Adapted sport effect on postural control after spinal cord injury.

STUDY DESIGN: Cross-sectional study. OBJECTIVE: The aim of this study was to compare trunk muscle activation during anterior and lateral reach in athletic and sedentary individuals with spinal cord injury (SCI) and able-bodied people. SETTINGS: University Hospital-UNICAMP, Campinas, Brazil. METHODS: Individuals with complete traumatic SCI and thoracic neurological level were separated into two groups: sedentary (SSCI: n=10) and physically active (PASCI: n=10). The control group (C: n=10) without SCI was assessed. Trunk muscle activation was recorded during reach and grasp tasks. The significant level was set at P<0.05. RESULTS: The control group showed a highest mean activation for left longissimus muscle during all activities (P<0.05). The PASCI group presented significant highest activation for left iliocostalis muscles during all activities, except in the anterior reach task of 90% maximum reach (anterior reach (AR) 75: P=0.02; right lateral reach (RLR) 75: P=0.03; RLR90: P=0.01). The SSCI group presented highest activation for the left iliocostalis during the right lateral reach task of 75 and 90% maximum reach and right iliocostalis during the anterior reach task of 75% maximum reach (AR75: P=0.007; RLR75: P=0.02; RLR90: P=0.03). A different pattern of muscle activation between the control group and the groups with SCI was observed. CONCLUSION: Our results indicated that sports practice did not affect the trunk muscle activation in people with paraplegia. However, the pattern muscle activation in individuals with SCI is different compared with people without SCI during anterior reach tasks.

VIP expression in vessels of the vaginal wall: relationship with estrogen status.

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a neuropeptide with elevated expression in regions that control urogenital functions. Estrogen appears to modulate VIP expression in various organs, but this effect has not been demonstrated in the vaginal wall. The aim of this study was to evaluate the influence of estrogen status on VIP expression in vessels of the vaginal wall. METHODS: Surgical specimens were removed from the vaginal walls of 18 premenopausal women and 12 postmenopausal women who were given surgery for genital prolapse grade I or II. Vaginal specimens were stained with estrogen receptor-alpha (ER-α) and VIP antibodies. Levels of follicle stimulating hormone (FSH), estradiol, prolactin, fasting glucose and serum thyroxine stimulating hormone were also measured. Estrogen status was assessed on the basis of FSH and ER-α scores. RESULTS: The vaginal walls of premenopausal women had significantly higher ER-α scores than those of menopausal women (premenopausal group, 3.6 ± 2.2; menopausal group, 1.4 ± 1.8; p = 0.01). Premenopausal women also had significantly higher levels of VIP in the vaginal wall than menopausal women (p = 0.02). Increasing age was associated with lower level of VIP staining (odds ratio 0.88; 95% confidence interval 0.78-0.99). CONCLUSION: Levels of ER-α and VIP expression in the posterior vaginal wall were higher in premenopausal than in menopausal women, but VIP expression was not associated with estrogen status. Age was an independent predictor of VIP staining in vaginal wall biopsies.

Giant prolapsed submucous leiomyoma: a surgical challenge for gynecologists.

We present a case of a 45-year-old woman who presented with irregular vaginal bleeding and menorrhagia for two months, with an episode of massive bleeding initiating 24 hours before with hemodynamic shock. Vaginal inspection showed a soft, rounded, friable mass in vaginal introitus. After hospitalization, blood transfusion and hydration, she was submitted to vaginal myomectomy with the withdrawal of a 12-cm white, solid, huge, pedunculated, leiomyoma; however, hysterectomy was performed due to persistent uterine bleeding. The postoperation period had no complications. Macroscopy showed a retraction of the myoma pedicle. Gynecologists should prioritize clamping of a pedicle before surgery, reducing its size if the tumor is large.

Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients.

