Routine completion angiography during carotid endarterectomy is not mandatory.

OBJECTIVE: Intraoperative quality control after carotid endarterectomy (CEA) has been advocated to improve the results of surgical treatment of extracranial carotid artery disease. The aim of this study was to evaluate the usefulness of completion angiography (CA) in prevention of stroke and restenosis after CEA in a single center experience. MATERIALS AND METHODS: Data concerning 914 consecutive CEAs performed in 3 years (2000-2002) were prospectively collected in a dedicated database. Patients were divided into two groups: in the first group (mandatory-CA group; 430 cases) CA was routinely carried out, except in presence of contraindications to iodinate contrast agents; in the second group (selective-CA group, 484 cases) CA was performed only in selected cases, at surgeon's discretion. RESULTS: There were no significant differences between the two groups in terms of neurological complications at awakening (0.5% in mandatory-CA group and 0.4% in selective-CA group; p=n.s.) and in 30-day stroke and death rate (1.9% and 1.4%, respectively; p=n.s.). A surgical revision on the basis of CA findings was performed in 5 cases in mandatory-CA group and in 2 cases in selective-CA group (1.2% and 0.4%, respectively; p=n.s.). In the second group, the conditions significantly associated with the need for CA examination were internal carotid near-occlusion, preoperative symptoms, shunt insertion, kind of surgical reconstruction, redo surgery. Estimated absence of ipsilateral stroke and absence of restenosis at 18 months was 98.9% and 89.7% in mandatory-CA group and 99.3% and 93.4% in selective-CA group (p=n.s.) respectively. CONCLUSIONS: Based on our experience, routine CA following CEA is not suggested. A policy of selected CA at the surgeon's discretion seems to make the intervention safe and durable as well.

Reducing the risk of intraoperative neurological complications during carotid endarterectomy with early distal control of the internal carotid artery.

AIM OF THE STUDY: To assess the feasibility and effectiveness of a modified surgical technique with early clamping of the distal internal carotid artery (ICA) during carotid endarterectomy in a single centre experience. STUDY DESIGN: Retrospective study, teaching hospital. MATERIAL AND METHODS: Between 1996 and 2002, 2235 CEAs were performed. Until April 1999, the intra-operative strategy consisted of standard isolation and dissection of the carotid bifurcation preliminary to ICA clamping (group 1; 1090 interventions). Starting from May 1999, we performed early isolation and clamping of the distal ICA, followed by dissection of the carotid bifurcation and clamping of the external and common carotid artery (group 2; 1145 interventions). RESULTS: The modified technique was feasible in all the patients of group 2. In group 2 there was a significantly lower incidence of neurological deficit on waking than in group 1 (0.4% and 1.8%, respectively; p=0.02). CONCLUSIONS: Early distal control of the internal carotid artery during CEA is feasible and could contribute to reducing intra-operative neurological events.

Carotid endarterectomy with contralateral carotid artery occlusion: is this a higher risk subgroup?

OBJECTIVE: to evaluate early and mid-term term results of carotid endarterectomy (CEA) in patient with and without contralateral carotid occlusion. METHODS: between 1996 and 1999, 1324 CEAs were performed. In 82 patients contralateral carotid artery occlusion was present (group I); 1242 patients had patent contralateral carotid (group II). All patients were operated under general anaesthesia, and selective shunting was based on somatosensory evoked potentials (SEPs). Ultrasonographic follow-up was performed at 1, 6 and 12 months and then once a year. Early results and follow-up data were analysed retrospectively. RESULTS: in group I there was a significantly higher incidence of SEPs reduction and shunt insertion; however, there were no differences in terms of perioperative complications. The cumulative stroke and death rate at 30 days in group 1 and group 2 were 2.4% vs 1.4% (p=n.s.), respectively. At a mean follow-up of 15 months there were no differences between the two groups in terms of cumulative symptom-free survival. CONCLUSIONS: the presence of contralateral carotid occlusion caused an increased use of shunt, but not in early complications rates.

Early and long-term results of surgical treatment of splenic artery aneurysms.

BACKGROUND: This retrospective study was conducted to describe the presentation, surgical treatment, and follow-up of patients with splenic artery aneurysms. METHODS: From 1982 to 2000, 1,952 patients with abdominal aneurysms were referred to our department; 15 had splenic artery aneurysms. None had ruptured. All were operated on. RESULTS: Fourteen complete and 1 partial aneurysmectomies were carried out. Arterial continuity was restored in 10, by end-to-end anastomosis, and 4 had splenectomies. In 1 patient the spleen was preserved without arterial reconstruction. There were no deaths. Morbidity was restricted to 1 patient with a limited, asymptomatic splenic infarction. Eleven patients were followed up for a mean 19.7 months. No deaths or major complications were recorded. Reconstructed splenic arteries were patent in all cases without atrophy or new cases of splenic infarction. CONCLUSIONS: Elective surgery for splenic artery aneurysms is safe. Arterial reconstruction allows good early and long-term results. In some cases splenectomy may be unavoidable.

