic PUJO treated at our institution with the laparoscopic Vascular Hitch procedure modified by Chapman. Surgical indications included presence of clinical symptoms, worsening of intermittent hydronephrosis, signs of obstruction on the MAG-3 scan, clear or suspected images of polar crossing vessels on CT scan or Uro-MRI. All procedures were completed laparoscopically. No complications occurred. Mean follow-up was 13 months with resolution of symptoms and PUJ obstruction and significant improvement of hydronephrosis in all cases. When blood vessels crossing lower pole represent the pure mechanical cause of UPJ obstruction the laparoscopic Vascular Hitch procedure represents an excellent alternative to dismembered pyeloplasty. It is less technically demanding then pyeloplasty and is associated with a lower complication rate. The main challenge is to intraoperatively ascertain the absence of associated intrinsic stenosis.

Laparoscopic nephrectomy in children with Wilms tumor. Considerations after 10 years of experience.

Despite laparoscopy in children is considered safe and is routinely used for several procedures, even in neonates and in pediatric oncology, its role in the treatment of pediatric renal tumors is still controversial. This study analyzes the results of laparoscopic nephrectomy for Wilms Tumor (WT) in pediatric age compared with open nephrectomy after 10 years of experience in a single centre. From 1993 in our center of reference for pediatric oncology, 30 patients with WT have been treated. We performed 21 open nephrectomy and in the last 10 years 9 laparoscopic nephrectomy. In all patients treated laparoscopically, the same technique made by the same equip was used. Compared with patients treated by open surgery, we did not find a significant difference in terms of outcome and survival. In the open surgery group, two patients had lung relapse while in the other group there was one local relapse. These three children obtained and maintained a second complete remission with chemotherapy. Open surgery complications were a tumor rupture in two cases, and an episode of pancreatitis 10 days after surgery. In the laparoscopic group, there were two conversions to open surgery not considered as complications but a surgical choice for cystic areas present in the tumor. As far as complications and oncologic outcomes are concerned, both techniques showed similar results. In experienced hands, laparoscopy proves to be an attractive alternative to open surgery for pediatric renal tumors.

Seed dormancy and control of germination in Sisymbrella dentata (L.) O.E. Schulz (Brassicaceae).

Seed germination responsiveness to environmental cues is crucial for plant species living in changeable habitats and can vary among populations within the same species as a result of adaptation or modulation to local climates. Here, we investigate the germination response to environmental cues of Sisymbrella dentata (L.) O.E. Schulz, an annual endemic to Sicily living in Mediterranean Temporary Ponds (MTP), a vulnerable ecosystem. Germination of the only two known populations, Gurrida and Pantano, was assessed over a broad range of conditions to understand the role of temperatures, nitrate, hormones (abscisic acid - ABA and gibberellins - GA) and after-ripening in dormancy release in this species. Seed germination responsiveness varied between the two populations, with seeds from Gurrida germinating under a narrower range of conditions. Overall, this process in S. dentata consisted of testa and endosperm rupture as two sequential events, influenced by ABA and GA biosynthesis. Nitrate addition caused an earlier testa rupture, after-ripening broadened the thermal conditions that allow germination, and alternating temperatures significantly promoted germination of non-after-ripened seeds. Primary dormancy in S. dentata seeds likely allows this plant to form a persistent seed bank that is responsive to specific environmental cues characteristic of MTP habitats.

Experimental new automatic tools for robotic stereotactic neurosurgery: towards "no hands" procedure of leads implantation into a brain target.

The use of robotics in neurosurgery and, particularly, in stereotactic neurosurgery, is becoming more and more adopted because of the great advantages that it offers. Robotic manipulators easily allow to achieve great precision, reliability, and rapidity in the positioning of surgical instruments or devices in the brain. The aim of this work was to experimentally verify a fully automatic "no hands" surgical procedure. The integration of neuroimaging to data for planning the surgery, followed by application of new specific surgical tools, permitted the realization of a fully automated robotic implantation of leads in brain targets. An anthropomorphic commercial manipulator was utilized. In a preliminary phase, a software to plan surgery was developed, and the surgical tools were tested first during a simulation and then on a skull mock-up. In such a way, several tools were developed and tested, and the basis for an innovative surgical procedure arose. The final experimentation was carried out on anesthetized "large white" pigs. The determination of stereotactic parameters for the correct planning to reach the intended target was performed with the same technique currently employed in human stereotactic neurosurgery, and the robotic system revealed to be reliable and precise in reaching the target. The results of this work strengthen the possibility that a neurosurgeon may be substituted by a machine, and may represent the beginning of a new approach in the current clinical practice. Moreover, this possibility may have a great impact not only on stereotactic functional procedures but also on the entire domain of neurosurgery.

[Essential levels of care and mode of admission: a trial of cost-volume contracts]. / LEA e riconversione dei ricoveri: sperimentazione di contratti per costo e volume.