We describe a method to postlabel, in vivo, biotinylated monoclonal antibodies pretargeted onto tumor deposits when most of the non-tumor-bound antibodies have already been cleared as avidin-bound complexes. The application of this principle to tumor detection by immunoscintigraphy was tested in 20 patients with histologically documented cancer and increased circulating carcinoembryonic antigen levels. One mg of biotinylated anti-carcinoembryonic antigen monoclonal antibody (FO23C5) was administered i.v. (first step). After 3 days, 4-6 mg of cold avidin were injected i.v. (second step), followed 48 h later by 0.2-0.3 mg of a biotin derivative labeled with 111In (2-3 mCi) (third step). No evidence of toxicity was observed. Whole body radioactivity distribution was measured in five patients at various intervals postinjection by the conjugate counting technique. Tumors and metastases were detected in 18 of 19 patients (the remaining patient was a true negative) within 3 h after administration of 111In-biotin by planar or single photon emission tomography imaging. At the time of imaging, tumor/blood pool ratio was 5.5 +/- 3.2, and tumor/liver ratio was 6.7 +/- 3.9. Blood clearance of 111In-biotin was multiexponential, with the fast component having a t1/2 of 5 +/- 3 min. Urinary excretion of radioactivity over 3 h was 63.5 +/- 4.9% of the injected dose. Radioactivity at 3 h was 6.5 +/- 1.8% in blood, 1.6 +/- 0.3% in the kidney, and 2.4 +/- 0.6% in the liver. This approach represents an improvement in immunoscintigraphic techniques for tumor localization. The potential use for radioimmunotherapy is discussed.

Diphenylene iodonium as an inhibitor for the hydrogenase complex of Rhodobacter capsulatus. Evidence for two distinct electron donor sites.

The photosynthetic bacterium Rhodobacter capsulatus synthesises a membrane-bound [NiFe] hydrogenase encoded by the H2 uptake hydrogenase (hup)SLC structural operon. The hupS and hupL genes encode the small and large subunits of hydrogenase, respectively; hupC encodes a membrane electron carrier protein which may be considered as the third subunit of the uptake hydrogenase. In Wolinella succinogenes, the hydC gene, homologous to hupC, has been shown to encode a low potential cytochrome b which mediates electron transfer from H2 to the quinone pool of the bacterial membrane. In whole cells of R. capsulatus or intact membrane preparation of the wild type strain B10, methylene blue but not benzyl viologen can be used as acceptor of the electrons donated by H2 to hydrogenase; on the other hand, membranes of B10 treated with Triton X-100 or whole cells of a HupC- mutant exhibit both benzyl viologen and methylene blue reductase activities. We report the effect of diphenylene iodonium (Ph2I), a known inhibitor of mitochondrial complex I and of various monooxygenases on R. capsulatus hydrogenase activity. With H2 as electron donor, Ph2I inhibited partially the methylene blue reductase activity in an uncompetitive manner, and totally benzyl viologen reductase activity in a competitive manner. Furthermore, with benzyl viologen as electron acceptor, Ph2I increased dramatically the observed lagtime for dye reduction. These results suggest that two different sites exist on the electron donor side of the membrane-bound [NiFe] hydrogenase of R. capsulatus, both located on the small subunit. A low redox potential site which reduces benzyl viologen, binds Ph2I and could be located on the distal [Fe4S4] cluster. A higher redox potential site which can reduce methylene blue in vitro could be connected to the high potential [Fe3S4] cluster and freely accessible from the periplasm.

A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

Abnormal agonist-stimulated cardiac parasympathetic acetylcholine release in streptozocin-induced diabetes.

We examined the effect of three distinct depolarizing conditions on [3H]ACh release from cardiac postganglionic parasympathetic neurons in age-matched controls and insulin-treated STZ-induced diabetic rats to determine whether alterations in neurotransmitter release were present in the diabetic group. The effect of TTX, which exerts a use- and voltage-dependent block of sodium channels, was examined on the release of ACh stimulated by SRIF14 (preferentially acts at the cell body). We also studied the effect of STZ-induced diabetes on [3H]ACh release by the relatively site-specific depolarizing agent VT (preferentially acts at the axon) and high potassium (non-site-specific). Basal, SRIF14-(10(-7) M), VT-(10(-4) M), and K+ (100 mM)-stimulated [3H]ACh release was similar in control and STZ-induced diabetic animals. However, in STZ-induced diabetic but not control rats, SRIF14-induced [3H]ACh release was resistant to TTX (2 x 10(-7) M). In addition, the response to submaximal K+ (25 mM) stimulation was greater in STZ-induced diabetic compared with control animals. Treatment with insulin corrected these abnormalities. These data indicate that in the acute STZ-induced diabetic rat, SRIF14-, VT-, and high K(+)-evoked release of ACH is not impaired, which suggests that the mechanisms associated with ACh storage and release in postganglionic cardiac parasympathetic neurons are not affected in this model.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of mitral valve area in mitral stenosis: four echocardiographic methods compared with direct measurement of anatomic orifices.

