Eduardo Katchburian

Changes in Cell Wall Synthesis and Ultrastructure during Paradoxical Growth Effect of Caspofungin on Four Different Candida Species

ABSTRACT Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, β-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the β-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Apoptosis in the early developing periodontium of rat molars

Development of the periodontium involves a series of complex steps that result in the formation of root dentine, cementum, bone and fibres of the ligament. These precisely controlled and timed events require the participation of the enamel organ derived epithelial cells of Hertwigs (HRS) and ectomesenchymal cells of the dental follicle. These events involve rapid turnover of the tissues and cells, including disappearance of epithelial cells of HRS. Thus, it seemed likely to us that programmed cell death (apoptosis) may play a role in the development of the periodontium.

The Low Level Laser Therapy Effect on the Remodeling of Bone Extracellular Matrix

The low level laser therapy (LLLT) has been used as an option to accelerate the regeneration of bone tissue. In this study, both femurs of male Wistar rats (30 animals) were injured with a drill and the effect of LLLT using a laser diode (100 mW at 660 nm) in the bone matrix on the left paw measured. LLLT effect on the healing bone tissue matrix was evaluated by a combination of immunohistochemical histomorphometry, confocal immunofluorescence microscopy and isolation and characterization of glycosaminoglycans. Histomorphometric analysis showed that LLLT increased bone matrix and showing more organized. Alcian Blue and PAS staining seems to suggest differential glycosaminoglycans and glycoproteins. The data showed increased expression of chondroitin sulfate and hyaluronic acid, after reduction as the LLLT and mature bone, resembling the expression of osteonectin and biglycan. The difference in expression of siblings (DMP‐1, OPN and BSP) is in accordance with the repair accelerated bone formation after the application of LLLT as compared with control. The expression of osteonectin and osteocalcin supports their role in bone mineralization protein, indicating that LLLT accelerates this process. The overall data show that LLLT bone changes dynamic array, shortening the time period involved in the bone repair.

Apoptosis in the early involuting stellate reticulum of rat molar tooth germs.

 When the enamel organ of the rat tooth germ is fully developed at the tip of the prospective cusp, amelogenesis begins, and at this site the overlaying stellate reticulum begins its involution. During the involution process, there is a gradual decrease in intercellular spaces, invasion by blood vessels, appearance of macrophage-like cells and reduction in the number of stellate reticulum cells. Since reduction or disappearance of cells during embryonic development in organs and tissues has been shown to occur by apoptosis, we decided to examine early involuting regions of the stellate reticulum in the hope of detecting apoptosis. For this purpose, upper first molars of Wistar newborn rats aged 1 and 3 days were fixed in formaldehyde for the TUNEL method and in glutaraldehyde-formaldehyde for light and electron microscopy. Paraffin sections revealed TUNEL-positive structures, i.e. brown-yellow-stained bodies, in the central portion of the stellate reticulum, and next to the outer enamel epithelium and stratum intermedium. Examination of ultrathin sections confirmed the TUNEL findings: some stellate reticulum cells showed nuclei containing crescent-like electron-opaque condensed masses of peripheral chromatin, typical of apoptosis. Also, apoptotic bodies of various sizes and appearances were frequently observed within stellate reticulum cells. We should like to suggest that apoptosis is associated with the reduction in the number of cells during regression of the reticulum.

Some aspects of the natural history of the casque-headed tree frog Corythomantis greeningi Boulenger (Hylidae)

Abstract Corythomantis greeningi , a casque-headed tree frog, was studied in the field and in captivity. This species belongs to a monotypic genus, and together with other species of anurans, possesses a differentiated head which is flat, very rough and presenting cranial co-ossification with the skin. These are characteristics that are usually associated with phragmotic behaviour which is supposed to have a role in water saving and protection against predators. Observations were focused on the characteristics of the species which make it adapted to life in the Brazilian semi-arid (“caatinga”). Data about the body and external anatomy of the head, integument morphology, life, reproduction, interest for sheltering and defense against desiccation is presented. Finally, it is proposed that adaptation of this species to the dry environment is probably the result of the synergism of a number of different strategies against water loss, including phragmosis.

Development of tight junctions between odontoblasts in early dentinogenesis as revealed by freeze-fracture

Mature odontoblasts possess junctional structures constituted by adherens, gap, and tight junctions. Although adherens and gap junctions appear early between odontoblasts, there is no information on the appearance and development of tight junctions between odontoblasts. In this study, we have examined freeze‐fracture replicas of early dentinogenesis to study the development of tight junctions between odontoblasts and to determine whether these junctions are of zonular or macular type.

