Singer’s Nodules: Investigating the Etiopathogenetic Markers Progressing Their Pathogenesis and Clinical Manifestations

Vocal nodules (or Singer’s nodules) are benign vocal cord structures which are commonly encountered by clinicians. Though phonetic trauma/abuse is thought to be the main cause of the development of vocal nodules, the exact etiopathogenesis remains unknown. Hence, we compared the immunohistochemical markers for proliferation (Ki-67), apoptosis (TUNEL), growth (EGFR), ischemia (VEGF), inflammation (IL-1α and 10), and immunoreactive innervation (PGP 9.5), in vocal nodule tissue samples obtained from 10 females (17–56 years) and vocal cord tissue from seven controls. A statistically significant increase in Ki-67, TUNEL, EGFR, VEGF and IL-1α expression was noted (p < 0.05) between nodule tissue and control tissue in both epithelial and subepithelial layers. However, the difference was non-significant for both IL-10 and PGP 9.5 (p > 0.05). All markers demonstrated moderate to strong positive correlations, except for IL-10. These findings suggest increased cellular growth and proliferation in vocal nodules coupled with a persistent presence of inflammatory and ischemic environment. Furthermore, global prevalence of apoptotic cells and decreased anti-inflammatory cytokines highlight the presence of underlying complex mechanisms in the etiopathogenesis of vocal nodules, with age having a negligible impact on the marker levels. Our results could potentially further our knowledge in understanding the effects of different treatment modalities available at the cellular level.

The Endometriotic Nodule Has Lower T-cadherin, E-cadherin, Progesterone Receptor and Oestrogen Receptor Than Endometrioma Tissue

Objective:To compare the T-cadherin, E-cadherin, PR and ER staining levels of endometriotic nodules, ovarian endometriomas and normal endometrial tissues.Methods:Endometriotic nodules of 24 cases, endometrioma of 30 cases and normal endometrial tissues of 30 cases were examined. T-cadherin, E-cadherin, ER-α and PR-α staining levels of endometriotic nodular tissues, endometrioma tissues and endometrial tissues were compared immunohistochemically. H-score was calculated to compare the expression of T-cadherin, E-cadherin, ER-α, PR-α in IHC staining based on the percentage of cells stained at each intensity level.Results:T-cadherin, E-cadherin, ER and PR H-score were found lowest in endometriotic nodule tissue and the highest in endometrial tissue (p <0.0001, <0.0001, <0.0001 and <0.0001, respectively). In correlation analysis, a positive correlation was found between T-cadherin, E-cadherin, PR and ER H-score (p <0.0001 for each). No correlation was found between age, BMI, VAS score, CA125, endometrioma size and the severity of dysmenorrhea, dyspareunia and dystonia (p> 0.05).Conclusions:T-cadherin, E-cadherin, ER and PR H-score were found lowest in endometriotic nodule tissue, the highest in endometrium tissue. The finding of lower expression of PR-α in endometriotic nodule in our study may be related to decrease in progesterone effect which could not inhibit the decrease in the expression of T-cadherin and E-cadherin, thus the invasiveness of endometriotic nodule. These findings suggest that endometriotic nodule and ovarian endometrioma tissues have a different biology.

Localization of Superoxide Dismutases and Hydrogen Peroxide in Legume Root Nodules

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to O2 and H2O2 and thus represent a primary line of antioxidant defense in all aerobic organisms. H2O2 is a signal molecule involved in the plant's response to pathogen attack and other stress conditions as well as in nodulation. In this work, we have tested the hypothesis that SODs are a source of H2O2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized by in situ RNA hybridization and immunogold electron microscopy, respectively, whereas H2O2 was localized cytochemically by electron microscopy of cerium-perfused nodule tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the meristem (I) and invasion (II) zones, interzone II-III, and distal part of the N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected cells. At the subcellular level, CuZnSOD was found in the infection threads, cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the bacteroids, bacteria within infection threads, and mitochondria. The distinct expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in nodules. Large amounts of H2O2 were found at the same three nodule sites as CuZnSOD but not in association with MnSOD. This colocalization led us to postulate that cytosolic CuZnSOD is a source of H2O2 in nodules. Furthermore, the absence or large reduction of H2O2 in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that at least some of the H2O2 originates by the sequential operation of an NADPH oxidase- like enzyme and CuZnSOD. Results also show that there is abundant H2O2 associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.

Effect of Mutations in Pisum sativum L. Genes Blocking Different Stages of Nodule Development on the Expression of Late Symbiotic Genes in Rhizobium leguminosarum bv. viciae

In this report, the expression of late symbiotic genes (fnrN, fixN, and nifA) of Rhizobium leguminosarum bv. viciae was studied in nodules of mutant pea lines blocked at four successive stages of nodule development. Bacterial gene expression was analyzed in situ with transcriptional gusA reporter gene fusions. As a control, a constitutively expressed gusA gene was included. In the nodules of Nop- (nodule persistence) mutants (mutant in gene sym13), which had not yet exhibited signs of premature senescence, the expression patterns observed were identical to those in wild-type nodules. Normal expression of fusions also occurred in nodules defective at the infection droplet differentiation stage (mutantin gene sym40) in which bacteria are endocytosed, but infection threads and infection droplets are hypertrophied. In contrast, in Itn- (infection thread formation inside the nodule tissue) mutants (mutant gene sym33), in which there is no endocytosis of bacteria, expression of the constitutive fusion was only in infection threads and no activity was shown for the other fusions. From this it can be concluded that functionality of the plant gene Sym33, i.e., bacterial endocytosis, is a prerequisite for the expression of late symbiotic genes in the microsymbiont. No morphologically distinct interzone II–III could be detected in nodules blocked at the bacteroid differentiation stage (mutants in gene sym31). The constitutive fusion was expressed equally throughout the nodule tissue (except for the meristem), and the activity of fusions to late symbiotic genes increased gradually with a maximal expression level at the base of the nodule. This is consistent with an altered oxygen barrier previously reported for these nodules. By including double mutants, earlier results on sequential functioning of gene pairs sym33-sym40 and sym31-sym13 could be confirmed and it could be demonstrated that the developmental epistasis found at the morphological level also is reflected in the expression pattern of late symbiotic genes in the microsymbiont.

Symbiotic Gene Sym31 Controls the Presence of a Lectinlike Glycoprotein in the Symbiosome Compartment of Nitrogen-Fixing Pea Nodules

The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum in nodule tissue from a symbiotically defective pea mutant, Sprint2Fix¯(sym31), and the parent line, Sprint2. Immunostaining of Western blots (immunoblots) revealed that, whereas wild-type nodules contained three antigenic isoforms of PsNLEC-1, nodule homogenates of mutant sym31 contained only one isoform, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was not associated with symbiosomes in either the mutant or the wild-type parent (unlike the other two isoforms from wild-type nodules). Light microscopy revealed that PsNLEC-1 antigen was more abundant in the infected tissues of wild-type nodules than in nodules of sym31. By contrast, in situ hybridization indicated that the PsNlec1 gene transcript was strongly expressed in infected cells of both Sprint2 and sym31 nodule tissues. At the ultrastructural level, most of the PsNLEC-1 antigen in sym31 nodule tissue was visualized as inclusion bodies in the vacuolar compartment of infected host cells but it was apparently absent from the symbiosome compartment. The results suggest an aberrant vesicle targeting pathway during symbiosome development in this mutant.