Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Differentiation of the epidermis in turtle: an immunocytochemical, autoradiographic and electrophoretic analysis

Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.     

Modulation of pro- and anti-apoptotic genes in lymphocytes exposed to bone cements

The ability of bone cements to modify the apoptotic program in activated immune cells and the mechanisms by which they act were evaluated. Mononuclear cells were collected from healthy individuals, cultured for 4 and 24 h with phytohemoagglutinina-P and cement extracts and then tested to assess: (a) cell viability; (b) early apoptotic events, by Annexin V/propidium iodide staining; and (c) the expression of pro- (p53, c-myc, ICE) and anti-apoptotic (bcl-2) genes. After 4 h three cements were able to increase significantly the percentage of apoptotic cells, while after 24 h no differences were found. The proportion of dead cells was not significantly changed at either culture time. The simultaneous expression of both pro-apoptotic (ICE, c-myc, p53) and antiapoptotic genes (bcl-2) was investigated only with regard to the materials which induced significant changes in apoptosis: two cements induced the p53 expression, while the third down-regulated bcl-2. As apoptosis regulates the balance of immune response, the authors recommend that the interaction between materials and immune cells should be assessed, so that the use of pro-apoptotic materials may be avoided in patients with immune defects.     

A standard dosimetry procedure for 192Ir sources used for endovascular brachytherapy

The experimental dosimetry of a high dose rate (HDR) 192Ir source used for the brachytherapy of peripheral vessels is reported. The direct determination of the reference air kerma rate Kr agrees, within the experimental uncertainty, with the results obtained by a well ionization chamber calibrated at the NIST and the manufacturer's certification. A highly sensitive (HS) radiochromic film (RCF), that presents only one active layer, was used for the source dosimetry in a water phantom. The adopted experimental set-up, with the source in its catheter positioned on the RCF plane, seems to have given better accuracy of the RCF optical density measurements. The agreement between the measurement of the dose rate constant DKr (10 mm, pi/2) and the literature data confirmed the coherence of the HS RCF calibration obtained by the kerma in air measurements. The RCF measurements supplied dosimetric information about the dose to water per reference air kerma rate D(r, theta)/Kr along the source transverse bisector axis, the radial dose function g(r) and the anisotropy function F(r, theta). The value D(2 mm, pi/2)/Kr = 22.4 +/- 1.2 cGy h(-1)/(microGy h(-1)) is supplied with a dose uncertainty that is essentially due to the indeterminacy of the source position in the catheter. The data of the radial and anisotropy functions have been compared with Monte Carlo determinations reported in the literature.     

Clinical characterization of depressive mixed state in bipolar-I patients: Pisa-San Diego collaboration

BACKGROUND: Although mixed states were classically described as various concomitant admixtures of depression and mania, the official current definitions in both DSM-IV and ICD-10 tend to restrict the concept to manic patients with full syndromal depression. Recent research has actually shown that mania with few depressive symptoms constitutes the most prevalent clinical presentation of mixed or dysphoric mania. Major depressive patients with few concomitant manic symptoms are not officially recognized within the current nosology. In this paper we attempt to delineate the clinical profile of such depressive mixed states in the context of bipolar I disorder. METHODS: In the Pisa day center, we studied 195 bipolar I patients who either met Pisa criteria for bipolar mixed state (n=159) or DSM-III-R criteria for major depressive episode (bipolar major depression or B-MD, n=36). Of the 159 patients identified by Pisa criteria as mixed state, 86 also met the criteria of the DSM-III-R for mixed episode (core mixed state or MS group), while 32 met the DSM III-R criteria for major depressive episode (provisionally defined as depressive mixed states, D-MS); the remaining patients (n=41, 25.7%) with predominatly manic picture were not included in the present comparisons. RESULTS: The three groups (B-MD, MS and D-MS) had close similarities in clinical and sociodemographic characteristics such as age, sex distribution, marital status, schooling, residence, age at onset, age of first treatment, age of first hospitalization, degree of chronicity of the index episode, stressor within the 6 months before the index episode, lifetime suicide attempts and premorbid temperament. First degree family history for bipolar illness and that for other mental disorders was also similar, except for major depression that was more common among the relatives of D-MS. MS and D-MS were further distinguished from B-MD by the fact that the latter followed a more 'cyclic' course with shorter yet greater number of episodes, and which began with a pure depressive episode; by contrast, MS and D-MS had fewer episodes of longer duration, less interepisodic remission, and tended to begin with a mixed episode. Incongruous psychotic features were more common in the two mixed groups compared to B-MD, and the most common features of the D-MS group were agitation, psychotic depression with irritable mood, pressured speech and/or flight of ideas. LIMITATION: It was not feasible to collect information blind to clinical status in patients with severe psychotic mood states. CONCLUSION: These data confirm the existence of psychotic agitated depressive mixed states with flight of ideas, distinct from cyclic retarded pure bipolar depressive states. The recognition of these affective states is clinically important to protect patients from the potentially harmful indiscriminate use of antidepressants and to provide them with the benefits of an anticonvulsant, a short-term neuroleptic, or ECT.     

