Distinctive features of tumor-infiltrating γδ T lymphocytes in human colorectal cancer.

γδ T cells usually infiltrate many different types of cancer, but it is unclear whether they inhibit or promote tumor progression. Moreover, properties of tumor-infiltrating γδ T cells and those in the corresponding normal tissue remain largely unknown. Here we have studied features of γδ T cells in colorectal cancer, normal colon tissue and peripheral blood, and correlated their levels with clinicopathologic hallmarks. Flow cytometry and transcriptome analyses showed that the tumor comprised a highly variable rate of TILs (5-90%) and 4% γδ T cells on average, with the majority expressing Vδ1. Most Vδ1 and Vδ2 T cells showed a predominant effector memory phenotype and had reduced production of IFN- Î³ which was likely due to yet unidentified inhibitory molecules present in cancer stem cell secretome. Transcriptome analyses revealed that patients containing abundant γδ T cells had significantly longer 5-year disease free survival rate, suggesting their efficacy in controlling tumor at very early stage.

High-dose rocuronium for rapid-sequence induction and reversal with sugammadex in two myasthenic patients.

The anesthetic management of patients affected by myasthenia gravis is usually challenging in elective surgery and even more so in emergency procedures. The difficulties involved are several-fold, ranging from the choice of an appropriate muscle relaxant (i.e. one that enables safe and rapid airway management) to neuromuscular monitoring and normal muscular recovery. Additionally, optimizing patient conditions - either pharmacologically or with plasmapheresis - before intervention is well beyond the realm of possibility. We discuss the anesthetic management of two myasthenic patients undergoing emergency surgery (for sigmoid perforation and upper gastrointestinal bleeding respectively). In both cases, we opted for rapid-sequence induction with high-dose rocuronium to prevent inhalation of gastric contents. We also report on the implication of neuromuscular monitoring. We found that the rocuronium-sugammadex combination was a useful and effective option in the emergency setting.

Reversal of rocuronium induced neuromuscular block with sugammadex or neostigmine: a large observational study.

BACKGROUND: This 'real-life' study aimed to analyze the time from the start of neostigmine or sugammadex administration to recovery to a train of four ratio (TOFr) of 0.9 in a real-life in patients receiving rocuronium. The secondary aims were to assess the proportion of patients: presenting TOFr < 0.9 after 5, 10, and 20 min from reversal agent administration, receiving opioids for intraoperative analgesia and extubated in the post-anesthesia care unit (PACU). METHODS: This was a multisite, prospective, nonrandomized, observational real-life study. Reversal agent was administered at either T2 reappearance or at a post-tetanic count of 1 or 2. Drugs dosages were free according to each investigator's usual practice. RESULTS: Three hundred fifty-nine patients were enrolled onto the study. Time from reversal administration to TOFr to 0.9 is significantly faster in the sugammadex group than in the neostigmine group (shallow block: 2.2 vs. 6.9 min, respectively; P < 0.0001; deep block: 2.7 vs. 16.2 min, respectively; P < 0.0001). The number of patients with TOFr < 0.9 at 5, 10, and 20 min post-reversal agent administration was higher in the neostigmine than in the sugammadex group. Just five patients did not receive opioids. All patients were extubated in the operative room except for a single patient in the sugammadex group who was extubated following PACU admission. CONCLUSIONS: This real-life study confirms that reversal time is faster in patients receiving sugammadex than in those receiving neostigmine. TOFr < 0.9 20 min after reversal was only present in patients treated with neostigmine.

S(+)-ketamine for control of perioperative pain and prevention of post thoracotomy pain syndrome: a randomized, double-blind study.

