Validation therapy (VT) in nursing home: a case-control study.

VT is a method for communicating with elderly people with dementia. It has been applied since 2001 at the "Istituto Giovanni XXIII" in Bologna, a public trust, housing over 500 not self-sufficient elderly people. Around 75% of these subjects suffer from cognitive impairment, associated to behavioral and psychological symptoms of dementia (BPSD) in over 35%. To assess the effectiveness of VT, we carried out a study involving 50 subjects divided in two groups, of cases and controls, made up by 27 and 23 patients, respectively. In both groups neuropsychiatric inventory (NPI) and the Bedford Alzheimer nursing severity scale (BANSS) were used before the start and after the end of the study; the case group underwent both individual and group VT. The results show a marked decrease of the average NPI symptom score in the case group (from 22.0 to 9.5) vs. a rise in the control group (from 21.7 to 24.1). Agitation, apathy, irritability and nighttime behaviors were the most improved NPI items among the subjects who underwent the VT. In these patients also the NPI distress score turned out reduced, vs. a small increase in the control group. In the case group an improvement occurred with BANSS too, even if much slighter changes were registered vs. the control group. Although the small number of subjects enlisted does not allow to draw firm inferences, the study suggests that VT is able to reduce the severity and frequency of BPSD, thus improving the relationship with and the management of patients having diagnosis of dementia without any side effects.

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients.

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.

Modifications in the metabolic pathways of benzene in streptozotocin-induced diabetic rat.

Benzene is a ubiquitous environmental pollutant primarily metabolized by a cytochrome P-450 (CYP-450) isoenzyme, CYP-450 IIE1. A consistent induction of CYP450 IIE1 has been observed in both rat and human affected by diabetes mellitus. The aim of this study was to evaluate whether streptozotocin (STZ)-induced diabetes determines modifications in the metabolic pathways of benzene in rat. Benzene (100 mg/kg per day, dissolved in corn oil) was administered i.p. once a day for 5 days. Urine samples were collected every day in STZ-treated and normoglycaemic animals, treated and untreated with benzene (n = 10). Urinary levels of trans,trans-muconic acid and of phenol, catechol and hydroquinone (free and conjugated with sulphuryl and glucuronic group) were measured by high-performance liquid chromatography (HPLC). In normoglycaemic rats during the 5 days of treatment with benzene we observed a progressive and significant decrement in the urinary excretion of phenol, phenyl sulphate and glucuronide, catechol, catechol glucuronide, hydroquinone, hydroquinone glucuronide and t,t-muconic acid (P < 0. 05). In the diabetic animals, conversely, the same metabolites showed progressively increasing urinary levels (P < 0.05). Catechol sulphate and hydroquinone sulphate levels were below the instrument's detection limit. In the comparison between diabetic and normoglycaemic benzene treated rats, the inter-group difference was significant (P < 0.05) from day 3 of treatment for t,t-muconic acid, and from day 1 for free and conjugated phenol, free and glucuronide catechol and free hydroquinone. In the normoglycaemic rat exposed to benzene the decreasing trend observed in urinary excretion of free and conjugated metabolites may be due to their capability to reduce cytochromial activity. Conversely, in the diabetic rat, urinary levels of benzene metabolites tended to increase progressively, probably due to the consistent induction of CYP-450 IIE1 observed in diabetes, which would overwhelm the inhibition of this isoenzyme caused by phenolic metabolites. Furthermore, the metabolic switch towards detoxification metabolites observed after administration of high doses of benzene is not allowed in the diabetic because of reduced glutathione-S-transferase activity. As a consequence, higher levels of hydroquinone, phenol and catechol, considered the actual metabolites responsible for benzene toxicity, will accumulate in the diabetic rat. Extrapolating these data to human, we may thus suggest that occupational exposure to benzene of a diabetic subject poses a higher risk level, as his metabolism tends to produce and accumulate higher levels of reactive benzene catabolites.

Antigenic characterization of recombinant, lymphoblastoid, and leukocyte IFN-alpha by monoclonal antibodies.