Initial stability of a new hybrid fixation hip stem: experimental measurement of implant-bone micromotion under torsional load in comparison with cemented and cementless stems

A new hybrid fixation stem, named cemented-locked uncemented (CLU), for total hip arthroplasty was developed to achieve good initial stability. Primary stability is guaranteed by the cement which is injected into two pockets in the lateral area. This leaves a large surface available for long-term biologic fixation (direct bone attachment on implant). This study evaluates in vitro the initial stability of the CLU prototype under torsional load, in comparison with cemented and cementless stems. The results show that the CLU stem is very stable in simulated stair climbing. Its micromotions are comparable to those of a cemented prosthesis, and significantly less (80-90% lower) than those for a cementless stem. These findings confirm the optimal initial stability expected from the CLU prototype. This new design, which employs hybrid fixation, should improve bone formation on the implant and reduce the risk of stem loosening.     

Effects of the sterilisation method on the wear of UHMWPE acetabular cups tested in a hip joint simulator

Ultra-high molecular-weight-polyethylene is the most commonly used bearing material in total joint replacement. Wear of polyethylene is a Serious Clinical problem that limits the longevity of orthopaedic implants. Information on degradative changes in the material properties and on the methods used for the sterilisation of polyethylene may help in the selection process of orthopaedic implants with the best wear resistance. This study was performed to investigate the effects of the sterilisation method (gamma irradiation and ethylene oxide treatment) on the wear and on the changes in physical properties of polyethylene acetabular cups. At this purpose, gamma-sterilised and ethylene oxide (EtO)-sterilised acetabular cups were tested against CoCr femoral heads in a hip joint simulator run for 5 million cycles in bovine calf serum. The crystallinity of the cups was evaluated by micro-Raman spectroscopy as a function of the inner surface position. The partial least square calibration was used to correlate the Raman spectra with the crystallinity of the polymer measured by differential scanning calorimetry. The analysis performed on soak control acetabular cups demonstrated that the gamma-sterilised cups are significantly more crystalline than the EtO-sterilised ones. The mean crystallinity values obtained for the gamma-sterilised and EtO-sterilised soak control cups were 65.0% and 63.4%, respectively. Weight loss measurements revealed that the gamma-sterilised acetabular cups exhibited a lower wear rate than that by EtO-sterilised. Thc Raman results obtained on gamma-sterilised and EtO-sterilised acetabular cups showed that the changes in surface crystallinity were mainly caused by irradiation rather than by the mechanical friction during the in vitro tests.     

Improvements to a method for determining leg length following total hip arthroplasty

Abstract

Discrepancy in leg length does frequently occur as a side effect of total hip arthroplasty and may lead to reduced patient satisfaction as well as injury in the sequalae. It is consequently important to reduce leg length discrepancy where-ever technically possible. This may be achieved by recording precise intraoperative measurements and using different sized implanted components. The aim of the given study was to improve the accuracy of a previously validated optic measurement system (OMS) to reduce leg length discrepancy. This pre-existing OMS was first trialled and based on these preliminary findings developed further. Using this improved system, measurements were taken in models and cadavers. Inter observer reliability of the improved OMS was assessed. The system is introduced in the given technical feasibility study. Its accuracy was greater in the model setup (swivel joint: 772.7?±?1.5?mm; ball joint: 770.0?±?3.7?mm; reference: 772?mm) compared to the trial using cadaveric tissues (588.8?±?5.7?mm; reference: 586?mm). Results of two examiners were similar. The third one measured significantly shorter values (p=.011). The results of the measurements with the OMS indicate that a significant increase in accuracy (p?=?2.076×¹⁰?⁶) has been achieved compared to the previously reported system, however, a further improvement to measurement accuracy is necessary for this to be applied clinically.     

Frequent spontaneous deletions at a shuttle vector locus in transgenic mice

Transgenic mice carrying multiple copies of a recoverable lambdaphage shuttle vector (     

Automated spoken dialogue system for hypertensive patient home management

Recent advances in automatic speech recognition and related technologies allow computers to carry on conversations by telephone. We developed an intelligent dialogue system that interacts with hypertensive patients to collect data about their health status. Patients thus avoid the inconvenience of traveling for frequent face to face visits to monitor the clinical variables they can easily measure at home; the physician is facilitated in acquiring patient information and cardiovascular risk, which is evaluated from the data according to noted guidelines. Controlled trials to assess the clinical efficacy are under way.