Miniaturized fiber-optic ultrasound probes for endoscopic tissue analysis by micro-opto-mechanical technology.

A new Micro-Opto-Mechanical System (MOMS) technology for the fabrication of optoacoustic probes on optical fiber is presented. The technology is based on the thermoelastic emission of ultrasonic waves from patterned carbon films for generation and on extrinsic polymer Fabry-Perot acousto-optical transducers for detection, both fabricated on miniaturized single-crystal silicon frames used to mount the ultrasonic transducers on the tip of an optical fiber. Thanks to the fabrication process adopted, high miniaturization levels are reached in the MOMS devices, demonstrating fiber-optic emitters and detectors with minimum diameter around 350 and 250 µm respectively. A thorough functional testing of the ultrasound emitters mounted on 200 and 600 µm diameter optical fibers is presented, in which the fiber-optic emitter with a diameter of 200 µm shows generated acoustic pressures with peak-to-peak value up to 2.8 MPa with rather flat emission spectra extended beyond 150 MHz. The possibility to use the presented optoacoustic sources in conjunction with the fiber-optic acousto-optical detectors within a minimally invasive probe is also demonstrated by successfully measuring the ultrasonic echo reflected from a rigid surface immersed in water with various concentration of scatterers. The resulting spectra highlight the possibility to discriminate the effects due to frequency selective attenuation in a very wide range of frequencies within a biological medium using the presented fiber-optic probes.

Which clinical factors delay proper treatment in panic disorder? A cross-sectional multicentric study.

AIM: The aim of the present study was to identify clinical and socio-demographic factors associated with duration of untreated illness (DUI) in patients affected by panic disorder (PD). METHODS: Data were collected from patients' medical records (N = 157) of two mental health services respectively located in Milan and in Monza (Italy). Correlation analyses and analysis of variance (ANOVAs) were run to analyse the relation between DUI and quantitative/qualitative variables respectively. Statistically significant variables in uni- variate analyses were then inserted in a linear multivariable regression model (backward procedure). RESULTS: Mean DUI was 27.33 (±50.56) months. Patients with an earlier age at onset (r = -0.270; p < .01), a longer duration of illness (r = 0.483; p < .01) and who received a lifetime psychotherapy (F = 6.86; p = .01) had a longer DUI. The final global model showed that a longer DUI was associated with pre-onset poly-substance misuse (p = .05) and a longer duration of illness (p < .01). CONCLUSION: The results of our study showed that a longer DUI was predicted by clinical factors such as the presence of a pre-onset poly-substance use disorder and that delayed proper treatment can lead to a chronicization of PD, as indicated by a longer duration of illness. Further studies are needed to in-depth investigate the role of DUI in influencing the course and outcome of anxiety disorders, including PD.

Quantitative variations in the vaginal bacterial population associated with asymptomatic infections: a real-time polymerase chain reaction study.

The real-time polymerase chain reaction (PCR) quantification of several vaginal bacterial groups in healthy women and patients developing asymptomatic bacterial vaginosis (BV) and candidiasis (CA) was performed. Statistical analysis revealed that the BV condition is characterised by a great variability among subjects and that it is associated with a significant increase of Prevotella, Atopobium, Veillonella and Gardnerella vaginalis, and a drop in Lactobacillus. On the contrary, the vaginal microflora of healthy women and patients developing CA was found to be homogeneous and stable over time.

A life-threatening paediatric case of acute autoimmune haemolytic anaemia (AIHA) successfully cured by plasma-exchange and combined immunosuppressive treatment.

A 3-year-old boy present with a severe autoimmune haemolytic anaemia, triggered by IgG-class auto-antibodies, with hemoglobin levels decreased to 2, 1 gr/dL. A combined immunosuppressive regimen was begun together with multiple plasma-exchanges and transfusions which sustained the cardio-vascular balance until the specific therapy became effective.

Early colonisation and temporal dynamics of the gut microbial ecosystem in Standardbred foals.

