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We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

Multi-photon driven photo-switching between dark and bright (fluorescent) states of a green fluorescent protein (GFP) mutant is demonstrated. A single-protein investigation shows the existence of two distinct bright states that display sharp two-photon cross-section bands peaked at 780 nm and at 870 nm. Fluorescence of these two species can be independently switched on and off. These results highlight a new photoconversion pathway for photochromic GFPs and can have significant applications in multi-photon confocal microscopy and in optical data-storage architectures.

Gate-voltage control of interedge tunneling at a split-gate constriction in the fractional quantum Hall regime is reported. Quantitative agreement with the behavior predicted for out-of-equilibrium quasiparticle transport between chiral Luttinger liquids is shown at low temperatures at specific values of the backscattering strength. When the latter is lowered by changing the gate voltage, the zero-bias peak of the tunneling conductance evolves into a minimum, and a nonlinear quasiholelike characteristic emerges. Our analysis emphasizes the role of the local filling factor in the split-gate constriction region., (Copyright 2004 The American Physical Society)

The regulation of transcription of the human immunodeficiency virus (HIV) is a complex event of significant pathological relevance, which recapitulates general concepts of cellular transcription with some peculiarities. The viral promoter is embedded in a chromatin structure that exerts powerful repression on transcription; activation of gene expression relies on the combined activity of a series of cellular factors that respond to different external stimuli, and on the function of a single viral regulatory protein, the Tat transactivator. Transcriptional activation is consequent to both chromatin remodeling and to the recruitment of elongation-competent RNA polymerase II complexes onto the integrated promoter, two events that require the coordinate, but transient, assembly of different protein complexes. Application of optical imaging techniques now allows us to appreciate the spatial and temporal evolvement of these reactions in vivo. The picture that is emerging is not only descriptive, but also relevant to the understanding of the regulation of the process. In particular, it appears that the confinement of biomolecules within specific subcellular compartments represents a way to control and coordinate the assembly of functional complexes that regulate viral gene expression.

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse cell membranes. This ability has generated great interest due to potential therapeutic applications. However, the relevant cellular pathway and its dynamics have not been elucidated yet. Here we unravel the intracellular fate of exogenously added Tat fused with green fluorescent protein (GFP) in live HeLa and CHO cells, from the early interaction with the plasma membrane up to the long-term accumulation in the perinuclear region. We demonstrate that the internalization process of full-length Tat and of heterologous proteins fused to the transduction domain of Tat exploits a caveolar-mediated pathway and is inhibited at 4 degrees C. Remarkably, a slow linear movement toward the nucleus of individual GFP-tagged Tat-filled caveolae with an average velocity of 3 micro m/h was observed. No fluorescence was observed in the nucleus, possibly suggesting that Tat fusion protein unfolding is required for nuclear translocation. In addition, early sensitivity to cytochalasin-D treatment indicates the essential role of the actin cytoskeleton in the displacement of Tat vesicles toward the nucleus. Our results imply that HIV-1 Tat mediates the internalization of protein cargos in a slow and temperature-dependent manner by exploiting the caveolar pathway.

Inelastic light-scattering measurements of low-lying collective excitations of electron double layers in the quantum Hall state at total filling nu(T)=1 reveal a deep magnetoroton in the dispersion of charge-density excitations across the tunneling gap. The roton softens and sharpens markedly when the phase boundary for transitions to highly correlated compressible states is approached. The findings are interpreted with Hartree-Fock evaluations that link soft magnetorotons to enhanced excitonic Coulomb interactions and to quantum phase transitions in the ferromagnetic bilayers.

Human cyclin T1, the cyclin partner of Cdk9 kinase in the positive transcription elongation factor b (P-TEFb), is an essential cellular cofactor that is recruited by the human immunodeficiency virus type 1 (HIV-1) Tat transactivator to promote transcriptional elongation from the HIV-1 long terminal repeat (LTR). Here we exploit fluorescence resonance energy transfer (FRET) to demonstrate that cyclin T1 physically interacts in vivo with the promyelocytic leukaemia (PML) protein within specific subnuclear compartments that are coincident with PML nuclear bodies. Deletion mutants at the C-terminal region of cyclin T1 are negative for FRET with PML and fail to localize to nuclear bodies. Cyclin T1 and PML are also found associated outside of nuclear bodies, and both proteins are present at the chromatinized HIV-1 LTR promoter upon Tat transactivation. Taken together these results suggest that PML proteins regulate Tat- mediated transcriptional activation by modulating the availability of cyclin T1 and other essential cofactors to the transcription machinery.