OBJECTIVES: This study sought to compare the mitral valve areas of patients with rheumatic mitral valve stenoses as determined by means of four echocardiographic and Doppler methods with those obtained by direct anatomic measurements. BACKGROUND: There has been no systemic comparison between Doppler-determined valve areas and the true anatomic orifice in a single cohort. METHODS: In 30 patients with mitral stenosis, the mitral valve areas determined by two-dimensional echocardiographic planimetry, pressure half-time, flow convergence region and flow area were compared with the values directly measured on the corresponding excised specimen by means of a custom-built sizer. RESULTS: The correlation coefficient was r = 0.95 (SE 0.06, p < 0.0001) for two-dimensional planimetry; r = 0.80 (SE 0.09, p < 0.0001) for pressure half-time; r = 0.87 (SE 0.09, p < 0.0001) for flow convergence region; and r = 0.54 (SD 0.1, p < 0.002) for flow area. Two-dimensional echocardiographic planimetry, pressure half-time, flow convergence region and flow area overestimated the actual anatomic orifice by > 0.3 cm2 in 2, 1, 6 and 0 patients, respectively, and underestimated it by > 0.3 cm2 in 0, 4, 1 and 8 patients, respectively. CONCLUSIONS: Mitral valve areas determined by two-dimensional planimetry, pressure half-time and proximal flow convergence region reliably correlated with size of the anatomic orifice. The flow area method provided a less reliable correlation.

Distortion-product otoacoustic emissions and selective sensorineural loss in IDDM.

OBJECTIVE: To provide information about possible subclinical damage of the cochlear outer hair cells (OHCs) by means of transiently evoked otoacoustic emissions (TEOAEs) and distortion-product otoacoustic emissions (DPOAEs) in subjects with IDDM. RESEARCH DESIGN AND METHODS: TEOAEs and DPOAEs were recorded in 47 IDDM patients with normal hearing and in age- and sex-matched nondiabetic subjects. Peripheral neuropathy was diagnosed by nerve conduction velocity (NCV) at the peroneal and surral nerves. RESULTS: A subclinical peripheral neuropathy was found in 15 diabetic patients. Mean TEOAE amplitude was found to be significantly reduced in diabetic patients with a reduced NCV (7.6 +/- 3.2 dB; Scheffé's test: P = 0.03), but not in those without neuropathy (9.5 +/- 4.3 dB), with respect to control subjects (11 +/- 3.1 dB). Neuropathic patients also showed mean reduced DPOAE amplitude values in the region of middle and high frequencies from 1,306 to 5,200 Hz (P < 0.05), whereas no difference was found at the lowest-frequency amplitudes. A frequency-selective reduction of DPOAEs was also found in non-neuropathic patients (P < 0.05) in the region of higher frequencies at 3,284, 4,126, and 5,200 Hz compared with control subjects. No correlations were found among duration of diabetes, HbA1c values, TEOAEs and DPOAEs. CONCLUSIONS: Our results suggest that IDDM patients show an early abnormality of the micromechanical properties of the OHCs. In IDDM patients without a subclinical peripheral neuropathy, damage is limited to the higher frequencies and can be detected only by DPOAEs, whereas in IDDM patients with neuropathy, damage also involves the middle range of frequencies and can be detected by TEOAEs and DPOAEs. Therefore, DPOAEs seem to be able to detect the earliest cochlear selective-frequency dysfunction in IDDM patients without peripheral neuropathy. DPOAEs appear to be of greater clinical interest than TEOAEs; the former seem to be frequency specific and can be recorded at any chosen frequency, including high frequencies.

In vivo evaluation of timing, degree, and distribution of bacterial translocation following experimental small bowel transplantation.

