Alberto Calligaro

A 12-week treatment with fractional CO2 laser for vulvovaginal atrophy: a pilot study

Abstract Objective This pilot study aimed to assess the efficacy and feasibility of fractional CO2 laser in the treatment of vulvovaginal atrophy (VVA) in postmenopausal women. Methods VVA symptoms were assessed before and after three applications of laser over 12 weeks in 50 women (age 59.6 ± 5.8 years) dissatisfied with previous local estrogen therapies. Subjective (visual analog scale) and objective (Vaginal Health Index Score, VHIS) measures were used during the study period to assess VVA. Quality of life was measured by using the SF-12. A subjective scale to evaluate the degree of pain related to the laser application and the degree of difficulty to perform the laser procedure was used. Results Fractional CO2 laser treatment was effective to improve VVA symptoms (vaginal dryness, vaginal burning, vaginal itching, dyspareunia, dysuria; p < 0.001) at 12-week follow-up, as well as the VHIS (13.1 ± 2.5 at baseline vs. 23.1 ± 1.9; p < 0.001). Both physical and mental scores of quality of life were significantly improved in comparison with baseline (p < 0.001). Satisfaction with the laser procedure was reported by 42 women (84%) and a minimal discomfort was experienced at the first laser application, mainly because of the insertion and the movements of the probe. Finally, the technique was very easy to perform in all women starting from the second application at week 4 and no adverse events were recorded during the study period. Conclusions A 12-week treatment with the fractional CO2 laser was feasible and induced a significant improvement of VVA symptoms by ameliorating vaginal health in postmenopausal women. Further controlled studies should be performed to confirm the present data and to assess the long-term effects of the laser procedure on vaginal tissues.

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

Histological study on the effects of microablative fractional CO2 laser on atrophic vaginal tissue: An ex vivo study

ObjectiveMicroablative fractional CO2 laser has been proven to determine tissue remodeling with neoformation of collagen and elastic fibers on atrophic skin. The aim of our study is to evaluate the effects of microablative fractional CO2 laser on postmenopausal women with vulvovaginal atrophy using an ex vivo model. MethodsThis is a prospective ex vivo cohort trial. Consecutive postmenopausal women with vulvovaginal atrophy managed with pelvic organ prolapse surgical operation were enrolled. After fascial plication, the redundant vaginal edge on one side was treated with CO2 laser (SmartXide2; DEKA Laser, Florence, Italy). Five different CO2 laser setup protocols were tested. The contralateral part of the vaginal wall was always used as control. Excessive vagina was trimmed and sent for histological evaluation to compare treated and nontreated tissues. Microscopic and ultrastructural aspects of the collagenic and elastic components of the matrix were studied, and a specific image analysis with computerized morphometry was performed. We also considered the fine cytological aspects of connective tissue proper cells, particularly fibroblasts. ResultsDuring the study period, five women were enrolled, and 10 vaginal specimens were finally retrieved. Four different settings of CO2 laser were compared. Protocols were tested twice each to confirm histological findings. Treatment protocols were compared according to histological findings, particularly in maximal depth and connective changes achieved. All procedures were uneventful for participants. ConclusionsThis study shows that microablative fractional CO2 laser can produce a remodeling of vaginal connective tissue without causing damage to surrounding tissue.

Peptidergic nerves in human dental pulp

SummaryThe peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.

Microscopic and ultrastructural modifications of postmenopausal atrophic vaginal mucosa after fractional carbon dioxide laser treatment

Vaginal atrophy occurring during menopause is closely related to the dramatic decrease in ovarian estrogens due to the loss of follicular activity. Particularly, significant changes occur in the structure of the vaginal mucosa, with consequent impairment of many physiological functions. In this study, carried out on bioptic vaginal mucosa samples from postmenopausal, nonestrogenized women, we present microscopic and ultrastructural modifications of vaginal mucosa following fractional carbon dioxide (CO2) laser treatment. We observed the restoration of the vaginal thick squamous stratified epithelium with a significant storage of glycogen in the epithelial cells and a high degree of glycogen-rich shedding cells at the epithelial surface. Moreover, in the connective tissue constituting the lamina propria, active fibroblasts synthesized new components of the extracellular matrix including collagen and ground substance (extrafibrillar matrix) molecules. Differently from atrophic mucosa, newly-formed papillae of connective tissue indented in the epithelium and typical blood capillaries penetrating inside the papillae, were also observed. Our morphological findings support the effectiveness of fractional CO2 laser application for the restoration of vaginal mucosa structure and related physiological trophism. These findings clearly coupled with striking clinical relief from symptoms suffered by the patients before treatment.