BACKGROUND: Even if horses strictly depend on the gut microbiota for energy homeostasis, only a few molecular studies have focused on its characterisation and none on the perinatal gut microbial colonisation process. OBJECTIVES: To explore the perinatal colonisation process of the foal gut microbial ecosystem and the temporal dynamics of the ecosystem assembly during the first days of life. STUDY DESIGN: Longitudinal study. METHODS: Thirteen Standardbred mare-foal pairs were included in the study. For each pair, at delivery we collected the mare amniotic fluid, faeces and colostrum, and the foal meconium. Milk samples and faeces of both mare and foal were also taken longitudinally, until day 10 post-partum. Samples were analysed by means of next-generation sequencing of the 16S rRNA gene on Illumina MiSeq. RESULTS: Our findings suggest that microbial components derived from the mare symbiont communities establishes in the foal gut since fetal life. After birth, an external transmission route of mare microorganisms takes place. This involves a rapid and dynamic process of assembling the mature foal gut microbiome, in which the founder microbial species are derived from both the milk and the gut microbial ecosystems of the mare. MAIN LIMITATIONS: The inability to discriminate between live and dead cells, the possible presence of contaminating bacteria in low biomass samples (e.g. meconium and amniotic fluid), the limits of the phylogenetic assignment down to species level, and the presence of unassigned operational taxonomic units. CONCLUSIONS: Our data highlight the importance of mare microbiomes as a key factor for the establishment of the gut microbial ecosystem of the foal.

T cells stimulated by CD40L positive leukemic blasts-pulsed dendritic cells meet optimal functional requirements for adoptive T-cell therapy.

Adoptive T-cell immunotherapy may provide complementary therapy for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this study, we have analyzed the functional characteristics of anti-BCP-ALL effector T cells generated by co-culturing T lymphocytes and dendritic cells (DC) from allogeneic human stem cell transplantation (HSCT) donors. After 21-day co-culture with DC pulsed with CD40L+ apoptotic BCP-ALL blasts, T cells presented with both effector and central memory phenotype, and showed high and specific cytotoxic activity against leukemic cells (average lysis = 77%), mostly mediated by CD8+ T cells. Noticeably, growth of CD4 T cells was maintained (45% of total cells), which actively produced Th1 cytokines (IFN-gamma, TNF-alpha, IL-2), but not IL-4, IL-5 and IL-10. Anti-BCP-ALL T cells expressed CD49d and CXCR4 (implicated in the recruitment to bone marrow), and CD62L and CCR7 (involved in the migration to lymphoid organs). In accordance with this profile, T cells significantly migrated in response to the chemokines CXCL12 and CCL19. In conclusion, stimulation of T cells with CD40L+BCP-ALL cells-loaded DC not only elicited the generation of potent and specific anti-leukemic cytotoxic effectors, but also the differentiation of specific and functional Th-1 CD4 lymphocytes. These effectors are fully equipped to reach leukemia-infiltrated tissues and have characteristics to support and orchestrate the anti-tumor immune-response.

Wide bandwidth fiber-optic ultrasound probe in MOMS technology: Preliminary signal processing results.

An ultrasonic probe consisting of two optical fiber-based miniaturized transducers for wideband ultrasound emission and detection is employed for the characterization of in vitro biological tissues. In the probe, ultrasound generation is obtained by thermoelastic emission from patterned carbon films in Micro-Opto-Mechanical-System (MOMS) devices mounted on the tip of an optical fiber, whereas acousto-optical detection is performed in a similar way by a miniaturized polymeric interferometer. The microprobe presents a wide, flat bandwidth that is a very attractive feature for ultrasonic investigation, especially for tissue characterization. Thanks to the very high ultrasonic frequencies obtained, the probe is able to reveal different details of the object under investigation by analyzing the ultrasonic signal within different frequencies ranges, as shown by specific experiments performed on a patterned cornstarch flour sample in vitro. This is confirmed by measurements executed to determine the lateral resolution of the microprobe at different frequencies of about 70µm at 120MHz. Moreover, measurements performed with the wideband probe in pulsed-echo mode on a histological finding of porcine kidney are presented, on which two different spectral signal processing algorithms are applied. After processing, the ultrasonic spectral features show a peculiar spatial distribution on the sample, which is expected to depend on different ultrasonic backscattering properties of the analyzed tissues.

