Evaluation of nutritional status in head and neck radio-treated patients affected by oral mucositis: efficacy of class IV laser therapy.

PURPOSE: To retrospectively evaluate the role of class IV laser therapy in the amelioration of nutritional status of patients affected by oral mucositis due to radiotherapy of the head and neck region during oncological treatment. METHODS: Sixty-three oncological patients were included in this study. All patients were affected by tumors in the head and neck region and had developed oral mucositis during radiotherapy. Forty-two patients had been treated by high-power laser therapy whereas 21 patients had been managed with traditional medications. Data collection included weight measurement (kilogram) and body mass index (BMI) calculation (mass (kilogram)/(height) (square meter)) on the first and last day of radiotherapy. In addition, gender, age, pathology, and the kind of oncological treatment have been considered. RESULTS: Laser-treated patients decreased less in BMI during radiotherapy (p=0.000). Patients treated by combined oncological treatments (radiotherapy and/or chemotherapy and/or surgery) had a higher weight loss during radiotherapy (p=0.015). According to a multivariate regression analysis, the only variable which significantly influenced the reduction of BMI was laser treatment (p=0.000). CONCLUSIONS: Laser therapy is actually considered one of the recommended remedies for the healing of oral mucositis due to cancer treatments. Healing of mucositis can deeply influence the feeding capacity of patients, through reduction of pain and improvement of chewing and swallowing capacities. It also allows lowering the costs for hospitalization and supportive care. Laser therapy should become part of nutritional interventions in oncological patients affected by oral mucositis.

Electron tomography of amplified nanogold immunolabelling: Improvement of quality based on alignment of projections with sinograms and use of post-reconstruction deconvolution.

Electron tomography of immunolabelled proteins identified with amplified nanogold particles imaged by Scanning and Transmission Electron Microscopy within thick sections is a powerful method to investigate the three-dimensional organization of complex cellular machineries. In order to increase the overall quality of the reconstructed cube, we have developed two methods that improve the tomographic reconstruction process. We first performed a very precise alignment of the projections before reconstruction with a technique using sinograms. After reconstruction, we propose to compute image restoration by calculating the Point Spread Function of the projection/back-projection system and to use it to deblur the reconstructed cubes. Improvement in the quality of the reconstructed cubes is demonstrated on images of nucleolar proteins tagged with EGFP and immunolabelled with nanogold particles.

Haploidentical bone marrow transplantation in leukemia and genetic diseases.

From 1986 to June 2000, sixty children suffering from acute and chronic leukemia (n = 42, 33 of which in resistant relapse), genetic diseases (n = 11), aplastic anemia (n = 2, one of which with platelet refractoriness and bleeding), myelodysplasia (n = 5) received an haploidentical bone marrow, mismatched for 2-3 HLA loci. The donor's marrow was treated in vitro with vincristine and methylprednisolone to obtain a functional T depletion (MLC and CTL inhibition, functional blockade of Th1 and Th2). The prevalence of infectious complications and GVHD was similar to that recorded in matched unrelated donor (MUD) transplants. In situations of high risk of rejection (chronic leukemia, genetic diseases) we infused immediately one half of the harvest and then frozen aliquots from the second week. Of the 25 ALL and 8 AML in resistant relapse, 3 survived, disease-free at 14, 8 and 1 years respectively. Of the 3 ALL, transplanted during remission, 1 is surviving at 18 months. Of the 6 CML, 1 had fractionated bone marrow and is surviving at 3 years, and 5 had standard single dose infusion and died of progression of their disease after rejection of the graft (4) or blast crisis after complete engraftment (1). The 2 patients with aplastic anemia, those with myelodysplasia, and 6 of the 10 with genetic disorders died of transplant-related complications or disease progression. 4 patients with osteopetrosis (n = 2), MLD (n = 1), Wiskott Aldrich dis. (n = 1) survive at 8, 2, 5 and 1.5 years respectively. In patients transplanted with fractionated marrow GVHD > 2nd grade occurred in 15%. Only one patient rejected the graft. Compared with MUD transplantation, mismatched BMT whenever performed in patients in good conditions provides similar outcome and widens the donor availability.

The three-dimensional study of chromosomes and upstream binding factor-immunolabeled nucleolar organizer regions demonstrates their nonrandom spatial arrangement during mitosis.

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.

Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of toxoplasma gondii.