A humanized anti-tumor necrosis factor-alpha monoclonal antibody that acts as a partial, competitive antagonist of the template antibody.

We have constructed several humanized versions of a monoclonal antibody (MAb78) against human tumor necrosis factor-alpha (huTNF-alpha) retaining the complementarity-determining regions (CDR) of the original mouse MAb with or without a variable number of original framework region (FR) residues. All versions, except one, showed a loss of binding affinity and neutralizing potency of at least 10-fold compared to the original mouse MAb or its chimeric equivalent. In some cases, however, the decrease in neutralizing potency was significantly greater than the decrease in binding affinity. Two humanized versions showing the greatest dissociation between these two parameters were studied for their capacity to inhibit the neutralizing activity of chimeric or murine MAb78 when used at concentrations that bound but only partially neutralized huTNF-alpha. One humanized version (MAb78D) was indeed able to do so, whereas the other (MAb78C) was not found to exert any inhibitory activity at all concentrations tested. The antagonistic effect of MAb78D was concentration dependent and could be overcome by increasing the concentrations of chimeric or murine MAb78. Two different models of MAb78-huTNF-alpha interaction that may help explain the antagonist activity of humanized MAb78D are discussed.

Idiotope determining regions of a mouse monoclonal antibody and its humanized versions. Identification of framework residues that affect idiotype expression.

The contribution of framework regions (FRs) of antibody-variable domains to idiotype expression was studied by examining the interaction of various "humanized" versions of a mouse anti-TNF alpha monoclonal antibody (mAb78) with polyclonal and two monoclonal antibodies (mAb1G3 and mAb9F1), generated against the mAb78 idiotype. Humanized mAb78, bearing human constant domains and mouse complementarity-determining regions (CDRs) inserted with human FRs, was found to be five to sevenfold less reactive than mAb78 with polyclonal anti-idiotype antibodies and 200 to 300-fold less active in neutralizing TNF alpha. The substitution of heavy-chain FRs residues of the humanized antibody with original mouse residues 28 to 30, 48 to 49, 67 to 68, 70 to 71, 78, 80 and 82 progressively restored the immunoreactivity with polyclonal immunoglobulin Gs to the level of a version having mouse heavy chain and human light chain FRs, and increased 10 to 20-fold the TNF alpha neutralizing activity. This suggests that at least some of these residues are critical for TNF alpha binding as well as for the expression of idiotopes that are strongly immunogenic in syngeneic animals. All antibody versions with either human or mouse FRs were able to bind to various extents mAb1G3, a gamma-type anti-Id antibody that inhibits mAb78/TNF alpha interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an alpha-type anti-Id antibody unable to block the access of TNF alpha to mAb78 paratopes. Substitution of heavy chain FR residues 28 to 30 markedly decreased the binding of mAb1G3 (100 to 1000-fold). This suggests that these antibodies recognize CDR and FR idiotopes, respectively, that can be drastically modified by changes in the FRs. In conclusion, the results suggest that CDRs as well as FRs markedly contribute to antibody Id expression. Although strongly immunogenic idiotopes are probably located within the CDRs, the results also suggest that some FR residues are critically involved in shaping antibody Id diversity by affecting the structure of CDR-related idiotopes.

Evidences that syngeneic alpha-type anti-idiotypic antibodies may non-competitively inhibit idiotype/oligomeric antigen interactions by affecting idiotype avidity.

The effects of syngeneic anti-Id antibodies on the multivalent interaction between human TNF-alpha, a homotrimeric Ag, and an anti-TNF mAb (mAb(1)78) have been studied. Eight anti-mAb(1)78 Ig secreting hybridoma, able to inhibit TNF binding in a competitive or non-competitive mode, have been generated. Two representative clones (mAb(2)1G3 and mAb(2)9F1) were selected for studying the inhibition mechanism of TNF-mAb(1)78 interaction. Idiotype-paratope topography studies indicated that mAb(2)1G3 (IgG2a) and mAb(2)9F1 (IgG1) bind two sterically distinct idiotopes on mAb(1)78 (IgG1) V regions. In particular, mAb(2)1G3 was found to bind an idiotope located within (or spatially close to) the Ag combining site suggesting that competitive inhibition of TNF binding to mAb(1)78 by mAb(2)1G3 occurs through paratope blockade. On the other hand, mAb(2)9F1 recognizes an idiotope located outside the paratope, being able to bind mAb(1)78 even in the presence of saturating amounts of TNF. mAb(1)78-TNF molar ratio in complexes, at stoichiometric equivalence, was unchanged in the presence of a large excess of mAb(2)9F1, suggesting that the functional bivalency of mAb(1)78 was not impaired by this anti-Id antibody. However, bivalent mAb(2)9F1 was able to partially inhibit the binding of bivalent mAb(1)78 to oligomeric TNF in liquid-phase as well as in solid-phase assays, whereas no inhibition was observed with monovalent mAb(2)9F1-F(ab) or mAb(1)78-F(ab). This suggests that inhibition is based on a decrease of the avidity of bivalent mAb(1)78 and not on allosteric effects on antigen binding sites. The effect of mAb(2)9F1 on mAb(1)78 arm flexibility and paratope orientation is discussed. In conclusion, the results indicate that anti-Id antibodies may inhibit Ag-antibody multivalent interactions by paratope blockade or by affecting the antibody avidity.