Vulvo-vaginal atrophy: A new treatment modality using thermo-ablative fractional CO2 laser

OBJECTIVE To evaluate the efficacy and feasibility of thermo-ablative fractional CO2 laser for the treatment of symptoms related to vulvo-vaginal atrophy (VVA) in post-menopausal women. METHODS From April 2013 to December 2013, post-menopausal patients who complained of one or more VVA-related symptoms and who underwent vaginal treatment with fractional CO2 laser were enrolled in the study. At baseline (T0) and 30 days post-treatment (T1), vaginal status of the women was evaluated using the Vaginal Health Index (VHI), and subjective intensity of VVA symptoms was evaluated using a visual analog scale (VAS). At T1, treatment satisfaction was evaluated using a 5-point Likert scale. RESULTS During the study period, a total of 48 patients were enrolled. Data indicated a significant improvement in VVA symptoms (vaginal dryness, burning, itching and dyspareunia) (P<0.0001) in patients who had undergone 3 sessions of vaginal fractional CO2 laser treatment. Moreover, VHI scores were significantly higher at T1 (P<0.0001). Overall, 91.7% of patients were satisfied or very satisfied with the procedure and experienced considerable improvement in quality of life (QoL). No adverse events due to fractional CO2 laser treatment occurred. CONCLUSION Thermo-ablative fractional CO2 laser could be a safe, effective and feasible option for the treatment of VVA symptoms in post-menopausal women.

PC10 monoclonal antibody to proliferating cell nuclear antigen as probe for cycling cell detection in developing tissues

Proliferating cell nuclear antigen (PCNA), also referred to as cyclin, is an auxiliary protein to DNA-polymerase delta and a proposed marker of replicating cells. We have investigated the applicability and limitations of PC10 monoclonal antibody to PCNA in a cell kinetics study of developing human and rat tissues by immunocytochemical and flow cytometric techniques. Our data demonstrate that the epitope recognized by PC10 antibody is resistant to wax embedding, but sensitive to aldehyde fixation; conversely, alcoholic fixative solutions preserve the immunoreactivity to PC10. Tissue distribution, DNA content and bromodeoxyuridine uptake confirm that PC10-immunoreactive cells in alcoholfixed tissues are cycling (G1-, S- and G2-phases traversing) cells. It is concluded that the PC10 antibody can be regarded as a powerful tool to study cell kinetics and differentiation in developing tissues, provided that the tissue processing is adeguate.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts.

Abstract. Prolidase, a ubiquitously distributed dipeptidase, is involved in the latter stage of degradation of endogenous and dietary proteins and is particularly important in collagen catabolism. It hydrolyzes dipeptides containing proline or hydroxyproline at the C-terminal position. Mutations in the gene encoding for prolidase cause prolidase deficiency (PD), an autosomal recessive disorder mainly characterized by skin lesions, mental retardation and recurrent infectious. In this work we reported the identification of the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a homozygous G>A transversion in two siblings leading to a G448R substitution. A heterozygous IVS11+1G>C transition causing the skipping of exon 11 and a null allele were detected in a third proband. In two unrelated patients, a homozygous IVS7-1G>A transversion was identified and shown to cause multiple alternative spliced transcripts. All the mutations result in loss of prolidase activity. Long-term cultured fibroblasts from these PD patients were used to develop an in vitro model that allowed investigation of the affected cells. Light and electron microscopy revealed that PD cells were more round and branched out than controls with increased cytosolic vacuolization, interruptions of the plasma membrane, mitochondria swelling, mitochondrial matrix and cristae modifications. JC-1 labeling showed decreased mitochondrial membrane potential. A significant intracellular accumulation of the Gly-Pro dipeptide was detected by capillary electrophoresis analysis. Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD.

Endothelin and nitric oxide synthase in lymphatic endothelial cells: immunolocalization in vivo and in vitro.

Endothelin (ET) is an endothelium‐derived multifunctional peptide that produces a potent, long‐lasting vasoconstriction. Nitric oxide (NO), besides being the most important endothelium‐derived relaxant factor in blood vessels, is supposed to be involved in regulating the interactions among endothelium, adhesive molecules, and leukocytes.