Evaluation in a clinical setting of the performances of a new rapid confirmatory assay for HIV1/2 serodiagnosis.

BACKGROUND AND OBJECTIVES: The performances of the new Geenius rapid confirmatory test (Bio-Rad) were evaluated with emphasis towards identifying acute infection (AHI) and discriminating HIV-1/2 in a clinical setting STUDY DESIGN: Serum samples from individuals attending the L. Spallanzani Institute in Rome, Italy, for HIV diagnosis (one year retrospective collection), repeatedly reactive at 4th generation HIV-1/2 screening assays, confirmed with HIV-1 and HIV-2 Western blot (New LAV I and II Bio-Rad), were retested with Geenius. RESULTS: Of 6,200 samples, 406 resulted repeatedly reactive at screening, including samples from clinically confirmed AHI. New LAV I identified 378 HIV-1-positive samples. Of these, Geenius found 377 HIV-1-positive and one unclassified HIV-positive. New LAV I classified as indeterminate 18 samples, including 14 from AHI. Among these 14, Geenius results were: 12 positive, 1 indeterminate and 1 negative. Of the remaining, 2 resulted Geenius negative (false-positive screening results) and 2 HIV-2. Ten samples were New LAV I-negative (5 AHI). Geenius results were: 1 (AHI) positive and 9 negative. Geenius detected 110 additional positive samples with no p31 reactivity with respect to New LAV I, with an almost similar prevalence of low avidity samples. Geenius confirmed 3 out of 4 HIV-2 infections identified by New LAV II (one coinfected with HIV-1), while rated as HIV-1 the remaining sample, classified as coinfection by New LAV I and II. CONCLUSIONS: Geenius allows fast, sensitive and accurate confirmation of HIV serodiagnosis, including AHI and HIV-2 infections. The high sensitivity, in particular towards AHI, could avoid additional sampling and molecular tests.

Clinical characteristics and associated comorbidities in diabetic patients with cranial nerve palsies.

BACKGROUND: Cranial mononeuropathy is one of the not so common forms of diabetic neuropathy that often appears to be a serious problem from a diagnostic and therapeutic point of view. AIM: Objective of this study was to determine the incidence, the clinical characteristics, and risk factors associated with cranial nerve palsies among persons with diabetes. METHODS: We have performed a retrospective study of all diabetic patients with cranial nerve palsies who were hospitalized in a metabolic department over a 12-yr period. RESULTS: During the period of the survey, a total of 8150 diabetic subjects were hospitalized and cranial nerve palsies were identified in 61 patients (0.75%). Isolated III nerve palsies accounted for the majority of patients (0.35%), with VII nerve palsies (0.21%) occurring more frequently than VI (0.15%) and multiple palsies (0.04%). Peripheral neuropathy was present in only 24% of patients. Patients with VII nerve palsies showed a tendency toward a lower coexistence of diabetic complications and cardiovascular risk factors than those with III and VI nerve palsies. CONCLUSIONS: Cranial nerve palsies are a not common problem among patients with diabetes; diagnosis of diabetic mononeuropathy should be considered even in the absence of peripheral neuropathy; the oculomotor nerve was most frequently affected in our case report. The coexistence of diabetic complications and cardiovascular risk factors was slightly lower in patients with VII nerve palsy: this fact is compatible with the hypotesis that this event is less closely related to diabetes and metabolic factors in its pathogenesis.

Severe hypoglycaemia leading to hospital admission in type 2 diabetic patients aged 80 years or older.

AIM: Evidence is mounting that hypoglycaemia among elderly diabetic patients is a very real and costly concern. Objective of this study was to determine the incidence and risk factors for developing severe hypoglycaemia leading to hospital admission, among type 2 diabetic subjects aged 80 years or older. METHODS: Hypoglycaemia was defined as a symptomatic event requiring treatment with i. v. glucose and confirmed by a blood glucose determination <50 mg/dl. RESULTS: During a eight-year period severe hypoglycaemia was identified in 99 subjects. These patients were found to have a reduced cognitive ability, a heavy burden of comorbid disease and a HbA1c values of 5.9%. Of the hypoglycaemic episodes, 76 occurred in patients taking glibenclamide. Diabetes therapy was prescribed by general practitioners in 85 of them. Only 26 patients performed regular home blood glucose self-monitoring. CONCLUSION: Severe hypoglycaemia is a serious and not uncommon metabolic emergency among patients with type 2 diabetes aged 80 years or older; it is more frequent in patients with considerable comorbidity undergoing aggressive diabetes management and in users of a long-acting sulphonylurea. In elderly subject, each patient's risk for hypoglycaemia should be considered and therapy should be individualized accordingly; in our opinion, a great number of episodes may be avoided by teaching the principles of blood glucose monitoring and involving general practitioners in outpatients management of diabetes mellitus.