Remarkable nonlinearities in the differential tunneling conductance between fractional quantum Hall edge states at a constriction are observed in the weak-backscattering regime. In the nu=1/3 state a peak develops as temperature is increased and its width is determined by the fractional charge. In the range 2/3

Background: Notwithstanding the presence of numerous examinations for screening coeliac disease, it may still escape timely diagnosis. For this reason we carried out an investigation to see whether simple haematochemical anomalies (as revealed in what are now routine examinations carried out during hospitalisation) might make diagnosis quicker or at least trigger the suspicion of coeliac disease., Methods: Retrospectively, of 21 adult patients admitted to our hospital for the first time and who were diagnosed with coeliac disease, we considered haemoglobin, iron, calcium, potassium, albumin and RDW (part of the normal blood count)., Results: We found that elevated RDW was the most frequent anomaly (67% of patients) of the six haematochemical parameters observed. In addition, it became normal in most patients after a gluten-free diet., Conclusions: Elevated RDW was more frequent than sideropenic anaemia in patients with coeliac disease. In addition, RDW indicates a response to diet therapy because it became normal after a gluten-free diet.

This work presents a computational model for bone remodelling around cementless stems. The problem is formulated as a material optimisation problem considering the bone and stem surfaces to be in contact. To emphasise the behaviour of the bone/stem interface, the computer model detects the existence of bone ingrowth during the remodelling; consequently, the contact conditions are changed for a better interface simulation. The trabecular bone is modelled as a strictly orthotropic material with equivalent properties computed by homogenisation. The distribution of bone relative density is obtained by the minimisation of a function that considers both the bone structural stiffness and the biological cost associated with metabolic maintenance of bone tissue. The situation of multiple load conditions is considered. The remodelling law, obtained from the necessary conditions for an optimum, is derived analytically from the optimisation problem and solved numerically using a suitable finite element mesh. The formulation is applied to an implanted femur. Results of bone density and ingrowth distribution are obtained for different coating conditions. Bone ingrowth does not occur over the entire coated surfaces. Indeed, we observed regions where separation or high relative displacement occurs that preclude bone ingrowth attachment. This prediction of the model is consistent with clinical observations of bone ingrowth. Thus, this model, which detect bone ingrowth and allow modification of the interface conditions, are useful for analysis of existing stems as well as design optimisation of coating extent and location on such stems.

In recent years, radioimmunotherapy (RIT) with beta(-) particle emitting radionuclides targeting the CD20 antigen on B cells in the treatment of non-Hodgkin's lymphoma has provided the most compelling human clinical data for the success of RIT. CD19, like CD20, is an antigen expressed on the surface of cells of the B lineage, and CD19 may provide an alternative target for radioimmunotherapy of B cell neoplasms. CD19 has been largely overlooked as a target for conventional 131I RIT, because the antigen rapidly internalizes upon binding of antibody, resulting in catabolism and significant release of 131I. Such modulation may be an advantage to RIT with radiometals such as 90Y, 177Lu, 213Bi and 225Ac. Herein, we have compared beta(-) particle RIT with antibodies targeting either CD19 or CD20. The anti-CD19 and anti-CD20 antibodies, B4 or C2B8, respectively, were appended with the SCN-CHX-A''-DTPA bifunctional chelating agent and labeled with 90Y. In the tumor model used, there were three times as many CD20 target sites on lymphoma cells as compared to CD19 sites (62000 vs 20000 binding sites, respectively). We compared the efficacy of the 90Y-labeled antibodies to reduce lymphoma in a nude mouse xenograft solid tumor model, after measurable lymphoma appeared. Reduction in tumor size began at day 3 in all three 90Y-treated groups, but tumor began to recur in many animals 9 days after the treatments. There was one cure in each specific treatment group. In contrast, the tumor in the two control groups showed no regression. There was a significant prolongation of median survival time from xenograft (P < 0.0001) in all the 90Y-labeled antibody construct-treated groups (32 days for 0.15 mCi 90Y-B4; 26 days for 0.20 mCi 90Y-C2B8, and 23 days for 0.15 mCi 90Y-C2B8) in comparison to the two control groups (11 days for 0.02 mg of C2B8 and 9 days for untreated growth controls). Specificity of the radioimmunotherapy was also shown. In conclusion, 90Y-labeled anti-CD19 antibody has efficacy comparable to 90Y-labeled anti-CD20 antibody in the treatment of mice bearing human lymphoma xenografts. These data suggest that CD19-targeted RIT merits further study.