To gain more insight into similarities of different interferon-alpha (IFN-alpha) species, we evaluated neutralization and immunoactivity of a variety of IFN preparations with various monoclonal antibodies (IFN-alpha mAb). Nine IFN-alpha mAb obtained through immunization with recombinant IFN-alpha (rmAb), lymphoblastoid IFN-alpha (LY mAb), and leukocyte IFN-alpha (LE mAb) were tested. The IFN-alpha mAb were evaluated for their ability to neutralize the antiviral activity of 11 recombinant IFN-alpha subtypes, two recombinant IFN-alpha hybrids, and lymphoblastoid and leukocyte IFN-alpha preparations. The same IFN-alpha mAb were also used in immunoblotting, and some of them were used in immunoaffinity chromatography. The results of the neutralization assay reveal that the IFN-alpha mAb significantly differ in their ability to neutralize the individual IFN-alpha species. Interestingly, none of the IFN-alpha mAb was able to neutralize all the IFN-alpha species. In particular, rmAb were unable to neutralize LE-IFN-alpha or LY-IFN-alpha, whereas LE mAb and LY mAb efficiently neutralized rIFN-alpha2. In some cases, the epitopes to which IFN-alpha mAb are directed were identified through the use of synthetic fragments of IFN-alpha2 or by evaluating the selectivity in binding to IFN-alpha subtypes.

The IFN-alpha-inducing capacity of Sendai Virus in human leukocytes is independent from the hemagglutinating and hemolytic activities and from the viral proteins of the Sendai Virus preparations.

About 30 Sendai Virus (SV) preparations, examined for their capacity to induce natural human interferon alpha from fresh human leukocytes (Le-IFN-alpha) of healthy donors, were characterized for hemagglutinating (HA) and hemolytic (HemA) activities and for SDS-PAGE proteic pattern. The SV preparations were produced by a single passage or by serial propagations through eggs in different conditions of multiplicity of infection (m.o.i.). The produced SV subpopulations showed variable IFN-inducing capacity, the values of which are distributed over a 6-fold range resembling a Gaussian distribution. No detectable difference was observed comparing the SV preparation obtained by serial propagations with those obtained by a single passage. The variability of the measured HA activity and HemA activity and as well as the SDS-PAGE proteic pattern of the SV preparations did not correlate with the induced amount of IFN per cell. In the same experimental conditions to produce Le-IFN-alpha, u.v.-treated SV preparations were unable to induce interferon depending on the u.v.-treatment. So it can be concluded that the distinct nHu-IFN-alpha-inducing capacity of SV subpopulation could be mainly associated with divergent compositions of the viral RNAs rather than with a different contents of viral proteins, among those detectable in SDS-PAGE and those responsible for HA activity and for HemA activity.

Variations in the interferon-inducing capacity of Sendai virus subpopulations.

Several Sendai virus (SV) preparations, propagated through eggs from the same viral seed, exhibited significantly different capacities to induce interferon (IFN) in human leukocytes (nHu-IFN-alpha). The amount of induced IFN and the numbers of SV IFN-inducing particles (IFP) per cell were determined in dose (SV concentration)-response (IFN yield) curves, kinetics of IFN production, and coinfection experiments with SV preparations that differed in IFN-inducing capacities. The possible role of leukocyte sources and the quality of the SV preparations and of allantoic fluids in affecting the IFN-inducing capacity of SV populations also were tested. The data indicate that different SV preparations induced different amounts of IFN per leukocyte and contained approximately the same concentrations of IFN-inducing particles. There was no apparent correlation between the IFN induced and the apparent quality of the SV preparations examined (EID50, HAU, and EID50/HAU). The leukocyte source and the allantoic impurities of SV preparations did not have any influence on the magnitude of the IFN yield. Similar shapes of the dose-response curves, the absence of any lag in the kinetics of IFN production, and the ability of a viral preparation that induced low yields of IFN to suppress partially a high-yielding inducer suggest that a common mechanism of induction is always present. Hence, propagation of SV in eggs from low multiplicity produced virus stocks that differed significantly in their inducing capacity, suggesting that genetic bottlenecks may be operative.

Structure-activity of type I interferons.

Type I IFNs constitute a family of proteins exhibiting high homology in primary, secondary, and tertiary structures. They interact with the same receptor and transmit signals to cellular nucleus through a similar mechanism, eliciting roughly homogeneous biological activity. Nevertheless, the members of that family, IFN alpha species, IFN beta and IFN omega, due to local differences in the structure sometime show distinct properties. From the reported data it results that even minute changes or differences in the primary sequences could be responsible for a significant variety of biological actions, thus inducing to the hypothesis that Type I IFNs, rather than to be the result of a redundant replication during the evolution play definite roles in the defense of living organisms to foreign agents.