Clinical characteristics and associated comorbidities in diabetic patients with restless legs syndrome.

AIM: RLS, despite being a common entity, is often underdiagnosed and appears to be a serious problem from a diagnostic and therapeutic point of view. There have been few studies primarily concerned with the clinical characteristics of RLS in diabetic subjects. Those published have emanated largely from neurological referral centres rather than metabolic departments. The objective of this study was to determine the clinical characteristics and risk factors associated with RLS among persons with diabetes. METHODS: We have performed a retrospective study of all diabetic inpatients with RLS who were seen in the Metabolic Division at "S. Biagio" Hospital, Marsala, over a 18 months period. A detailed history, blood laboratory profile and an electrophysiological evaluation were obtained for each patient. RESULTS: During the period of the survey RLS was identified in 23 patients. These patients were found to have a poorly controlled diabetes and had increased associated comorbidities: they showed an high prevalence of diabetic neuropathy (96%) and metabolic syndrome (74%). Moreover, median duration of RLS symptoms onset was 2.8 years. CONCLUSIONS: RLS is a serious and not uncommon problem among patients with diabetes mellitus. This syndrome is closely related to diabetic neuropathy and probably to metabolic factors in its pathogenesis. Diagnosis of RLS is often delayed and because it can be effectively treated, a better education of the general medical community toward greater awareness of the syndrome is necessary.

Comparison of Versant HBV DNA 3.0 and COBAS AmpliPrep-COBAS TaqMan assays for hepatitis B DNA quantitation: Possible clinical implications.

We compared two commercial assays for HBV DNA quantitation, Versant HBV 3.0, System 340 (bDNA; Bayer Diagnostics) and COBAS AmpliPrep-COBAS TaqMan HBV Test (TaqMan; Roche Diagnostics). Analytical sensitivity, calculated on WHO International Standard, predicted 95% detection rate at 11.4 and 520.2IU/ml for TaqMan and bDNA, respectively. Specificity, established on 50 blood donor samples, was 100% and 84% for TaqMan and bDNA, respectively. When using clinical samples, HBV DNA was detected by TaqMan in 21/55 samples negative to bDNA. Mean values of HBV DNA obtained with bDNA were higher than those obtained with TaqMan (4.09log(10)+/-1.90 versus 3.39log(10)+/-2.41, p<0.001), and 24.4% of samples showed differences in viral load values >0.5log(10), without association with HBV genotype. There was a good correlation for HBV DNA concentrations measured by the two assays (r=0.94; p<0.001) within the overlapping range, and the distribution of results with respect to relevant clinical threshold recently confirmed (20,000 and 2000IU/ml) was similar. Approximately 50% of samples with low HBV DNA, appreciated by TaqMan but not by bDNA, were successfully sequenced in pol region, where drug resistance mutations are located.

Modulation of costimulatory molecules CD80/CD86 on B cells and macrophages by stress proteins GroEL, GroES and DnaK.

The aim of this study was to evaluate the effect of the Heat Shock Proteins GroES, GroEL and DnaK on the expression of the costimulatory molecules CD80/CD86 in B cells and macrophages. The interactions among these molecules are able to highly influence the immune response through the regulation of cytokine liberation which, on their own, are able to regulate the immunological response by a feedback mechanism. Our results showed that, on B cells, GroES and GroEL stimulated the expression of CD86 but did not induce the increase of the CD80 expression. CD86 peak expression showed a peak after 24-48 h of culture and decreased 60h after the stimulation. GroES and GroEL also stimulated the expression of CD80 and CD86 on macrophages. The same HSPs did not modify the expression of CD80 and CD86 on cells having characteristics of activated macrophages, the A-THP-1 cell line. DnaK did not induce any increase in the expression of CD80 and CD86 on lymphocytes or macrophages.

Lipoteichoic acid and muramic acid modulate the expression of CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells.

The aim of this study is to evaluate the effect of lipoteichoic acid (LTA) and muramic acid (MA) on costimulatory molecules CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells. The interactions between these molecules strongly influence the immune response through the regulation of cytokine release which, on its own, is able to regulate the immunological response by a feedback mechanism. Our results show that LTA and MA regulate expression of CD86 on macrophages while the expression of CD80 remains unmodified. LTA and MA increase the expression of CD86 on THP-1 cells, a macrophage cell line. MA increased Jurkat T cells CD152 expression.