A single, high linear energy transfer alpha particle can kill a target cell. We have developed methods to target molecular-sized generators of alpha-emitting isotope cascades to the inside of cancer cells using actinium-225 coupled to internalizing monoclonal antibodies. In vitro, these constructs specifically killed leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at becquerel (picocurie) levels. Injection of single doses of the constructs at kilobecquerel (nanocurie) levels into mice bearing solid prostate carcinoma or disseminated human lymphoma induced tumor regression and prolonged survival, without toxicity, in a substantial fraction of animals. Nanogenerators targeting a wide variety of cancers may be possible.

Human cyclin T1, a component of the P-TEFb kinase complex, was originally identified through its biochemical interaction with the Tat transactivator protein of human immunodeficiency virus type 1 (HIV-1). Current understanding suggests that binding of Tat to P-TEFb is required to promote efficient transcriptional elongation of viral RNAs. However, the dynamics and the subnuclear localization of this process are still largely unexplored in vivo. Here we exploit high resolution fluorescence resonance energy transfer (FRET) to visualize and quantitatively analyze the direct interaction between Tat and cyclin T1 inside the cells. We observed that cyclin T1 resides in specific subnuclear foci which are in close contact with nuclear speckles and that Tat determines its redistribution outside of these compartments. Consistent with this observation, strong FRET was observed between the two proteins both in the cytoplasm and in regions of the nucleus outside of cyclin T1 foci and overlapping with Tat localization. These results are consistent with a model by which Tat recruits cyclin T1 outside of the nuclear compartments where the protein resides to promote transcriptional activation.

The coherent dynamics of vibronic wave packets in the green fluorescent protein is reported. At room temperature the nonstationary dynamics following impulsive photoexcitation displays an oscillating optical transmissivity pattern with components at 67 fs (497 cm(-1)) and 59 fs (593 cm(-1)). Our results are complemented by ab initio calculations of the vibrational spectrum of the chromophore. This analysis shows the interplay between the dynamics of the aminoacidic structure and the electronic excitation in the primary optical events of green fluorescent proteins.

A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.

Inelastic light scattering by low-energy spin-excitations reveals three distinct configurations of spin of electron double layers in gallium arsenide quantum wells at even-integer quantum Hall states. The transformations among these spin states appear as quantum phase transitions driven by the interplay between Coulomb interactions and Zeeman splittings. One of the transformations correlates with the emergence of a spin-flip intersubband excitation at vanishingly low energy and provides direct evidence of a link between quantum phase transitions and soft collective excitations in a two-dimensional electron system.

Background: Cryptogenic fibrosing alveolitis (CFA) is an uncommon disorder of unknown aetiology characterised by interstitial fibrosis which typically shows a restrictive pattern on pulmonary function testing. Some patients with CFA and relative preservation of lung volumes have been described and it has been suggested that their volume preservation may be due to concomitant emphysema. In a retrospective study the relative frequency of preserved lung volumes in CFA, its relationship to emphysema determined by CT scanning, its clinical features, and its subsequent natural history were investigated., Methods: Using predefined characteristics 48 patients with CFA were identified from pulmonary function records over a three year period. Volume preservation was defined as a forced vital capacity (FVC) of > 80% predicted at presentation. Patients with relative volume preservation were compared with those with more typical pulmonary restriction and clinical data at presentation, and details of their subsequent prognosis, treatment and loss of lung function with time were obtained. Where available, computed tomographic (CT) scans for the two groups were compared in a blinded fashion to score the extent of fibrosis and the presence of concomitant emphysema., Results: Twenty one (44%) of the patients with CFA had a FVC of > 80% predicted. They were more likely to be male (76% versus 48%) and to be current smokers (57% versus 22%) with a heavier life time cigarette consumption than the restricted patients (mean (SE) 38 (4.6) versus 25 (4.5) pack years). There were no significant differences in prognosis and subsequent treatment between the groups. Comparable HRCT scans were available in 23 subjects (seven preserved, 16 restricted). They showed no difference in extent of the pulmonary fibrosis but patients with volume preservation were more likely to show concomitant emphysema (86% versus 19%). Patients with emphysema on HRCT scans were heavier smokers (41(10) versus 21(17) pack years) than those without emphysema but there was no difference in the extent of CFA score between the two groups., Conclusions: In this area of high smoking prevalence a significant number of patients with CFA presented with relative preservation of lung volumes and FEV1/FVC ratio. In many of these subjects this appears to reflect coincidental emphysema. This may make interpretation of gas transfer factor used to monitor progression in CFA difficult. However, there was no evidence that lung volumes at presentation were of prognostic significance.