Gut microbiota and hematopoietic stem cell transplantation: where do we stand?

Advances in biological techniques have potentiated great progresses in understanding the interaction between human beings and the ∼10 to 100 trillion microbes living in their gastrointestinal tract: gut microbiota (GM). In this review, we describe recent emerging data on the role of GM in hematopoietic stem cell transplantation, with a focus on immunomodulatory properties in the immune system recovery and the impact in the development of the main complications, as GvHD and infections.

Vaccination to improve the persistence of CD19CAR gene-modified T cells in relapsed pediatric acute lymphoblastic leukemia.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

CD40 ligand-stimulated B cell precursor leukemic cells elicit interferon-gamma production by autologous bone marrow T cells in childhood acute lymphoblastic leukemia.

Childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, collected from bone marrow (BM) at diagnosis, were cultured, after thawing, on allogeneic human bone marrow stroma (HBMS) for 48 h in the presence of a soluble trimeric CD40 ligand (stCD40L) molecule. HBMS maintained leukemic cells viability in all tested cases (mean viability 85%). Under these culture conditions we noticed upregulation or de novo expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) in 22/22, 15/23 and 21/23 cases, respectively. Upregulation, in terms of fluorescence intensity, was also observed in the expression of MHC I, MHC II, CD54 (ICAM 1) and CD58 (LFA 3) molecules. HBMS alone, although to a lesser extent, was able to induce modulation of these molecules, but not CD80, in a similar proportion of cases. Neither stCD40L nor HBMS induced modulation of CD10 and CD34 molecules. Moreover, in 4/4 tested cases, stCD40L-stimulated ALL cells were able to induce allogeneic T cells proliferation. To evaluate whether leukemia-reactive T cells were detectable in the BM of ALL patients at diagnosis, stCD40L-stimulated ALL cells were co-cultured with autologous T cells (ratio 1:1), isolated from BM at diagnosis, for 4 days and a 24 h ELISPOT assay was applied to detect the presence of interferon-gamma (IFN-gamma)-producing cells. In four of seven cases IFN-gamma-producing cells were detected with frequencies of 1/900, 1/1560, 1/2150 and 1/1575 autologous T cells. These data confirm that stCD40L exposure can activate the antigen-presenting cell (APC) capacity of BCP-ALL cells cultured on HBMS and that ELISPOT assay can be used to measure the frequency of leukemia-reactive autologous T cells in the BM of ALL patients even after short-term culture with stCD40L-stimulated ALL cells.

Cell-based strategies to manage leukemia relapse: efficacy and feasibility of immunotherapy approaches.

When treatment fails, the clinical outcome of acute leukemia patients is usually very poor, particularly when failure occurs after transplantation. A second allogeneic stem cell transplant could be envisaged as an effective and feasible salvage option in younger patients having a late relapse and an available donor. Unmanipulated or minimally manipulated donor T cells may also be effective in a minority of patients but the main limit remains the induction of severe graft-versus-host disease. This clinical complication has brought about a huge research effort that led to the development of leukemia-specific T-cell therapy aiming at the direct recognition of leukemia-specific rather than minor histocompatibility antigens. Despite a great scientific interest, the clinical feasibility of such an approach has proven to be quite problematic. To overcome this limitation, more research has moved toward the choice of targeting commonly expressed hematopoietic specific antigens by the genetic modification of unselected T cells. The best example of this is represented by the anti-CD19 chimeric antigen receptor (CD19.CAR) T cells. As a possible alternative to the genetic manipulation of unselected T cells, specific T-cell subpopulations with in vivo favorable homing and long-term survival properties have been genetically modified by CAR molecules. Finally, the use of naturally cytotoxic effector cells such as natural killer and cytokine-induced killer cells has been proposed in several clinical trials. The clinical development of these latter cells could also be further expanded by additional genetic modifications using the CAR technology.

Gut microbiota trajectory in pediatric patients undergoing hematopoietic SCT.