Porins and lipopolysaccharide from Salmonella typhimurium regulate the expression of CD80 and CD86 molecules on B cells and macrophages but not CD28 and CD152 on T cells.

OBJECTIVE: The aim of this study was to evaluate the effect of porins from Salmonella typhimurium on costimulatory molecules such as CD80/CD86 and CD28/CD152. The interactions between these molecules are able to influence the immune response through the regulation of cytokines release which, on their own, are able to regulate the immunological response by a feedback mechanism. METHODS: S. typhimurium strain SH5014 (a rough lipopolysaccharide (LPS) producing strain) was used as the source of porins and LPS. Peripheral blood mononuclear cells were obtained from healthy adult donors. THP1 cells were obtained from ATCC (Rockville, MD, USA). Immunofluorescence and flow cytometry were performed using a FACS IV (Becton-Dickinson, Mountain View, CA, USA). RESULTS: Our results show that porins of S. typhimurium increase the expression of CD86 and the expression of CD80 both on B lymphocytes and macrophages, while the expression of CD28 and CD152 on T lymphocytes was unaltered. The expression of CD80 and CD86 is dose-dependent and starts after 24 h post treatment, peaks at 48 h and goes back to the basal value after 72 h. CONCLUSIONS: S. typhimurium porins are able to induce a high expression of costimulatory molecules (CD80 and CD86) on lymphocytes and macrophages.

Coinfection with BHV-1 modulates cell adhesion and invasion by P. multocida and Mannheimia (Pasteurella) haemolytica.

Viruses are thought to facilitate bacterial infections of the respiratory tract. The present study shows the effect of BHV-1 on Pasteurella multocida and Mannheimia haemolytica adherence and invasion of MDBK cells. The virus-infected MDBK cells become more susceptible to the adherence of both species of Pasteurella. The observed adherence increase depends on the length of virus pre-incubation time and on virus concentration. When MDBK cells are not infected with virus, they are only invaded by P. multocida, while M. haemolytica is not able to penetrate. The viral infection favours also the invasion by M. haemolytica.

Synthetic undecapeptide (NTX10-20) of noxiustoxin blocks completely the I(A) potassium currents of cerebellum granular cells.

Native noxiustoxin (NTX) and synthetic peptides corresponding to its primary sequence, from positions 1-9, 1-14, 1-20, 10-20, 21-39 and 30 39, were prepared and assayed on the K+ currents of cerebellum granular cells, using the patch-clamp technique in the whole-cell configuration system. Native toxin has a reversible inhibitory effect (IC50 = 360 nM), whereas synthetic peptides NTXI-20 and NTX1-9 had a half-effective dose IC50 of approximately 2 and 10 microM, respectively, which correlates with their biological effects in vivo. Synthetic peptide NTX10-20 was quite remarkable in having a preference for the IA current, which was completely inhibited at high peptide concentration. The effects of the other peptides (NTXI 14, NTX21-39 and NTX30-39), although positive and reversible, required higher concentrations (50 200 microM) to block both currents, suggesting no affinity or, at least, much lower specificity for the channels responsible for the potassium currents in the granular cells studied.

Interactions between bovine endothelial cells and Pasteurella multocida: association and invasion.

We investigated the association and the invasion of a bovine aortic endothelial cell (BAEC) line by Pasteurella multocida to study the potential role of internalized bacteria and possible intracellular survival during Pasteurella infections. Our data indicate that P. multocida is able to adhere to and to invade BAECs. The density of the bacterial population plays a defined role for an optimal mechanism of interaction between bacteria and cells, as does the incubation period of association and invasion. The optimal bacteria/cells ratio was found to be 100/1, while the optimal infection time was approximately 4 h of incubation. Bacterial internalization was dependent on microfilament and microtubule stability. The invasion ability of P. multocida in the presence of cytochalasin D was reduced by 60%; in the presence of colchicine it was reduced by 97% and in the presence of nocodazole it was reduced by 95%. Our data show that internalized P. multocida did not induce mortality of invaded endothelial cells. Some Pasteurella cells were able to survive and undergo exocytosis.

Fast K(+) currents from cerebellum granular cells are completely blocked by a peptide purified from Androctonus australis Garzoni scorpion venom.