Capillary electrophoresis of heparin and dermatan sulfate unsaturated disaccharides with triethylamine and acetonitrile as electrolyte additives.

Capillary electrophoresis at constant voltage with the addition of triethylamine as electrolyte to a running buffer containing borate using fused-silica capillaries permits the complete resolution in less than 30 min of 11 standard heparin and 8 standard dermatan sulfate disaccharides, which represent degradation products of heparin and dermatan sulfate by specific lyases. Triethylamine influences the migration time of disaccharides by reducing both their electrophoretic mobility towards the anode and the electroosmotic flow towards the cathode. A modulated combination of these effects together with borate-disaccharide complex formation is responsible for separation, especially in the case of isomers which differ in the position of the sulfate groups. The addition of acetonitrile did not introduce any favourable effect in the separation of disaccharide mixtures. Under these conditions different dermatan sulfates were analysed to assess the source of the preparations.

Sendai virus and herpes virus type 1 induce apoptosis in human peripheral blood mononuclear cells.

Recent reports suggest that several viruses, besides human immunodeficiency virus, induce apoptosis in infected cells. We report here that Sendai virus or Herpes simplex virus type 1 (HSV-1), two potent inducers of interferon-alpha, caused cell death in a consistent number of human peripheral blood mononuclear cells. A careful analysis of infected cells by different techniques, such as optical and electron microscopy, DNA agarose gel electrophoresis, and cytofluorimetric analysis of DNA content, showed that cell death was of apoptotic type. Sendai virus was more apoptogenic than HSV-1, and it was further studied to understand the mechanism(s) by which it induced apoptosis. Physical (uv and heat) and chemical (beta-propiolactone) inactivation reduced or abolished the apoptogenic power of Sendai virus. The use of a novel technique, which allows the study of mitochondrial membrane potential (MMP) in intact cells by flow cytometry, showed that a decrease of MMP is concomitant with the appearance of the hypodiploid peak. These results suggest that Sendai virus and HSV-1 can be added to the list of viruses causing apoptosis, which appears to be a general mechanism occurring during viral infection.

Use of electrocochleography to monitor antigenic challenge in Menière's disease.

Allergy has been reported as a cause of Menière's disease. King et al. have established the validity of the provocative food test (PFT) for the diagnosis of food allergy. When the PFT is used to test patients with Menière's disease, the test is considered positive if the patient develops aural fullness, hearing loss, increased tinnitus, or dizziness during challenge with the offending food and relief of these symptoms during neutralization. Ferraro et al. have shown that electrocochleography (ECoG) provides an objective indication of subjective symptoms in Menière's disease by demonstrating an increased SP/AP amplitude ratio when the symptoms of aural fullness and hearing loss are present. We present several patients with Menière's disease in whom measurement of the SP/AP amplitude ratio was compared with symptom production during antigenic challenge and neutralization.

Purification of recombinant human interferon-beta by immobilized antisense peptides.

Synthetic antisense peptides encoded in the antisense strands of DNA corresponding to the 1-14, 42-54 and 103-115 fragments of the human interferon-beta sequence were applied in the purification of recombinant human interferon-beta from a mammalian cell culture. The protein fragments were selected on the basis of their computer-predicted exposure on the surface of the protein. The antisense peptides were synthetized by the solid-phase method directly on the resin used as the stationary phase in affinity chromatography. All the tested antisense peptides showed a selective affinity for human interferon-beta, permitting a ten-fold purification of the protein.

Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification.

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

Large-scale purification of the synthetic peptide fragment 163-171 of human interleukin-beta by multi-dimensional displacement chromatography.

Multi-dimensional chromatography has been used successfully in the displacement mode for the purification of the synthetic peptide H-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the fragment 163-171 of human interleukin-beta. This peptide can mimic several of the in vivo and in vitro immunostimulatory activities of the entire protein, except for the inflammatory effect. A large-scale procedure has been developed to purify the synthetic peptide by reversed-phase (RP) and ion-exchange (IE) displacement chromatography (DC) in a single run without any pretreatment. Masses from 100 mg to about 35 g of the unpurified compounds synthesized by a solid-phase technique on a Merrifield-type resin and obtained by acidolytic cleavage from the solid support, can be purified in this way. In the RP-DC mode the carrier and the displacer were aqueous solutions of 0.1% trifluoroacetic acid and 50 mM benzyltributylammonium chloride, respectively, whereas in the IE-DC mode the carrier was water and the displacer 50 mM ammonium citrate solution. RP-DC and IE-DC were also performed in series by directing the effluent of the RP column onto the IE column. Peptide purities and recoveries greater than 96 and 90%, respectively, were obtained.