Acute GvHD (aGvHD) is the main complication of hematopoietic SCT (HSCT) during the treatment of hematological disorders. We carried out the first longitudinal study to follow the gut microbiota trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. Gut microbiota trajectories and short-chain fatty acid production profiles were followed starting from before HSCT and through the 3-4 months after transplant in children developing and not developing aGvHD. According to our findings, HSCT procedures temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of aGvHD is associated with specific gut microbiota signatures both along the course of gut microbiota reconstruction immediately after transplant and, most interestingly, prior to HSCT. Indeed, in pre-HSCT samples, non-aGvHD patients showed higher abundances of propionate-producing Bacteroidetes, highly adaptable microbiome mutualists that showed to persist during the HSCT-induced ecosystem disruption. Our data indicate that structure and temporal dynamics of the gut microbial ecosystem can be a relevant factor for the success of HSCT and opens the perspective to the manipulation of the pre-HSCT gut microbiota configuration to favor mutualistic persisters with immunomodulatory properties in the gut.

Long-term effects of bone marrow transplantation on dental status in children with leukaemia.

Minimal data about oral and dental health in long-term survivors after BMT are available. We studied the dental status of 27 children (19 males, eight females) with leukaemia, followed up with a routine oral examination, panoramic tomogram and, when necessary, an endoral radiograph at a median of 2 years (range 1-10) after BMT. Community periodontal index treatment necessity (CPITN), dental caries, missing or filled permanent teeth (DMFT) and dento-facial alterations according to WHO criteria were registered and evaluated. Median age of the patients at BMT was 9 years (range 1.1-17.9). The mean DMFT score ranged from 1.6 to 12.4 according to age at examination and was slightly higher than that which we previously reported in children who received chemotherapy alone. CPITN showed the presence of soft deposits in 77.7%, serious gingivitis in 59.2% and parodontal involvement in 3.7% of cases. Dento-facial abnormalities were found in 55.5% of patients, while 62.9% of the patients had tooth abnormalities or agenesis. Nine out of 27 patients (33%) had root hypoplasia. A negative impact on DMFT index due to multiple post-BMT factors was found. Age is the crucial factor in determining a developmental defect of enamel and root. The follow-up of long-term survivors after BMT should include regular dental examination.

Comments on "Frequency decomposition and computing of ultrasound medical images with wavelet packets".

In this paper, errors and discrepancies in the subject paper [Cincotti et al., (2002)] are highlighted. A comment, concerning the axial resolution associated to the adopted processing procedure is also reported.

Intrathecal chemotherapy with antineoplastic agents in children.

Intrathecal chemotherapy with antineoplastic agents is mainly utilised in children with leukaemia and lymphoma, and in selected brain tumours. In these diseases, intrathecal use is restricted to methotrexate (MTX), cytosine arabinoside (Ara-C) and corticosteroids. A number of other agents are, at the present time, under evaluation. Intrathecal MTX administered sequentially with systemic high dose MTX infusion prolongs therapeutic cerebral spinal fluid (CSF) levels of the drug. Prolonged therapeutic CSF levels can also be achieved by giving repeated small intrathecal doses of MTX over an extended period in selected patients, with an implanted Ommaya reservoir. In the CSF, the metabolic inactivation of Ara-C is significantly lower than in plasma with a CSF clearance similar to the rate of CSF bulk flow. A slow-release formulation of Ara-C may be given intrathecally, resulting in a prolonged cytotoxic concentration in the CSF. CNS relapse and neurotoxicity in patients with acute lymphoblastic leukaemia, especially younger children, may be reduced by using age-related dosing of intrathecal MTX and Ara-C. Hydrocortisone is used in combination with MTX and Ara-C for so-called 'triple intrathecal chemotherapy' in the treatment of meningeal leukaemia. Intrathecal thiotepa does not appear to be advantageous over systemic administration in patients with brain and meningeal leukaemia. Monoclonal antibodies, reactive with tumour-associated antigens, can be used as delivery systems for chemotherapeutic agents and radionuclides. However, the development of this new approach is currently under evaluation in larger clinical studies. Neurological adverse effects may be expected with intrathecal chemotherapy and are increased by high dose systemic therapy, concomitant cranial radiotherapy or meningeal infiltration by neoplastic cells. Inadvertant intrathecal administration of antineoplastic agents that are indicated for systemic administration only, is dangerous and may result in a fatal outcome.