BACKGROUND: Post-thoracotomy pain syndrome (PTPS) often complicates the long term outcome of patients; its appearance has been related to perioperative acute pain. The main goal of this study was to evaluate a possible role of S(+)-ketamine in the prevention of PTPS up to 6 months and secondarily its efficacy in the control of perioperative pain when added to thoracic epidural analgesia (TEA) and adjuvants. METHODS: Sixty-six patients underwent thoracotomy under general anesthesia. A thoracic epidural catheter was placed for levobupivacaine and sufentanil administration. Thirty-three patients received an i.v. infusion of S(+)-ketamine (Group S(+)K) for 60 hours and 33 patients received i.v. placebo (Group PLAC). Pain was evaluated by Numeric Rating Scale (NRS) during the whole study. All patients had supplementary doses of analgesics, as needed, to have NRS targeted to a value of ≤3 in the 1st and <3 in the following days. Neuropathic Pain Symptom Inventory (NPSI) was evaluated at 1, 3 and 6 months. RESULTS: All patients had NRS ≤3 in the early postoperative period and NPSI was less or equal to 1 in the follow-up control for each group with no significant difference at three (P=0.67, OR 0.8 [IC95% 0.3-2.2]) and at six months (P=0.23, OR 1.9 [0.7-5.4]). Incidence of moderate PTPS was 24.6% at 3 and 21.1% at six months while severe PTPS was 6.6% at 3 and 1.8% at six months. No difference was detected in NRS and NPSI at 3 and 6 months between groups. CONCLUSION: S(+)-ketamine had no effects in respect to placebo in the prevention of PTPS at 3 and 6 months but had a significant role in maintaining a NRS≤3 in the early postoperative period. A tight control of perioperative pain seems to be associated with a low incidence of moderate and severe PTPS.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti-tumour activities of gammadelta T cells have prompted the development of protocols in which gammadelta-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vgamma9Vdelta2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vgamma9Vdelta2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vgamma9Vdelta2 T cell numbers emerged, as seven patients who failed to sustain Vgamma9Vdelta2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vgamma9Vdelta2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vgamma9Vdelta2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatment-refractory patients with advanced breast cancer.

Thoracic epidural analgesia in post-thoracotomy patients: comparison of three different concentrations of levobupivacaine and sufentanil.

BACKGROUND: Relative effects of dosage, volume and concentration of local anaesthetics used for postoperative thoracic epidural analgesia are still under debate. In this randomized, prospective, double-blinded study, we evaluated the incidence of side-effects such as changes in arterial pressure, postoperative nausea, vomiting, and pruritus in patients admitted for thoracic surgery during continuous thoracic epidural infusion using levobupivacaine and sufentanil mixture in three different volumes. METHODS: We studied 150 patients who underwent thoracotomy with a thoracic epidural catheter placed between T4 and T7. The patients were randomized into three groups which received 10 mg h(-1) of levobupivacaine at three different concentrations (0.5%, 0.25%, and 0.15%), in combination with sufentanil at 2.6 microg h(-1). Haemodynamic effects, pruritus, nausea, vomiting, sensory and motor block, pain score, additional analgesic requirement, sedation, and patient satisfaction were registered immediately after the surgical operation and on the first, second, and third postoperative days. RESULTS: We did not detect any differences in the incidence of side-effects such as changes in arterial pressure, and also postoperative nausea, vomiting, and pruritus. The three groups were also similar with regard to patient characteristics, sensory and motor block, pain score, analgesic rescue dose, sedation, and patient satisfaction. CONCLUSIONS: The same dose of a mixture of levobupivacaine and sufentanil administered in three different volumes and concentrations during continuous thoracic epidural infusion for thoracotomy provided an equal incidence of adverse haemodynamic effects, nausea, vomiting, or pruritus.

Cerebral cavernomas and seizures: a retrospective study on 163 patients who underwent pure lesionectomy.

The objective was to evaluate the outcome of microsurgical "pure" lesionectomy in patients with supratentorial cavernous angiomas presenting with seizures. For this retrospective study 163 patients with cavernoma-related epileptic seizures were selected. They all underwent surgery in a single institution between 1988 and 2003. A microsurgical frame/frameless guided minimally invasive transulcal "pure" lesionectomy was performed. The haemosiderin stained gliotic brain parenchyma that was usually found surrounding the lesion was not removed. Among the 99 patients with epilepsy and longer clinical history, 68 (68.7%) were found completely to be seizure-free, 10 (10.1%) presented sporadic and less frequent seizures and 17 (17.1%) remained unchanged. Sixty-three out of 64 (98.4%) patients who experienced only single or sporadic seizures were found to be completely seizure-free after surgery. Five patients were lost at follow-up (mean 48 months, range 0.5-14 years). Long-term morbidity was 1.8%. Mortality was null. No haemorrhagic episodes were observed during follow-up. Pure lesionectomy prevents bleeding and development of epilepsy in patients that receive early surgery after the epileptic onset. In most of the epileptic patients with a good concordance between the electroclinical data and the location of the angioma, good results can be achieved by this kind of surgery so that more invasive and costly studies to find and remove the epileptogenic cerebral parenchyma seem justified only after lesionectomy fails.