These studies were designed to evaluate the correlation between morphologic and functional changes after heterotopic auxiliary small bowel isograft with systemic venous drainage and two ostomies in 20 Lewis rats. Morphologic damage of the graft was scored by full-thickness biopsies before surgery and 1, 3, 5, and 7 days after transplant. Functional evaluation of the graft was done, at the same time points, by urinary excretion of lactulose and mannitol injected in the proximal ostomy. The intestinal permeability was also studied by injecting Escherichia coli labeled with indium-111 oxine in the proximal ostomy. Translocation of radiolabeled bacteria was quantitated in extraintestinal tissues by radionuclide counts and number of viable organisms and in vivo by scintigraphic imaging. One day after transplant, significant graft damage (score 17.2 +/- 4.2) was observed when compared with the pretransplant value (7.3 +/- 2.6). The degree of tissue injury was similar on days 3 (15.8 +/- 3.5) and 5 (16.1 +/- 3.9) after transplant and remained high on day 7 (11.8 +/- 2.8). The lactulose to mannitol ratio showed a significantly increased permeability on day 1 (17.5) versus pretransplant values (2.6), remained high on day 3 (8.6), and returned to normal values on day 5 (2.8). Translocation of bacteria to distant organs, as measured by both radionuclide counts and number of viable organisms, was strikingly enhanced on day 1 after transplantation, compared with control animals, but returned to the pretransplant value on day 3. A good qualitative and quantitative correlation was observed between radionuclide counts in the extraintestinal organs and in vivo images obtained by scintigraphic scanning. In conclusion, in this model, timing and degree of bacterial translocation do not seem to correlate well, with the exception of the acute post-transplantation phase, with morphologic and permeability changes of the graft. Evaluation of translocation by scintigraphic imaging appears a suitable approach to study in vivo the kinetics and distribution of this process.

Radioimmunoguided surgery using iodine-125-labeled biotinylated monoclonal antibodies and cold avidin.

UNLABELLED: One of the limitations of intraoperative tumor detection with radiolabeled monoclonal antibody (Mab), by means of a gamma-detecting probe (GDP), is the long time interval needed between Mab injection and surgery to obtain low blood-pool activity. Such an interval can be shortened considerably, exploiting the high affinity between avidin and biotin. METHODS: Twenty patients with colorectal cancer were injected with 1 mg of biotinylated 125I monoclonal antibodies followed, 48 hr later, by a chase of cold avidin. During surgery, the GDP was used to detect radioactive emissions from the tumor and normal tissue. Tumor tissue samples were analyzed in vitro by immunohistochemical tests for the presence of tumor antigens and in vivo antibody localization. RESULTS: At the time of surgery (average 7 days postinjection), the mean value of circulating radioactivity was 6% +/- 3% of the injected dose. Of 20 patients studied, tumors were localized in 13 cases (65%). Subclinical tumors were detected in 3 patients (15%). CONCLUSION: The use of 125I-labeled biotinylated Mabs followed by avidin as a chase enhances the applicability and effectiveness of radioimmunoguided surgery technology and will allow the use of radioisotopes with a shorter half-life than 125I.

Imaging osteomyelitis with streptavidin and indium-111-labeled biotin.

UNLABELLED: Animal studies of infection imaging by a two-step protocol have shown that important improvements in target to nontarget ratios are possible. In this protocol, unlabeled streptavidin is administered and allowed sufficient time to accumulate in the lesion, probably by nonspecific processes, and to clear elsewhere. Thereafter, 111Inbiotin is administered. A fraction of the labeled biotin may be retained in the lesion because of biotin's high affinity for streptavidin while most of the activity is cleared through the kidneys. METHODS: Radioscintigraphy with unlabeled streptavidin followed with 111Inlabeled biotin was performed in 15 patients with chronic osteomyelitis. As controls, each patients received either 111In-labeled biotin without the preadministration of streptavidin or 111In-labeled nonspecific IgG. RESULTS: Regions of focal uptake were identified in all patients receiving streptavidin followed by radiolabeled biotin as early as 10 min postadministration of radioactivity, and retention of label was evident through 24 hr. Coincident regions of abnormal accumulation were apparent with 111In-IgG, but only in delayed images. Moreover, with 111In-biotin alone, without the preadministration of streptavidin, focal accumulations were detected in areas similar to that identified with the two-step protocol. Although, these observations were only in the earliest images. CONCLUSION: The results of this preliminary clinical investigation suggest that a two-step protocol with unlabeled streptavidin and radiolabeled biotin may be an alternative for the detection of infection.