A novel peptide was purified from the venom of the scorpion Androctonus australis Garzoni (abbreviated Aa1, corresponding to the systematic number alpha KTX4.4). It contains 37 amino acid residues, has a molecular mass of 3850 Da, is closely packed by three disulfide bridges and a blocked N-terminal amino acid. This peptide selectively affects the K(+) currents recorded from cerebellum granular cells. Only the fast activating and inactivating current, with a kinetics similar to I(A)-type current, is completely blocked by the addition of low micromolar concentrations (K(i) value of 150 nM) of peptide Aa1 to the external side of the cell preparation. The blockade is partially reversible in our experimental conditions. Aa1 blocks the channels in both the open and the closed states. The blockage is test potential independent and is not affected by changes in the holding potential. The kinetics of the current are not affected by the addition of Aa1 to the preparation; it means that the block is a simple 'plugging mechanism', in which a single toxin molecule finds a specific receptor site in the external vestibule of the K(+) channel and thereby occludes the outer entry to the K(+) conducting pore.

Chloride channels activated by hypotonicity in N2A neuroblastoma cell line.

By using the patch-clamp technique we have shown that, in hypotonic extracellular solutions, the mouse neuroblastoma cells Neuro2A (N2A) develop ionic currents mediated by a chloride-selective channel which is also permeable to other anions in accordance with the permeability sequence: I->Br->Cl->gluconate->glutamate-. The currents persist for several hours when Mg-ATP is present in the recording pipette but occur only transiently in the absence of Mg-ATP. Typical blockers of anions channels such as La3+ and Zn2+ do not affect the hypotonicity-activated channel; conversely, the stilbene sulfonate-derivatives, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibit the channel in a voltage-dependent manner. Also intact cells exposed to hyposmotic solutions activate volume-regulation mechanisms which decrease the transient volume increase that develops immediately after the application of the hyposmotic challenge. Since N2A neurons have been used as an expression system of exogenous channels, the presence of osmolarity-regulated channels in these cells is an important aspect that deserves the attention of researchers who may wish to express and study the properties of transport proteins in this cell line.

Interaction between dihydropyridines and phospholipid bilayers: a molecular dynamics simulation.

Interaction of the calcium-channel antagonist dihydropyridines (DHPs), lacidipine and nifedipine, with a phospholipid bilayer was studied using 600 ps molecular dynamic simulations. We have constructed a double layer membrane model composed of 42 dimirystoyl-phosphatidylcholine molecules. The DHP molecules locate at about 7 A from the centre of the membrane, inducing an asymmetry in the bilayer. While lacidipine did not induce significant local perturbations as judged by the gauche-trans isomerisation rate, nifedipine significantly decreased this rate, probably by producing a local rigidity of the membrane in the vicinity of the DHP.

A novel toxin form the scorpion Androctonus australis blocks Shaker K+ channels expressed in Xenopus oocytes.

The Shaker B potassium channel expressed in Xenopus laevis oocytes is blocked, in a total reversible manner from the outside part, by a new toxin (Aa1) composed of 40 amino acid residues, purified from the venom of the North African scorpion Androctonus australis Garzoni. The experiments were performed with patch-clamp technique in the outside-out configuration. The half blocking concentration is approximately 4.5 microM with a 1:1 stoichiometry. The activation and inactivation kinetics of the current are not modified by the blocking mechanism. The binding affinity is not voltage dependent. These results suggest a simple bimolecular mechanism of blockade by which the toxin occludes the external vestibule of the channel and thereby inhibits the K+ ions conduction.

The Androctonus australis garzoni scorpion venom contains toxins that selectively affect voltage-dependent K(+)-channels in cerebellum granular cells.

A purified peptide from Androctonus australis Garzoni venom (AaG) affects selectively a K(+)-current recorded from cerebellum granular cells. This current is characterized by fast activating and inactivating kinetics similar to an IA-type current. Addition of 2 microM peptide Aa1 (from Androctonus australis, toxin 1) to the external side of the channel suppressed completely and in a selective manner the IA-type current, with an IC50 value of 130 nM, whereas in the same conditions, the other potassium current, identified as delayed rectifier (Id), was not affected. Additionally, we show that another partially purified peptide (III-12) from the same venom was able to block reversibly both K(+)-currents.

[Synchronous carcinoma of the colorectum]. / Il carcinoma sincrono del colon-retto.

The incidence of synchronous carcinoma of the large intestine is rising in relation to a greater oncogenic environmental charge and increased average life expectancy. There is also a constant risk of not recognising the disease, especially in the case of small carcinoma and, to a greater extent, in patients operated during the occlusive phase. Having underlined the diagnostic value of a correct preparation of the colon prior to instrumental tests, the authors emphasise the importance of a careful intraoperative exploration of the viscera, its preliminary confinement in occluded subjects and repeated surgery in the event of doubts regarding the monolocation of the tumour. Lastly, they underline the importance of postoperative radiological and endoscopic controls since these tests mark both the successful outcome of treatment and the start of follow-up.