Efficient laser-ultrasound generation by using heavily absorbing films as targets.

An efficient all-fiber optic source is presented; it adopts absorbing films, deposed directly over the fiber tip, as targets. It is demonstrated that the use of absorbing films made of pure graphite, or graphite powder mixed with epoxy resin, has produced a conversion efficiency increase of two orders of magnitude with respect to metallic materials. It is observed that the conversion efficiency increases monotonically as thickness is reduced down to the material optical penetration depth. Moreover, the conversion efficiency rises with the concentration of graphite powder. Principal advantages of this kind of source are the ease of production and miniaturization, the excellent electromagnetic compatibility, wide ultrasonic bandwidth and, consequently, high spatial resolution. The ultrasonic bandwidth can be controlled by varying the laser pulse duration. The possibility of generating ultrasonic signals with high frequency and flat spectral distribution makes the proposed device suitable for biological tissue spectral characterization.

Bone marrow transplantation for childhood hematological disorders: a global pediatric approach in a twelve year single center experience.

One hundred and 43 consecutive pediatric patients (June 1985-December 1996) with at least 18 months of follow-up, were considered: most of the patients (111/143, 77.6%) underwent allogeneic BMT. The median follow-up was 5.7 years. Overall survival and 5 years EFS were 48.6% and 46.9%, respectively. For patients who underwent allogeneic BMT from HLA-identical siblings, the 5 years EFS for ALL was 75% in 1st CR, 60.4% in 2nd CR, 22.3% in > 2nd CR and 86.7% for AML in 1st CR. The EFS for Allo-BMT in "good" and "poor" prognosis patients was 68.6% and 21.8%, respectively (p value = 0.001). Early mortality in Allo-BMT patients was 17.7% between 1985-1990 and 10.3% between 1991-1996. Early treatment-related organ complications occurred mostly in patients who underwent BMT from an unrelated or a mismatched family donor. Late toxicity was evaluated in 57 patients (median follow-up of 82 months): none of the patients complained of significant late cardiac or respiratory dysfunction. With regards to growth, 18/57 patients (31.6%) lost more than two height centile channels. Three cases of thyroid neoplasms were observed. Evaluation of psychosocial functioning, studied in 39 patients who had at least 2 years of follow-up in CR, did not reveal any evident quality of life impairment. The possibility of curing childhood hematological malignancies is based on a global pediatric and multidisciplinary approach. A continuous need to improve results in terms of EFS and quality of life suggests that further multicenter prospective studies should be carried out.

Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo.

As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitor cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.

A probiotics-containing biscuit modulates the intestinal microbiota in the elderly.

OBJECTIVES: Evaluation of the impact of a biscuit containing the probiotics Bifidobacterium longum Bar33 and Lactobacillus helveticus Bar13 on the intestinal microbiota in the elderly. DESIGN: Randomized double-blind placebo-controlled trial. PARTICIPANTS: Thirty-two elderly volunteers living in Italy. The group was composed of 19 women and 13 men aged between 71 and 88 years (mean 76). INTERVENTION: Subjects were randomized in two groups consuming one dose of the probiotics-containing biscuit or placebo once a day for 30 days. MEASUREMENTS: For each subject the intestinal microbiota was characterized using the phylogenetic microarray platform HTF-Microbi.Array before and after intervention. RESULTS: Our data demonstrated that one-month consumption of a probiotics-containing biscuit was effective in redressing some of the age-related dysbioses of the intestinal microbiota. In particular, the probiotic treatment reverted the age-related increase of the opportunistic pathogens Clostridium cluster XI, Clostridium difficile, Clostridium perfringens, Enterococcus faecium and the enteropathogenic genus Campylobacter. CONCLUSION: The present study opens the way to the development of elderly-tailored probiotic-based functional foods to counteract the age-related dysbioses of the intestinal microbiota.