Ultrastructural localization of a P29 protein of Toxoplasma gondii was examined on thin sections by an immunogold technique using a P29 antigen-specific monoclonal antibody (5-241-178). Immunolocalization of the P29 protein in extracellular tachyzoites demonstrated that this antigen was present in the dense granules. Thus, we have identified this P29 antigen as the seventh protein (GRA7) to be localized to the dense granules of T. gondii. P29 immunolocalization in intracellular tachyzoites demonstrated association of this antigen with the parasite membrane complex, tubular elements of the intravacuolar network, and with the parasitophorous vacuolar membrane. Our immunolabeling data suggest trafficking of the P29 (GRA7) antigen from the dense granule via the intravacuolar network to the parasitophorous vacuolar membrane on invasion of the tachyzoite into the host cell. (J Histochem Cytochem 46:1411-1421, 1998)

Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization.

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60-80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Membrane-type matrix metalloproteinase-1 expression at the site of human placentation.

Human trophoblast implantation is a highly regulated process of invasion that requires action of proteolytic enzymes to degrade extracellular matrix components of the endometrium. Among these enzymes, matrix metalloproteinases (MMPs) seem to be particularly important in this degradative process. We previously showed that gelatinase A is extensively expressed in vivo in the human placenta. A new MMP, MT-MMP-1 (membrane-type matrix metalloproteinase-1), which is thought to activate progelatinase A, has recently been described. In this study, we examined the expression of MT-MMP-1, by immunohistochemistry and in situ hybridization, in human placental bed biopsies taken during the first trimester of gestation. Human first trimester intermediate trophoblasts synthesized MT-MMP-1 mRNAs and the protein. The MT-MMP-1 pattern of distribution in placental beds was similar to that of gelatinase A, suggesting a pivotal role for MT-MMP-1 in placentation, perhaps by activating progelatinase A.

Kinetics study of the localization and quantitation of target antigens of immunoglobulin a antibodies in acquired and congenital toxoplasmosis.

The cellular distribution (localization and quantitation) of the target parasite's antigens in the tachyzoite along the IgA kinetics was determined in the course of acquired toxoplasmosis and congenital toxoplasmosis. In the case of acquired toxoplasmosis, throughout the IgA kinetics a correlation was noted between the membrane and submembrane immunolabeling and the results of the immunocapture and enzyme-linked immunosorbent assay IgA (ELISA-A) tests. The rhoptries' immunolabeling remained higher. The immunolabeling evolution and the results of the immunology tests were not closely related to the treatment (Rovamycin). From the congenital toxoplasmosis cases it was observed that membrane immunolabeling correlated with the results of the serology tests and with the treatment (Fansidar). The rhoptry antigens were recognized throughout the IgA kinetics; even when the serology tests became negative, immunolabeling persisted. Rhoptries appeared as secretory organelles of antigens recognized during acute, chronic, and congenital stages of Toxoplasma infection.

Identification of antileucoprotease in remodelled human adult nasal surface epithelium.

Antileucoprotease (ALP) is generally considered as a specific marker for glandular serous cells, and plays a major role in the defence of the respiratory tract against proteolytic damage. Nevertheless, several studies have identified ALP in bronchial and bronchiolar surface epithelial cells, and also an increased number of ALP-containing cells in bronchiolar tissue during the development of pulmonary diseases. In order to define more clearly whether the surface epithelium might be involved in the defence of the respiratory mucosa, we have investigated the expression of ALP by cells of the nasal surface epithelium. Indirect immunocytochemistry and in situ hybridization for ALP were performed on human nasal polyp sections. The height of the surface epithelium, its morphology, and the degree of local inflammation were assessed in parallel. Surface epithelium morphology was highly heterogeneous. ALP-containing cells were identified, but only in remodelled areas of the surface epithelium (foldings, basal cell and/or mucous cell hyperplasia), with no association to the degree of inflammation. These results demonstrate that the surface epithelial cells of the human adult nasal mucosa can express ALP in remodelled surface epithelium, and may be actively involved in the biochemical defence of the airways.

[Interstitial brachytherapy in carcinoma of the lip. Case histories and results]. / Brachiterapia interstiziale nel carcinoma del labbro. Casistica e risultati.