Craniotomies without burr holes using an oscillating saw.

OBJECTIVE: The object of this study is to describe a new method for performing craniotomies which obviates the need for burr holes, improves bony reconstruction, and reduces post-operative cosmetic deformities. Moreover, this technique provides excellent exposure of skull base structures and dural venous sinuses. METHODS: Craniotomies in varied locations are created with the use of a micro-oscillating saw and chisel. No burr holes are used and reconstruction with plates and screws is unnecessary. RESULTS: We initially applied our technique to approaches to the anterior skull base with combined craniofacial tumour resections. We have since performed over 2000 craniotomies of any size and shape in all supratentorial locations using the oscillating saw. CONCLUSIONS: We have found that our method creates better cosmetic results than standard techniques and is safer for craniotomies spanning dural venous sinuses. With experience, operating time was significantly reduced and costs were lowered because reconstruction with fixation devices was not needed.

Expression of a catalytically inactive H118Y mutant of nm23-H2 suppresses the metastatic potential of line IV Cl 1 human melanoma cells.

Nm23-H1 and nm23-H2 are putative metastasis suppressor genes that encode nucleoside diphosphate kinase (NDPK) A and B. NDPKs form oligomers distributed between soluble and particulate fractions of cells and therefore may exert their effects as either soluble or bound proteins. To determine whether metastasis-related functions of NDPKs are mediated by their catalytic activity in membrane bound or soluble complexes, we have stably transfected highly metastatic human melanoma Line IV Cl 1 cells with wild-type and catalytically inactive (H118Y) nm23-H1 and nm23-H2 genes and assayed their metastatic potential in nude mice. Transfection with wild-type nm23-H1 and nm23-H2 genes and catalytically inactive nm23-H1 did not significantly (all p > 0.10) alter the metastatic potential of Line IV Cl 1 cells while transfection with catalytically inactive nm23-H2 significantly (p < 0.01) reduced their metastatic potential. The lack of effect of transfection with wild-type and catalytically inactive nm23-H1 suggests that neither soluble nor membrane bound NDPK A affect the metastatic potential of Line IV Cl 1 cells. The metastasis suppressive effect of catalytically inactive NDPK B overexpression suggests that competition with bound complexes containing catalytically active NDPK B inhibits metastasis of Line IV Cl 1 cells. These results imply that bound NDPK B promotes metastasis and suggest that inhibition of its function or of its binding to critical sites may be a useful approach to limit the development of metastases in human melanoma.

Overexpression of NM23-1 enhances responsiveness of IMR-32 human neuroblastoma cells to differentiation stimuli.

BACKGROUND: Aggressiveness of neuroblastoma is associated with increased expression of the putative metastasis suppressor genes, nm23-1 and nm23-2. These genes encode nucleoside diphosphate kinases A and B that form free or bound homo- and heteromers, which are distributed between soluble and particulate fractions of cells and display catalytic and non-catalytic activities. MATERIALS AND METHODS: In order to establish which forms and activities of nm23 proteins are operative in neuroblastoma we stably transfected IMR-32 human neuroblastoma cells with constructs encoding wild type and catalytically inactive nm23-1 and nm23-2 proteins. RESULTS: Overexpression of wild type nm23-1 proteins stimulated spontaneous neurite outgrowth and enhanced differentiation in response to serum starvation and retinoic acid. In contrast, overexpression of the catalytically inactive nm23-1T mutant enhanced TPA-mediated inhibition of differentiation. CONCLUSION: Our findings suggest that differentiation associated functions of nm23 proteins in IMR-32 neuroblastoma cells are carried out by bound nm23-1 proteins docked in a limited number of nm23-1 specific sites.