Mode of interaction between tumor necrosis factor alpha and a monoclonal antibody expressing a recurrent idiotype.

Tumor Necrosis Factor alpha (TNF alpha) is an inflammatory cytokine which exists mainly as a 51kD complex built up of 3 identical, noncovalently-linked polypeptide subunits. We have raised monoclonal antibodies (mAb) against human TNF alpha (huTNF alpha). One of these mAb (mAb78, mouse IgG1k) was studied in detail. mAb78 expresses a recurrent idiotype typical of the BALB/c anti-huTNF alpha antibody response. HuTNF alpha bound to mAb78 with an affinity constant (Kobs) of 3.2 x 10(10)M-1. The number of huTNF alpha-binding sites per mAb78 molecule was approximately 0.7. At concentrations higher than the Kobs mAb78 neutralized huTNF alpha at a approximately 1.3:1 molar ratio. mAb78 precipitated huTNF alpha in a double immunodiffusion assay in agar. Gel-filtration experiments of mAb78-huTNF alpha mixtures, that had been set up in large antigen excess, detected complexes of 570 kD as the smallest ones formed under these conditions. We propose that these results are accommodated best by a model according to which cyclic complexes built up of 3 mAb78 and 2 huTNF alpha molecules are the smallest units formed upon interaction of the reagents. In view of this model we discuss how huTNF alpha and mAb78 can undergo a precipitin reaction.

Oligomeric tumour necrosis factor alpha slowly converts into inactive forms at bioactive levels.

The stability of oligomeric human tumour necrasis factor alpha (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37 degrees C) at 0.12 nM, whereas no apparent dissociation was detected at 4 nM. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nM by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.

Effects of lead on hepatocyte ultrastructure in Carassius carassius (L.) var. auratus.

Subcellular modifications in hepatocytes of Carassius carassius var. auratus subjected to 24 hr and 48 hr sublethal acute lead (5mg.1-1) exposure were studied by electron microscopy. Cytological alterations were observed after 24 hr of treatment and became more evident after 48 hr. Lead induced an increase in nuclear heterochromatin and alterations in mitochondria, endoplasmic reticulum and Golgi complex ultrastructure. Glycogen granula decreased, and secondary lysosomes and lipid droplets increased. Furthermore, intracytoplasmic lumina with microvilli-bearing surfaces and numerous autophagic vacuola were observed after 48 hr of exposure.

Rearrangement and expression of the antigen receptor alpha, beta and gamma genes in suppressor antigen-specific T cell lines.

The rearrangement and transcription of the antigen receptor alpha, beta and gamma genes were investigated in murine antigen-specific suppressor T cell lines, to establish whether the suppressor T cell subset expresses the same antigen receptor transcripts previously found in helper and cytotoxic T lymphocytes. The genomic organization of the alpha, beta and gamma chain loci was investigated using probes representative of the entire gene or fragments from variable, joining and constant regions. The present results show that in functional suppressor T cells the three antigen receptor genes are all rearranged. The beta gene is expressed in all the tested cell lines, while the expression of the alpha and gamma genes is variable. In one cell line (LH8) alpha and gamma genes are not efficiently transcribed; in the other cell line (LA41) the gamma mRNA is found in amounts similar to beta mRNA, whereas the alpha gene is expressed at low levels. These data suggest that in suppressor T cells no direct correlation exists between the expression of alpha, beta and gamma antigen receptor genes and the effector function.

Immunosuppression by cell-free translation products from monoclonal antigen-specific suppressor T cell mRNA.

Polypeptides synthesized in a rabbit reticulocyte lysate system directed by mRNA from the T cell line LH8-105, obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific suppressor T lymphocytes, are able, when injected into mice, to specifically suppress the antibody response and delayed-type hypersensitivity to HEL. The suppressive activity exerted by in vitro translated proteins appears to be independent from post-translational modifications. These in vitro translated polypeptides display fine antigenic specificity in immunosuppression and bind to HEL but not to the closely related ring-necked pheasant egg-white lysozyme immunosorbents. Suppressive molecules obtained by cell-free translation of LH8-105 mRNA or by culture supernatant of LH8-105 cells display, by gel filtration, a similar molecular mass of about 82-90 kDa.