Quantitative comparison of direct antibody labeling and tumor pretargeting in uveal melanoma.

UNLABELLED: SPECT radioimmunoscintigraphy with 99mTc-labeled anti-melanoma monoclonal antibodies (MAbs) 225.28S is being used to detect uveal melanoma. Recently, pretargeting methods have been described to reduce background activity and perform imaging in a shorter time interval. METHODS: We compared the three-step pretargeting method with conventional radioimmunoscintigraphy in 15 patients with a clinical and laboratory diagnosis of uveal lesion. High-resolution SPECT radioimmunoscintigraphy was performed in all patients with directly labeled MAbs and, 1 wk later, with the three-step pretargeting technique. Eleven patients underwent eye enucleation and specimens of uveal melanoma were available for histology, whereas four patients underwent conservative therapy. The percent injected dose (%ID) delivered to the tumor and the tumor-to-background ratio were calculated. RESULTS: In all three-step radioimmunoscintigraphy studies, there was a reduction of nonspecific nasopharyngeal background. The three-step radioimmunoscintigraphy tumor-to-nontumor ratio was 3.1 +/- 1.3 versus 1.5 +/- 0.5 of conventional radioimmunoscintigraphy, while the percent injected dose on the tumor was similar for the two methods (4.4 +/- 3.0 versus 3.8 +/- 2.8) x 10(-3). CONCLUSION: Improved SPECT imaging with the three-step radioimmunoscintigraphy results from reduced background and from higher counting statistics due to reduction of time interval between radiotracer administration and imaging, whereas the absolute amount of tracer delivered to the tumor by the two methods is comparable.

Comparison of dual-head coincidence PET versus ring PET in tumor patients.

UNLABELLED: This study compared the multiring detector (Ring-PET) and the dual-head coincidence imaging system (DH-PET) for staging/ restaging neoplastic patients before or after surgery or radiochemotherapy. METHODS: Seventy patients with suspected tumor recurrence or metastatic dissemination received an intravenous dose of 18F-fluorodeoxyglucose (FDG) under overnight fasting and were studied in sequence with a dedicated positron emission tomograph with Ring-PET and a DH-PET. Ring-PET studies were performed 45-75 min postinjection and were followed by a DH-PET scan approximately 3 h postinjection. Number and location of the hypermetabolic lesions detected on DH-PET and Ring-PET reconstructed images were blindly assessed by three independent observers. RESULTS: DH-PET identified all 14 head lesions detected by Ring-PET, 53 of 63 thoracic lesions and 36 of 45 abdominal lesions. Of the 19 lesions not identified by DH-PET, 6 were smaller than 10 mm, 8 were between 10 and 15 mm and 1 was 18 mm; dimensions of 4 bone lesions were not available. A concordant restaging, based on location and number of lesions detected, was found in all 14 patients with head tumors, in 28 of 30 patients with thoracic tumors and in 24 of 26 patients with abdominal tumors. CONCLUSION: We found a good agreement between Ring-PET and DH-PET assessment of oncologic patients in detecting hypermetabolic lesions > or = 10-15 mm.

Three-step immunoscintigraphy with anti-chromogranin A monoclonal antibody in tumours of the pituitary region.

In the present paper we evaluated the ability of pretargeted immunoscintigraphy (ISG) with antichromogranin A (CgA) monoclonal antibody (Mc-ab A11) in visualizing pituitary masses. The results obtained in 23 patients are described along with those of [111In]pentetreotide scintigraphy (Octreoscan) in 18 cases. Positive ISG was obtained in 9/11 (82%) non-functioning, 1/4 growth hormone-, 1/2 prolactin-, 1/2 thyrotropin-, 1/1 follicle-stimulating hormone-, 0/1 adrenocortico-trophin-secreting pituitary adenomas. In one patient with a positive ISG scan of a non-functioning pituitary adenoma, an associated neurinoma of the acoustic nerve was not imaged. The same occurred in one patient with a pituitary deposit from a mammary carcinoma and in another one with a cyst of the Rathke's pouch. Chromogranin A immunohistochemistry, carried out in 10 tumours, was positive in eight pituitary adenomas and negative in two non-adenomatous lesions. A concordant ISG occurred in all cases except for two pituitary adenomas: one cystic and necrotized and one post-operative remnant very small in size. In 18 patients with pituitary adenoma both ISG and Octreoscan were positive in 61% of cases but with a different distribution among tumours. At variance with ISG, Octreoscan visualized only 5/10 (50%) non-functioning pituitary adenomas and all (4/4) somatotropinomas. In conclusion, ISG is able to image pituitary tumours and particularly non-functioning pituitary adenomas. In this respect, it may be helpful in discriminating non-neuroendocrine masses of the pituitary region from non-functioning pituitary adenomas.