An unusual case of multiple myeloma is reported in a 61-year-old man presenting with hepatomegaly and multiple hypoechoic lesions in the liver seen on ultrasound. Following cytotoxic chemotherapy the size and number of the lesions within the liver had significantly reduced.

Chlamydia pneumoniae is the third strain of Chlamydia and transmission is presumed to be by droplet spread from symptomatic patients. The authors show a literature scientific review on subject-matter emphasizing several sporadic and epidemic case of illness in every age, first months excepted. The described cases are few and therefore clinical characteristics are shadowy. Certain clinical features are suggesting of severe pneumonia with fever and pharyngitis without exudate. In the childhood and in the adolescence the clinical evolution is favourable. Tetracyclines or erythromycin is recommended for therapy.

The authors show a scientific literary review on Ch. trachomatis pneumonia. A distinctive syndrome pneumonia has been reported in infants infected by Chlamydia (at 2-3 months of age) in genital-urinary infected mothers. The infection may be preceded by conjunctivitis, in apyrexia followed by attacks of coughing pertussis-like. Blood eosinophilia is present. Although favorable prognosis in infancy, erythromycin is the drug of choice shortening the clinical course erythromycin is also recommended in woman infected prevention.

The authors show a literary review on Mycoplasma pneumonia which represents a fifth of human pneumonia. M. pneumoniae is the major cause of illness in school-age children (5-9 years) and young adults and the peak incidence of illness occurs in this age. The infection appears mainly as an interstitial pneumonitis and the onset of illness is gradual, influence-like with few clinical symptoms and typical roentgenographic and bronchoscopic findings. Cold hemagglutinins appear in approximately 50% of patients. A severe prognosis can be associated with extrarespiratory symptoms; Erythromycin (as others macrolides) is the drug of choice.

Pneumonia due to L. pneumophila occurs frequently in children even though "legionnaires' disease" in adults in usual. The authors show a literary scientific review of almost one hundred cases, sporadic generally, in childhood. In children legionnaires' disease remembers the adults infection, beginning with bronchiolitis. The extrarespiratory symptoms appears more frequently in adults. Chest roentgenograms obtained early reveal patchy infiltrates that become nodular areas of consolidation bilateral also, with pleural effusion. In children prognosis is poor (exitus in 15 about 97 described cases). The infection responds to erythromycin; it is administered intravenously, but oral treatment may also be used.

The authors report a case of hemiballismus in an old patient with diabetes and hypertension, developed after a non ketotic hyperosmolar coma episode. In agreement with the recent literature, the syndrome is not always caused by a single Luys body lesion, but also by complex scattered lesions demonstrable only by RMN. A possible pathogenetic role of the recent non ketotic hyperosmolar diabetic coma and the good prognosis indicate that the vascular aetiology, even if prevalent, could not be the only one. The role of institutionalization of the patient during the development of the syndrome has also been discussed.

A hepatitis A vaccine was prepared by formaldehyde inactivation of purified hepatitis A virus (HAV) LSH/S strain grown on human diploid MRC-5 cells. The vaccine was devoid of residual infectivity in vitro and failed to induce in marmoset monkeys any pathological features or variations of haematological and clinical chemistry values. Infectious HAV particles were not detected in faeces and sera of the vaccinated primates by ELISA or after passages in MRC-5 cells. The immunogenicity of the vaccine was evaluated by injecting guinea-pigs with 0.8, 0.2 or 0.05 micrograms of HAV antigen adsorbed onto 0.5 and 1 mg of Al (OH)3 or 0.3 mg of AlPO4. The antibody response, measured by a competitive radioimmunoassay, was dose- and adjuvant-dependent. One injection of 0.2 micrograms of AlPO4-adsorbed HAV antigen induced seroconversion in 100% of animals and high levels of specific and neutralizing serum antibodies. A further increase of antibody titres was observed after the second and third inoculations. These results show that this vaccine formulation is safe and immunogenic in animal models, and suggest that it should be evaluated further by human clinical studies.