Preparative purification of Plasmodium falciparum circumsporozoite protein synthetic polypeptides by displacement chromatography.

Displacement chromatography was used for the preparative purification of a synthetic polypeptide that is a promising malaria vaccine. It was prepared by solid-phase synthesis and contains two important epitopes of circumsporozoite (CS) protein of Plasmodium falciparum sporozoite. With apparatus typically employed in analytical high-performance liquid chromatography (HPLC) and on a 250 x 4.6 mm I.D. reversed-phase column, up to 50 mg of crude polypeptide were purified in a single run and with a yield higher than 95%. The results demonstrate that displacement chromatography is suitable for the isolation of several milligrams of a pure polypeptide from a complex mixture that is difficult to separate even by analytical HPLC. In such a preparative application, displacement appears to be superior to elution chromatography as used traditionally.

Fast atom bombardment mass spectrometry and selective acid hydrolysis for the analysis of partially modified retro-inverso peptide analogues.

Fast atom bombardment (FAB) mass spectrometry has been successfully applied to the analysis of partially modified retro-inverso peptide isomers. The spectra are characterized by abundant protonated molecular ions and also by sequence ions due to fragmentation of the inverted bonds. Unambiguous information, as to the nature and the position in the backbone of the amino acids involved in the partial modification of the structure, are given by using a combination of FAB mass spectrometry and partial, selective acid hydrolysis, without separation of the resulting peptide mixtures.

High-performance displacement chromatography of melanotropins and their derivatives.

Mixtures containing derivatives of the biological active peptide alpha-melanocyte stimulating hormone (alpha-MSH) or beta-melanocyte stimulating hormone (beta-MSH) were purified by using reversed-phase chromatography in the displacement mode. On a 250 x 4.6 mm octadecyl silica column and with instrumentation used in analytical high-performance liquid chromatography about 30 mg of the peptide mixture were separated by using an aqueous solution of benzyldimethyldodecylamonium bromide as the displacer in a single chromatographic run. The results demonstrate the advantages of displacement over elution in preparative chromatography of peptides on the scale of tens of milligrams that is typical for physiological tissue extracts and in solid-phase peptide synthesis.

Fallopian tubal patency assessed by ultrasound following fluid injection. Work in progress.

The ability of ultrasound to detect patency of at least one fallopian tube by demonstrating free fluid in the cul-de-sac was evaluated in 35 infertile women. The results were compared with conventional hysterosalpingograms, which had been obtained simultaneously. Ultrasound demonstrated bilateral occlusion with a sensitivity of 100%, and showed tubal patency with a specificity of 96%. The ability to diagnose tubal occlusion or patency using this ultrasound technique, which the authors have designated "sonosalpingography," eliminates unnecessary exposure of the female pelvis to ionizing radiation and avoids the use of iodinated contrast material.

Strategy for the use of biliary scintigraphy in non-iatrogenic biliary trauma.

Biliary scintigraphy was used to examine 21 patients who had suspected non-iatrogenic biliary trauma. Seven patients (33%) had scintigraphic evidence of biliary leakage. Ultimately, surgical biliary repair was required for only three of these patients. Visualization of the gallbladder did not occur in eight trauma patients, but only one patient was shown to have cholecystitis. In this series, 16 patients had Tc-99m sulfur colloid scans that offered no significant advantage over cholescintigraphy in the detection of hepatic parenchymal defects. Biliary scintigraphy provides clinically useful information in cases both of blunt and penetrating trauma.

Scrotal masses with a uniformly hyperechoic pattern.

Eight scrotal masses which appeared homogeneously hyperechoic on ultrasound were studied; 5 were testicular and 3 were extratesticular. Pathologically, there was no evidence of malignancy, and most lesions consisted of scar tissue or fibrosis. Two benign adenomatoid tumors were encountered. In this series, a homogeneously hyperechoic scrotal lesion was benign regardless of its location.