Glucose transporters in rat peripheral nerve: paranodal expression of GLUT1 and GLUT3.

Peripheral nerve depends on glucose oxidation to energize the repolarization of excitable axonal membranes following impulse conduction, hence requiring high-energy demands by the axon at the node of Ranvier. To enter the axon at this site, glucose must be transported from the endoneurial space across Schwann cell plasma membranes and the axolemma. Such transport is likely to be mediated by facilitative glucose transporters. Although immunohistochemical studies of peripheral nerves have detected high levels of the transporter GLUT1 in endoneurial capillaries and perineurium, localization of glucose transporters to Schwann cells or peripheral axons in vivo has not been documented. In this study, we demonstrate that the GLUT1 transporter is expressed in the plasma membrane and cytoplasm of myelinating Schwann cells around the nodes of Ranvier and in the Schmidt-Lanterman incisures, making them potential sites of transcellular glucose transport. No GLUT1 was detected in axonal membranes. GLUT3 mRNA was expressed only at low levels, but GLUT3 polypeptide was barely detected by immunocytochemistry or immunoblotting in peripheral nerve from young adult rats. However, in 13-month-old rats, GLUT3 polypeptide was present in myelinated fibers, endoneurial capillaries, and perineurium. In myelinated fibers, GLUT3 appeared to be preferentially expressed in the paranodal regions of Schwann cells and nodal axons, but was also present in the internodal aspects of these structures. The results of the present study suggest that both Schwann cell GLUT1 and axonal and Schwann cell GLUT3 are involved in the transport of glucose into the metabolically active regions of peripheral axons.

NA+/K+ ATPase impairment and experimental glycation: the role of glucose autoxidation.

Non enzymatic glycation could be involved in the early impairment of Na+/K+ ATPase that occurs in sciatic nerve of diabetic rats. In fact, decrease of Na+/K+ ATPase activity is one of the first alterations showed in experimental diabetic neuropathy. In this respect, it is known that in the presence of transition metals under physiological conditions, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free radical intermediates, which, in turn, inhibit the cation pump. Our experiments were designed to determine if glucose autoxidation has any relevance in the early steps of Na+/K+ ATPase experimental glycation. Compared experiments with and without the sodium borohydride (NaBH4) reduction step demonstrated that incubation of brain Na+/K+ ATPase with glucose 6-phosphate (G 6-P) and trace metals induced a significant decrease in enzyme activity dramatically enhanced by addition of copper (Cu2+). A concomitant production of H2O2 was noticed. The presence of diethylenetriaminepentaacetic acid (DTPA), a strong metal chelator, completely prevented Na+/K+ ATPase impairment and hydrogen-peroxide formation. No gross structural and conformational alterations of the enzyme can be demonstrated by intrinsic and extrinsic fluorescence measurements. Our results suggest that during the exposure of brain NA+/K+ ATPase to glucose 6-phosphate in vitro (experimental glycation), the decrease in activity can be correlated, at lease in the early phases, to metal-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the enzyme. Since plasma hydroperoxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complications of diabetes like neuropathy.

Immunoscintigraphy with three step monoclonal pretargeting technique in diagnosis of uveal melanoma: preliminary results.