Assuring the complete inactivation of hepatitis A virus (HAV) vaccine commonly requires prolonged tissue culture amplification, followed by detection of virus antigen in cell lysates. A reliable, but faster, alternative procedure is highly desirable since it will permit the prescreening of experimental batches of killed HAV, prior to tissue-culture amplification. We established experimental conditions for simultaneous, polymerase chain reaction (PCR)-based amplification of viral and cellular mRNA sequences from infected cell RNA (compound PCR). Under these conditions, the presence of virus-specific amplified sequences, as detected by Southern blot, allows the identification of incompletely inactivated vaccine batches with a threshold practically identical to that of the more time-consuming subculture and ELISA. Compound PCR is, by its nature flexible enough for adaption to different requirements and it should prove useful for rapid prescreening of vaccine batches and pilot studies for improvement of inactivation protocols.

The authors describe, in a patient suffering from IDDM, a case of suspected EHS the beginning of which was concealed by serious ketoacidosis. The clinical suspicion, strengthened by the tests TC and RMN, was not confirmed by CSF analysis. In spite of the severity of the documented lesion and neurological findings at the onset and the outcome has turned out favourably.

A reduction in the number of radiographs for many investigations has been advocated. This study aims to assess the need for control views in sialography and whether the post-contrast lateral oblique radiograph would be satisfactory, at least initially, as the sole standard post-contrast film.

The lumbar spine radiographs of 400 orthopaedic outpatients were reviewed to determine if the absence of the coned lateral lumbosacral junction radiograph would lead to a loss in diagnostic accuracy. In 84 (21%) patients the antero-posterior and lateral lumbar spine radiographs failed to visualize adequately the lumbosacral junction. In these patients the coned lateral lumbosacral junction radiograph resulted in 17 (4.5%) diagnoses which would otherwise have been missed. The diagnoses were spondylosis (11), spondylolisthesis (3), spondylolysis (1), Paget's disease (1) and infection (1). Absence of the coned lateral lumbosacral radiograph will result in inadequate demonstration of the lumbosacral junction and a loss in diagnostic accuracy.

We describe a patient with the typical features of Hajdu-Cheney Syndrome. The previously undescribed cranial computerised tomographic (C.T.) findings in this condition are discussed. In addition we describe a possible association with bowel malrotation.

A novel isolate of hepatitis A virus, obtained from a clinical sample, has been adapted to grow on cultured human diploid cells. Growth and purification parameters have been optimized to obtain conditions suitable for the development of an inactivated vaccine. The entire viral genome was molecularly cloned, and the gene encoding the VP3 capsid protein was expressed in Escherichia coli. The resulting recombinant VP3 was used to obtain rabbit antisera which recognize the denatured protein in purified virion preparations. Nucleotide sequencing data are presented and compared to known sequences of different strains.

Radiation therapy has been shown to prevent heterotopic bone formation in high risk patients undergoing total hip replacement. A number of doses have been used without a randomized trial comparing one dose regimen against another. A prospective randomized trial was undertaken comparing 10 Gy in 5 fractions versus 8 Gy in 1 fraction. Forty-seven patients have been randomized at the time of this evaluation with 37 patients eligible for analysis. The pre-operative, immediate post-operative and 2 month post-operative radiographs were graded. At the time of this analysis, 17 patients were randomized to the 8 Gy arm with 20 patients in the 10 Gy arm. Patients were treated with limited fields so as to only cover the area at risk for development of heterotopic bone to prevent adverse effects on biologic fixation of uncemented implants. When comparing the pre-operative, operative, and 2 month post-operative radiographs, only four patients (1 patient in the 8 Gy arm and 3 patients in the 10 Gy arm), had an increase in the score. No patient had an increase in score to a clinically significant level, usually grade 3 or 4. These preliminary results appear to show that 8 Gy in a single fraction can be as effective as 10 Gy in 5 fractions in preventing heterotopic bone in susceptible individuals. Further follow-up of the remaining patients may confirm this.

The preparation of a hybrid molecule obtained by chemical glycosilation of pertussis toxin (PT) is reported, with the purpose of obtaining a semi-synthetic glycoprotein antigen with bivalent antigenicity. The chemical glycosilation was performed using an oligosaccharide hapten derived from the purified capsular polysaccharide of Neisseria meningitidis group C. The semi-synthetic molecule was investigated by chemico-physical and immunochemical analysis. The oligosaccharide haptens appeared exposed on the surface of the carrier protein PT, since the glycoprotein inhibited the immunoprecipitate between a specific polyclonal antiserum and the native bacterial capsular polysaccharide. By contrast, the main antigenic regions recognized in the native protein PT by specific polyclonal antibodies appeared lost after the coupling procedure involving PT as the carrier protein.