Nm23-transfected MDA-MB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: a 31P nuclear magnetic resonance study in vivo and in vitro.

Nm23 genes are involved in the control of the metastatic potential of breast carcinoma cells. To understand the impact of nm23 genes on tumor physiology and metabolism, a 31P nuclear magnetic resonance (NMR) spectroscopic study was performed on tumors formed in the mammary fat pad of severe combined immunodeficiency mice by MDA-MB-435 human breast carcinoma cells transfected with cDNA encoding wild type nm23-H1 and nm23-H2 proteins. Tumors formed by MDA-MB-435 cells transfected with vector alone were used as controls. All transgene tumors exhibited significantly higher levels of phosphodiester (PDE) compounds relative to phosphomonoester (PME) compounds in vivo compared with control tumors. Similar differences in PDE and PME also were observed for spectra obtained from cells growing in culture. Intracellular pH was significantly lower and extracellular pH was significantly higher for transgene tumors compared with control tumors. Histologic analysis of lung sections confirmed reductions in incidence, number, and size of metastatic nodules for animals bearing transgene tumors. These results suggest that nm23 genes may affect suppression of metastasis through phospholipid-mediated signaling and cellular pH regulation.

Total intravenous anesthesia with propofol and remifentanil for elective non-cardiac surgery.

BACKGROUND: Remifentanil is a highly effective mu opioid agonist with predictable pharmacokinetics and a close concentration-effect relationship. Moreover, studies on anesthetic drugs interactions show that optimal propofol concentrations decrease more significantly with remifentanil as compared with other opioids and recovery appears to be much faster than when propofol is combined with other opioids combinations. This intervention study was designed to evaluate the efficacy of propofol combined with remifentanil in elective non cardiac inpatient surgery. METHODS: N. 405 patients undergoing intraperitoneal, head-neck, intrathoracic, major orthopaedics, breast and major vascular surgery received: remifentanil (1 microgram.kg-1 at induction; 0.50 microgram.kg-1.min-1 at laryngoscopy; 0.25 microgram.kg-1.min-1 at skin incision; 0.25-0.30 microgram.kg-1.min1 from skin incision to end of skin suture) and propofol (0.5-1 mg.kg-1 at induction; 5 mg.kg-1.h-1 at laryngoscopy; 5 mg.kg-1.h-1 at skin incision and 5 mg.kg1.h1 thereafter). Intraoperative end-points included somatic responses, tachycardia and hypertension to laryngoscopy and surgery. Incidence of intraoperative bradycardia, hypotension and muscle rigidity were also recorded. Postoperative end-points included Aldrete score > or = 9, pain immediately following emergence and PONV. RESULTS: Propofol-remifentanil combination effectively controlled responses to laryngoscopy and surgical stress. Drug related adverse events were transient bradycardia (< 50 bpm) and hypotension (SBP < or = 80 mmHg) respectively: at prelaryngoscopy 11.60-1.48% and at pre-skin incision 10.61-0.98%. N. 365 patients were discharged from PACU and the median time to first Aldrete score > or = 9 was 22.3 min. The most frequent postoperative event was shivering recorded in n. 46 patients (12%). Postoperative analgesic medication was requested by n. 16 patients (4.4%) and PONV was noted in n. 6 patients (1.6%). CONCLUSIONS: When combined with propofol, remifentanil effectively provided for profound analgesia during surgery, stable anesthetic conditions, simplicity of use and predictable recovery.

Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase.

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.

The crystal structure of a human nucleoside diphosphate kinase, NM23-H2.

The 2.8 A resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription of the c-myc oncogene in vitro. NM23 has also been reported to be a suppressor of metastasis in some types of tumours. Whereas the M. xanthus NDP kinase is a tetramer, NM23-H2 is a hexamer. The fold of NM23-H2 is identical to the fold of other NDP kinases. Two antiparallel helices joined by a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein-protein contacts within the oligomers. The only protein-protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitro and in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.