Several problems still limit the full use of the diagnostic potential of immunoscintigraphy (IS) with technetium-99m labelled monoclonal antibodies (MoAbs) 225-28S directed to high molecular weight melanoma associated antigen (HMW-MAA). The principal problem is the unfavourable ratio of tumour to non-tumour activity (T/nT), due to the poor tumour uptake and the high aspecific uptake of the tissue surrounding the tumour. Recently, it was demonstrated that using the tumour pretargeting technique based on the injection of monoclonal antibody and the avidin/biotin system (three step immunoscintigraphy), an improvement in the T/nT ratio can be obtained in patients with carcinoembryonic antigen secreting tumours. The aim of this study was to compare the diagnostic sensitivity of traditional immunoscintigraphy with that of three step immunoscintigraphy in seven patients with uveal melanoma. All the patients underwent immunoscintigraphy with MoAb 225.28S radiolabelled with technetium-99m, and a three step immunoscintigraphy 1 week later. No patients demonstrated immediate toxic effects after receiving the reagents, no matter which of the two methods was used. The traditional immunoscintigraphy had a diagnostic sensitivity of 71.4%, diagnosing five out of seven melanomas tested. The three step study detected all the melanomas examined (7/7) with a diagnostic sensitivity of 100% and showed a drastic reduction in background. The preliminary results confirm the feasibility of visualising the uveal melanoma and show that the three step immunoscintigraphy is more diagnostically sensitive than traditional immunoscintigraphy, particularly in small lesions.

Lower limb arterio-venous shunts, autonomic neuropathy and diabetic foot.

We have quantitatively assessed the percentage of lower limb arterio-venous (a-v) shunting using a radioisotopic technique and correlated it with autonomic neuropathy evaluated by cardiovascular tests. We have studied three groups of diabetic patients: Group A, 12 non-neuropathic subjects without foot lesions; Group B, 12 neuropathic subjects without foot lesions; Group C, 12 neuropathic subjects with recurrent foot ulcers. Shunting was higher in Group C (10.4 +/- 2.7%) than in Group B (6.8 +/- 2.3%, P less than 0.01) and Group A (3.8 +/- 1.2%, P less than 0.001). Shunts in Group B were higher than in Group A (P less than 0.05). All the tests exploring autonomic function were more impaired in Groups B and C than in Group A, with no difference between Groups B and C. A direct correlation was found between a-v shunting and the following cardiovascular tests: postural hypotension (PH) (r = 0.41, P less than 0.02), sustained handgrip (SH) (r = 0.56, P less than 0.001), deep breathing (DB) (r = 0.40, P less than 0.005) and lying to standing (LS) (r = 0.44, P less than 0.01). A positive correlation was also found between a-v shunts and duration of the disease (r = 0.62, P less than 0.001). Arterio-venous shunting was found to be directly related to autonomic neuropathy even if the higher shunting found in the patients with foot ulcers was not related to a higher degree of autonomic involvement; in addition, this group of patients was characterized by having a more advanced sensory and motor neuropathy. In conclusion, autonomic neuropathy, through its influence on a-v shunts, may play a role in the pathogenesis of diabetic foot, but peripheral neuropathy probably plays a key role in conditioning the development of the overt clinical manifestations of diabetic foot.

Pretargeting of carcinomas with the avidin-biotin system.

Diagnosis and experimental therapy of cancer have been performed with encouraging results using radiolabelled monoclonal antibodies. However, the high background due to non-specific uptake by normal tissue and blood is a major drawback in antibody-guided tumor detection. Various strategies have been proposed to overcome this problem, such as computed background subtraction, use of a second antibody, and local delivery. An antibody is a slow "bullet" for tumor targeting, since in many lesions it requires two or three days to accumulate. The use of fragments such as F(ab')2 or Fab, which display a faster blood clearance than whole antibody, improves tumor localization to a sufficient extent (hours) to allow the use of the most suitable radionuclides, e.g. 99m-Tc. In therapeutic applications we are still far away from the optimal condition in terms of the absolute amount of radioactivity delivered to the tumor. The high specificity of antibodies could be exploited at its best by delaying the delivery of the label to a time when the ratio tumor-bound to non-tumor-bound antibody has reached its maximum value. To obtain this goal, the label should display a fast clearance and should be captured by the antibody already targeted onto tumor cells. These considerations have led to strategies of tumor pretargeting where antibody and label are administered separately. One of these strategies, based on the avidin-biotin system, has already been used extensively for several years in immunohistochemistry and in ELISA. Due to the flexibility of this system, several alternative protocols are possible.(ABSTRACT TRUNCATED AT 250 WORDS)