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.

Enzymatically dissociated hamster tracheal epithelial (HTE) cells were cultured on collagen coated Millicell filters. Within 3-5 days after being placed in culture large numbers of ciliated and mucus cells began to appear. By 1 week the HTE cells closely resembled those seen in vivo, i.e. columnar morphology and organelle polarity. After 4 weeks in vitro the HTE cultures were still able to maintain the polarity and overall columnar morphology found in in vivo tissue. There was, in addition, no apparent degradation of the collagen substrate. Auto-radiographic data indicated that there was an initial period of high DNA synthesis during the first 3 days in vitro. This was followed by a second phase in which, by day 6, the amount of DNA synthesis was greatly reduced. Analysis of the numbers of ciliated cells relative to non-ciliated cells demonstrated that between days 5 and 8 there was an increase in the percentage of ciliated cells, suggesting that cellular differentiation (i.e. ciliogenesis) follows cellular proliferation. The results of this study show that when HTE cells are grown on collagen-coated Millicell filters there is a significant improvement in cell growth and morphology yielding cells that are very similar to those present under in vivo conditions. Moreover, since there is no degradation of the collagen substrate, HTE cultures may be suitable for long-term studies of respiratory tract epithelia.

Errors in the childhood feeding administration are commonly met, particularly during the first and second year of life, according to the poor or vague knowledge among paediatricians. A first proof of this assumption is given when, as a consequence of digestive disturbance, caused by a wrong nourishment, the paediatrician suggests remedies that, substantially, make the same primitive mistakes. A second proof is given when a particular diet, due to the presence of intestinal disturbance and already seen to be inefficacious, is reproposed, even more than once, with industrial aliments which differ from the ones used in the name and the brand but not in the composition. Finally, another proof is given when in the exclusion therapy due to an intolerance towards gluten, towards cow's milk, towards disaccharides, the paediatrician inserts, in the diet, products containing, respectively, the above mentioned components. These and other mistakes are reported in the present casuistry (121 cases), the majority of which (75 cases) is accompanied by specific examples. Between the cases that mostly stand out we find the so called "precocious weaning" as cause of digestive disturbance and, frequently, as initial moment of a chain of errors responsible, in their turn for the protraction and the chronicization of the aforementioned intestinal pathology.

The terms "spondyloarthropathy" and "spondyloarthritis" have been recently introduced to indicate a group of syndromes associated in clinical and genetical peculiarities. The "ankylosing spondylitis with childhood onset", that is usually considered very rare, is, as a matter of fact, a disease difficult to diagnose because of the partial overlap with the "pauciarticular juvenile rheumatoid arthritis type II" for clinical and laboratory data. The Authors, after a recapitulation of the "pauciarticular juvenile rheumatoid arthritis type II" and of the "ankylosing spondylitis with childhood onset" diagnosis definition elements, propose a description of a clinical case that came to their observation. The clinical picture described, even though it presented some clinical peculiarities (particularly a primitive affection of the lumbosacral spine), some radiographic peculiarities (the precocious demineralization of the affected segments) and laboratory characteristics (positive F.A.N. to diffuse pattern) seemed a form of "ankylosing spondylitis with childhood onset", apparently primitive.

An artificial molecule was synthesized by covalently linking the oligosaccharide haptens derived frm Streptococcus pneumoniae type 6A and Neisseria meningitidis group C capsular polysaccharides to the non-toxic mutant protein CRM197, serologically related to diphtheria toxin. Immunochemical analysis using polyclonal and monoclonal antibodies showed in the glycoprotein the presence of specific immunodeterminants of the native polysaccharides and of the carrier protein. The immunological activity of this hybrid molecule tested in two animal models gave evidence for anamnestic induction of serum antibodies specifically directed to the three distinct native molecules. They neutralized the toxicity of diphtheria toxin, recognized the polysaccharide capsule of S. pneumoniae type 6A and 6B (group 6) strain and killed the N. meningitidis group C bacteria by complement-mediated bacterial lysis. These findings support the possibility of using in humans a multivalent antigen with immunogenic activity for several epidemiologically significant Gram-positive and Gram-negative encapsulated bacterial strains.