Differential expression and mutation of NME genes in autologous cultured human melanoma cells with different metastatic potentials.

The putative metastasis suppressor genes, NME1(nm23-1) and NME2(nm23-2), were examined in a model system we developed to approximate the dissemination of melanoma from a primary skin tumor. We utilized two autologous human melanoma cell lines, IV Cl 1 and IV Cl 3, which displayed qualitatively different metastatic phenotypes following subdermal inoculation into nude mice. Highly metastatic IV Cl 1 cells expressed approximately 5 fold lower levels of protein encoded by NME genes than non-metastatic IV Cl 3 cells. Similar differences in NME protein levels were observed in tumors induced by the two cell lines in nude mice. There were no differences in NME mRNA levels between these two cell lines, suggesting that expression of these proteins is regulated at a post-transcriptional level. We found a ser122-pro mutation in the NME2 gene of metastatic IV Cl 1 cells. A similar ser120-gly mutation in NME1 has been found in human neuroblastoma, suggesting that mutation in this region may be a general phenomenon related to tumor progression. These mutations may have functional consequences since they eliminate potential phosphorylation sites and may affect the tertiary structure of mature protein complexes.

Effects of 5 alpha-dihydrotestosterone (DHT) on the transcription of nm23 and c-myc genes in human prostatic LNCaP cells.

Proliferation of the androgen-dependent human prostate LNCaP cells was increased by the androgen DHT. Changes in the steady state level of the nm23 and c-myc mRNA in LNCaP cells, with or without 10 nM DHT, showed the nm23 mRNA to change rapidly, beginning to rise after 2 h and reaching its peak by 4 h of DHT treatment. In contrast, increases in the c-myc gene only became apparent after 4 h of treatment. Maximal increase of nm23 mRNA was observed at 10(-9) M DHT. The basal expression of c-myc and nm23 mRNAs was between 30-70% lower in the LNCaP cells, as compared with the androgen-independent PC-3 and JCA-1 human prostatic human carcinoma cells. Thus nm23 may be classified as a member of the early androgen-responsive genes.

Loss of heterozygosity and decreased expression of NME genes correlate with teratomatous differentiation in human male germ cell tumors.

Embryonal carcinoma in the human male is a pluripotential germ cell tumor (GCT), which is suggested to further differentiate to teratoma which displays somatic differentiation representing all three germinal layers. In a panel of 37 GCTs we determined frequency of loss of heterozygosity (LOH) and the level of expression of nucleoside diphosphate kinase (NDPK) genes NME 1 and NME 2. The frequency of LOH in teratomas (86%) was found to be highly significant (P < 0.01) compared to embryonal carcinomas (17%). We also found that the NME encoded proteins are expressed at a 4-5 fold lower level in teratomas compared to embryonal carcinomas. These findings lead us to hypothesize that a critical level of NDPK may be necessary for suppression of aberrant somatic differentiation.

[Foreign body inhalation in children. Report of a case]. / Inalazione di corpo estraneo in età pediatrica. Descrizione di un caso.

A case of inhalation of a foreign body in a child is reported. The case was recalcitant to treatment (antibiotics, bronchodilators, corticosteroids) and long-term follow-up is recommended.

Chromosomal localization and nucleoside diphosphate kinase activity of human metastasis-suppressor genes NM23-1 and NM23-2.

Human metastasis-suppressor genes nm23-1 (NME1) and nm23-2 (NME2) are implicated in control of the metastatic potential of malignant cells. Using somatic cell hybrid analysis and fluorescence in situ hybridization we co-localized both genes to 17q21.3. The 17q21 region carries the locus responsible for early-onset familial breast-ovarian cancer and several other genes that are involved in tumorigenesis and differentiation and undergo frequent rearrangements during neoplastic development. Thus, our mapping places the NME genes in a region that may be subjected to multiple selection pressures. NME1 and NME2 genes were expressed as soluble proteins in a T7 bacterial expression system. Both proteins are independently active nucleotide diphosphate kinases and readily form intra- and intermolecular disulfide bonds. The biochemical properties of these proteins may explain the diversity of mature eucaryotic nucleoside diphosphate kinases.