Squamous cell carcinomas of the lip can be treated by surgery or radiation therapy. Since local control rates exceed 90% with both treatment modalities, therapy is chosen according to functional and cosmetic results, to the patient's wishes and to the physician's medical policy. This study was aimed at retrospectively reviewing our local control results and patterns of failure relative to survival as well as evaluating the results according to technical surgical variables. From 1982 to 1991, sixty-nine patients with squamous cell carcinoma of the lip received a course of interstitial brachytherapy with 192Ir. The disease stage was T1 in 36 cases, T2 in 12 cases and T3 in 2 cases and 19 recurrences; 3 patients only had clinically detectable lymph nodes at diagnosis. Interstitial brachytherapy was used as postoperative treatment, at a dose of 60 Gy, in 47 cases. As an exclusive curative approach brachytherapy was given at a reference dose of 65 Gy, with a medium dose rate of 62.88 cGy; a single plane was used in 55 cases, a triangular plane in 11 and a double plane in 3 cases. In one patient only the disease persisted and no local failures were observed. Overall actuarial survival at 5 years is 76.81% (91.3% when corrected for disease). In fact, 6 patients died of disease progression: 3 with lung metastases, 1 with bone metastases and 3 with lymph node metastases. Five patients died of cancer in other sites--i.e., ovary, lung, prostate, hypopharynx, stomach--and 4 of non-neoplastic diseases. Tolerance rates were excellent, with only one mucosal necrosis which resolved spontaneously; no sequelae were observed in gums, teeth and jaws. The cosmetic result was also excellent in most of the cases with a worsening trend for multiplanar disposition. In conclusion, interstitial brachytherapy can be considered the treatment of choice for early lip cancer.

Subcellular localization and quantitative analysis of Toxoplasma gondii target antigens of specific immunoglobulins G, M, A, and E.

The target antigens of specific immunoglobulins G, M, A, and E from patients with acquired acute toxoplasmosis were determined using immunocytochemistry. The relative repartition of these antigens in four cellular compartments of Toxoplasma (membrane complex, apical area, rhoptries, and dense granules) was quantitatively evaluated. Rhoptry antigens mainly react positively with IgA. Membrane, submembrane area (membrane complex), and rhoptry antigens are immunodominant for IgA and IgM. Apical area antigens are recognized by IgM two times more than IgG and IgA. IgE recognized only rhoptry antigens. The localization of pathogenetically antigenic components and their identification by the immune system appeared to be of importance for selection of immunodominant or recombinant antigens. Such localization would improve laboratory diagnosis of serious congenital toxoplasmosis or in immunocompromised patients with toxoplasmic complications after cyst reactivation.

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections.

Scanning transmission electron microscopy at 300kV enables the visualization of nucleolar silver-stained structures within thick sections (3-8 microns) of Epon-embedded cells at high tilt angles (-50 degrees; +50 degrees). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-microns-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e-/A2/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-microns-thick sections with a spot-size of 5.6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 microns. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.

Digital three-dimensional visualization of cellular organelles studied by medium- and high-voltage electron microscopy.

Tilted thick sections (one-half to several micrometers) of biological specimens observed with medium- to high-voltage electron microscopes are extremely useful for the study of the three-dimensional (3-D) structure of organelles. If high resolution in 3-D visualization and 3-D reconstruction is needed, many images corresponding to various angles of rotation and tilt must be recorded. This necessitates very time-consuming work--including eventual photographic processing--before good positioning of the object is defined. We have developed software which permits very rapid and precise determination of the tilt-axis, the registration of tilted views, 3-D measurements and 3-D visualization. Images are digitized either from negative films or directly with a camera fitted to the microscope. The application of the software is performed in minutes and allows for a rapid check of the quality of the tilt-series and of the features of interest of the object. Application of the software to the study of the 3-D structure of active components of the nucleolus stained with silver is shown.

An afterloading technique suitable for carcinomas of the oral cavity.

The authors describe an afterloading brachytherapy to treat oral cavity carcinomas. Catheters for arterial/venous catheterization are inserted percutaneously in the target volume. The internal needles are then removed and replaced with iridium wires inside tubes to form wire loops. This technique has proven to be simple, quick and safe.

[Influence of radiotherapy on lymphocyte subpopulations]. / Influenza della terapia radiante sulla popolazione linfocitaria.

The authors investigated the effects of radiation therapy on the immune system by studying lymphocyte subsets and other parameters in 32 patients undergoing radiation therapy for solid cancer. With monoclonal antibody techniques, we studied both T- and B-lymphocytes; cell suspensions were analyzed by means of a Facs Spectrum III Ortho (Ortho-Diagnostic) unit. The first control was performed right after the beginning of radiotherapy, when the dose to the patients was 50 Gy or higher. The second control was performed at 40 Gy because all patients received this dose. 30% of the patients exhibited lymphopenia from the beginning of the study; at 40 Gy the number of T-lymphocytes was low and helper/suppressor ratio was altered. A variable response of B-cells was observed, although all patients exhibited restoration of normal values at 6 months. Four patients only suffered from side-effects: a patient with tongue cancer presented oral mycosis, and a woman--treated for breast cancer--presented vaginal mycosis. Two cases of cystitis were also observed, after 18 Gy, in patients with uterine carcinoma undergoing pelvic irradiation. Disease progression was observed in 2 patients with head and neck cancer, while 3 patients died from lung cancer progression. Another one, with head and neck cancer, died because of heart failure.