Within a research project funded by the Ministry of Health, the purpose of this work was to define the procedures of a contract for the provision of health services (hospital admissions), between the regional health administration (the "buyer") and the University teaching hospital (the "provider"), with the aim of improving efficiency. The contract concerned a few DRGs, among those identified as being "at risk of inappropriateness", in the national decree on "essential levels of care" (LEA). The contract defined production levels (number and percentage of admissions in day hospital), financing rules and methods for evaluation of results and improvement of performance within the hospital. This trial, even if run for a limited time and for demonstration purposes, showed that some results can be attained, provided some organizational adjustments are made.

Lipophilic methotrexate conjugates with antitumor activity.

Lipophilic methotrexate (MTX)-lipoamino acid conjugates coupled with amide or ester linkages (1a-1r) were synthesised. The inhibitory activity of the conjugates was evaluated on bovine liver DHFR. The in vitro growth inhibitory effect against MTX-sensitive human lymphoblastoid CCRF-CEM cells and an MTX-resistant sub-line (CEM/MTX), which displays defective intracellular transport of MTX, was determined under short-term and continuous (120-h incubation) exposure conditions. The alpha, gamma, or alpha,gamma amide conjugates showed different activity in inhibiting the growth of parent cells. CEM/MTX cells were much less susceptible than CCRF-CEM cells to inhibition by alpha or alpha,gamma-substituted lipoamino acid conjugates, whereas both cell lines were almost equally sensitive to the MTX-gamma conjugates. Although less potent than MTX, they could partially circumvent the impaired transport system. These findings confirm that lipophilic MTX conjugates may be good lead compounds on the drug development for the treatment of some MTX-resistant tumors. Ester-type conjugates displayed an interesting activity against parent CCRF-CEM cells, although they were less potent against the transport-resistant sub-line. Stability studies on these molecules indicated that they are not degraded into MTX in the culture medium, thus suggesting that they are not able to over-cross cell resistance despite of their lipophilicity.

Synthesis and preliminary in vitro screening of lipophilic alpha, gamma-bis(amides) as potential prodrugs of methotrexate.

As part of a program aimed at studying the feasibility of amide derivatives of methotrexate (MTX) as lipophilic prodrugs, with the aims of increasing passive cellular uptake and obtaining prolonged-release agents, we describe the synthesis of five long-chain alkyl bis(amides) of MTX, from decyl- to octadecylamide, by direct transamidation to the MTX diethyl ester. Compounds were subjected to a preliminary biological screening, to assess their inhibitory activity against bovine liver dihydrofolate reductase (DHFR) and in vitro antitumor activity against human leukemia CCRF-CEM cells. As a general trend, an increase in lipophilicity led to a linear reduction of enzyme inhibition; however, the bis(decyl)amide derivative showed a good intrinsic affinity for DHFR (IC50 6.41 nM), comparable to that of MTX diethyl ester and close to that of MTX (IC50 2.90 nM). In the antitumor assay, lower homologues (C10-C14) displayed an interesting activity profile, suggesting the desirability of additional studies with these and similar compounds.

Efficient one-step direct labelling of recombinant antibodies with technetium-99m.

High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99mTc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability.

Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.

Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.

Expression of ornithine decarboxylase gene in elderly human monocytes.

The proliferative capacity of the immune system is impaired in elderly subjects and the expression of various genes involved in cell cycle progression is reduced in PHA stimulated lymphocytes during the aging process. Macrophages play a fundamental role in the immune system response. It has recently been demonstrated that the process of macrophage activation is accompanied by a rapid, transient rise of ornithine decarboxylase (ODC) mRNA levels. In fact, the ODC gene seems to be involved in macrophage activation and differentiation. The authors demonstrated that the steady-state levels of ODC mRNA and the correlated superoxide anion production are lower in the monocytes of elderly subjects with respect to those in young subjects used as control. These results confirmed the impaired immune function of the elderly.

Polyamine involvement in functional activation of human macrophages.

Polyamines naturally occur in all living beings and play an important role in the regulation of cell proliferation, differentiation, and functional stimulation of terminally differentiated cells. Our studies, using specific inhibitors of polyamine biosynthesis such as alpha-difluoromethylornithine and methylglyoxal-bis[guanylhydrazone] to prevent polyamine accumulation, have indicated that polyamines are associated with functional activation of human macrophages. Both inhibitors diminished the respiratory burst activity of macrophages induced by lipopolysaccharide and interferon gamma. The methylglyoxal-bis]guanylhydrazone] inhibitory effect was concentration-dependent and could be reversed by spermine, which is the final product of polyamine biosynthesis.

AO 1535 inhibits O2- production by human macrophages.

AO 1535 is a semisynthetic monoglycosylceramide derived from O-glycosilated sphingosine, with a chemical structure similar to the glycolipids present in many mammalian tissues. In the epidermis monoglycosylceramides contribute to consolidate the structure of cutaneous layers. It has been recently shown that sphingosine and its derivatives are potent inhibitors of Protein kinase C, and block the 'respiratory burst' of phagocitic cells. In macrophages, like in neutrophils, the reactive oxygen intermediates are produced by a membrane associated enzymatic complex, NADPH-oxidase, which is activated by Protein kinase C. This study demonstrates that AO 1535 is able to inhibit the production of reactive oxygen intermediates in human monocytes and macrophages stimulated by phorbol ester and chemotactic tetrapeptide, suggesting a potential clinical application of AO 1535 in the treatment of inflammatory dermatoses.