Combined Therapy with Steroids and Antioxidants Prevents Ultrastructural Damage Surrounding Chronic Radiofrequency Lesions

We evaluated the effects of combined therapy with anti‐inflammatory and antioxidant drugs on the healing of radiofrequency (RF) lesions (70°C, 60 s) produced in the right atrium and each ventricle of 21 adult dogs. Three groups were studied: acute (n= 7, sacrificed 1 hour after RF); control (n = 7, sacrificed 54 ± 29 days after RF); and dogs (n = 7, sacrificed 32 ± 5 days after RF) receiving combined therapy with allopurinol (400 mg po 24 and 2 hours before RF); verapamil (200 μg/kg IV 15 min before and after RF); hydrocortisone (10 mg/kg IV after RF) and prednisone (20 mg po for 29 days). Atrial (n = 37) and ventricular (n = 70) lesion dimensions were determined and 21 right ventricular apex lesions (1 per dog) were selected for light and electron microscopy analysis. Histological and ultrastructural (US) characteristics in three zones extending from the visible lesion border, A (0–3 mm); B (3–6 mm); and C (6–9 mm), were assessed. In chronic groups, atrial and ventricular lesion dimensions were similar, but delayed scar formation was noted in treated animals. Acutely and at follow‐up, significant US abnormalities occurred in zones A and B and chronic lesions exhibited type I collagen proliferation. In Zone A, the extent of US injury and collagen proliferation was significantly less in treated dogs. Further, Zone B was normal in 5/7 (71%) treated dogs and in 0/7 (0%) controls. Zone C was essentially normal in all groups. Combined therapy with steroids, verapamil, and allopurinol is effective in limiting US damage surrounding chronic RF lesions. (PACE 2004; 27:65–72)

Structural and functional changes in the alveolar bone osteoclasts of estrogen-treated rats

This study investigated structural and functional features of apoptotic alveolar bone osteoclasts in estrogen‐treated rats. For this purpose, 15 female rats 22 days old were divided into three groups: Estrogen (EG), Sham (SG) and Control (CG). The rats of EG received daily intramuscular injection of estrogen for 7 days. The SG received only the oil vehicle. Maxillary fragments containing alveolar bone were removed and processed for light and transmission electron microscopy. Area (OcA) and number of nuclei (OcN) and bone resorption surface per TRAP‐positive osteoclasts (BS/OC) were obtained. Vimentin, caspase‐3 and MMP‐9 immunoreactions, TUNEL/TRAP and MMP‐9/TUNEL combined reactions were performed. In EG, the OcA, OcN and BS/Oc were reduced. Moreover, osteoclasts showed cytoplasm immunolabelled by caspase‐3 and a different pattern of vimentin expression in comparison with CG and SG. MMP‐9 expression was not affected by estrogen and the TUNEL‐positive osteoclasts were MMP‐9‐immunolabelled. In EG, ultrastructural images showed that apoptotic osteoclasts did not exhibit ruffled borders or clear zones and were shedding mononucleated portions. TRAP‐positive structures containing irregular and dense chromatin were partially surrounded by fibroblast‐like cells. In conclusion, the reduction in the BS/Oc may be due to reduction in OcA and OcN; these effects seem to be related to vimentin disarrangement rather than to an interference of estrogen with osteoclast MMP‐9 expression. Osteoclast apoptosis involves caspase‐3 activity and vimentin degradation; these cells release portions containing one apoptotic nucleus and, subsequently, undergo fragmentation, giving rise to apoptotic bodies.

Light microscopy and computer three-dimensional reconstruction of the blood capillaries of the enamel organ of rat molar tooth germs

We performed a light microscope and a computer three‐dimensional reconstruction study of serial sections of the molar enamel organ of 3‐ and 5‐day‐old rats perfused with Indian ink through the arterial system. The tooth germs were fixed in Bouins solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin. For the three‐dimensional reconstruction, light micrographs of the serial sections were digitized, and aligned using the serial EM Align software downloaded from http://synapses.bu.edu/tools/. After alignment, the boundaries of the India‐ink‐filled blood vessels were manually traced with a mouse using the software IGL trace (version 1.26b), also downloaded from the above website. After tracing, a three‐dimensional representation of the blood vessel contours was generated in a VRML format and visualized with the help of the software Cortona Web3D viewer (version 4.0) downloaded from http://www.parallelgraphics.com/products/cortona/. Our results showed that in regions where ameloblasts are polarized the capillaries are arranged in three distinct levels: (1) penetrating and leaving capillaries in relation to the outer enamel epithelium; (2) capillaries crossing and branching inside the stellate reticulum; and (3) capillaries branching and anastomosing profusely within the stratum intermedium, thereby forming an extensive capillary plexus intimately associated with the cells of the stratum intermedium. The existence of a conspicuous capillary plexus intermingled with cells of the stratum intermedium, as shown in our results, suggests that some molecules produced by cells of the stratum intermedium could be released into the capillary plexus and thereafter carried to the dental follicle.

Macrophages and apoptosis in the stellate reticulum of the rat enamel organ

Tooth germs of upper first molars of 1, 3, and 5‐d‐old rats, fixed in formaldehyde, were stained for the detection of apoptosis by the TUNEL method, and by the azo‐dye method for the demonstration of acid phosphatase. For conventional light and electron microscopy the specimens were fixed in glutaraldehyde‐formaldehyde and embedded in glycol methacrylate and Araldite. Results showed that macrophages, present in the stellate reticulum, contained basophilic bodies and TUNEL‐positive globules, i.e. apoptotic bodies, in their interior. Macrophages also possessed strong acid phosphatase activity. Electron microscopy showed the presence of large vacuoles inside the macrophages containing dense fragmented material. Taken together these results suggest that the intra‐epithelial macrophages of the stellate reticulum engulf apoptotic bodies.