Inhibitors of polyamine biosynthesis block tumor necrosis factor-induced activation of macrophages.

The activation of polyamine biosynthesis, dependent on increased gene expression of ornithine decarboxylase, has been found to play an important role in the control of cell proliferation and differentiation. In this report it has been found that accumulation of ornithine decarboxylase mRNA also follows stimulation of human monocytes/macrophages by tumor necrosis factor. Human recombinant tumor necrosis factor (100 units/ml) also evoked an enhanced respiratory burst of macrophages. The respiratory burst response was inhibited in a dose-dependent manner with difluoromethylornithine, an inhibitor of ornithine decarboxylase, and methylglyoxal-bis(guanylhydrazone), an inhibitor of the formation of spermidine and spermine. The data presented in this paper suggest that polyamines may play a functional role in tumor necrosis factor-driven macrophage activation, and they are discussed in the context of their possible use as inhibitors of polyamine metabolism in tumor chemotherapy.

Transcription factor activation and functional stimulation of human monocytes.

Activation of expression of genes encoding transcription factors: c-fos and c-jun and formation of AP1 transcriptional complex in human monocytes was investigated. It was found that lipopolysaccharide induced strongly both c-fos and c-jun expression as well as AP1 formation. Interferon gamma activated strongly c-fos and weakly c-jun and AP1. Tumor necrosis factor induced slightly c-fos and had almost no effect on c-jun and AP1. The data suggest that differences in functional responses elicited in monocytes by all three factors may be dependent on different routes on nuclear signalling employed by the factors.

Accumulation of ornithine decarboxylase mRNA accompanies activation of human and mouse monocytes/macrophages.

Accumulation of ornithine decarboxylase (ODC) mRNA was investigated in human monocytes and mouse peritoneal macrophages. Treatment of both populations of mononuclear phagocytes with bacterial lipopolysaccharide induced a marked and rapid increase in the accumulation of the ODC gene transcript. A similar phenomenon, albeit less pronounced, was also observed following treatment of human monocytes with human recombinant interferon-gamma. These results suggest a role for ODC, and therefore polyamines, in the regulation of mononuclear phagocyte functions.

Glial fibrillary acidic protein messenger RNA and glutamine synthetase activity after nervous system injury.

The level of the mRNA for glial fibrillary acidic protein (GFAP), the major protein of the intermediate filaments of astroglial cells, and the activity of glutamine synthetase (GS), an enzyme selectively localized in astrocytes, were measured at different times after a unilateral mechanical lesion in the rat cerebral cortex. A rapid and early increase (6 hours post-lesion) in GFAP mRNA was observed; GFAP mRNA level reached a peak at 1-3 days and then decreased. Moreover, an astrocytic activation in cortical zones far from the injury site and in the contralateral hemisphere was detected. No change of GS activity was observed in the same model of brain injury, showing that this astroglial marker is not modified during the reactive gliosis obtained with this experimental model. GFAP mRNA has also been detected in the rat sciatic nerve; however, its level was not modified after nerve transection, suggesting a different regulation of GFAP expression in the peripheral nervous system.

Immunochemical and biological characteristics of a human autoantibody to human chorionic gonadotropin and luteinizing hormone.

Anti-hCG/LH autoantibodies were found in the serum of an infertile woman a few days after an abortion which occurred after 46 days of amenorrhea. The antibody titer increased for approximately 4 more weeks, and then declined to low levels during a 14-month anovulatory period, after which regular menses resumed. Immunoglobulins isolated from a pool of serum obtained during the postabortion period neutralized the activity of both hCG and LH in an in vivo bioassay, and the binding affinity of the antibodies toward both hormones was high. When menses were resumed, there was a considerable reduction of the affinity toward LH. The variations in antibody titers and/or affinities can explain the sequence of fertilization, abortion, anovulatory period, and normalization of menses.

Substantial amounts of gonadotrophin-like materials in sera of normal subjects.

Ultrafiltration of human sera resulted in increased levels of human chorionic gonadotrophin (hCH), LH and FSH measured by radioimmunoassay. The effect was greatly enhanced by chromatography of serum through Sepharose-protein A (which specifically binds immunoglobulin G) followed by ultrafiltration of the unretarded fractions. Serum from a normally menstruating women was treated by ultrafiltration and the retained fraction from 200 mul serum further chromatographed on a Sephadex G-150 Superfine column. The amounts of LH and FSH measured in the eluate by radioimmunoassay were higher by three orders of magnitude than those found in the untreated serum. Comparable amounts of hCG were also found in the eluate by two specific radioimmunoassays, and a value of 45 i.u. hCG was recovered in the eluate by bioassay. Serum from another normally menstruating women was applied to DEAE-cellulose and eluted stepwise using an elution gradient. The ultrafiltrate of one eluted fraction led to separation of a low molecular weight material which was able to neutralize the biological activity of a large dose of hCG. The data support the hypothesis that substantial amounts of gonadotrophins are normally present in serum but that binding to inhibitor(s) prevents their assay in untreated sera.