"Virtual patch clamp analysis" for predicting the functional significance of pathogenic variants in sodium channels.

OBJECTIVE: Opening of voltage-gated sodium channels is crucial for neuronal depolarization. Proper channel opening and influx of Na+ through the ion pore, is dependent upon binding of Na+ ion to a specific amino-acid motif (DEKA) within the pore. In this study we used molecular dynamic simulations, an advanced bioinformatic tool, to research the dysfunction caused by pathogenic variants in SCN1a, SCN2a and SCN8a genes. METHOD: Molecular dynamic simulations were performed in six patients: three patients with Dravet syndrome (p.Gly177Ala,p.Ser259Arg and p.Met1267Ile, SCN1a), two patients with early onset drug resistant epilepsy(p.Ala263Val, SCN2a and p.Ile251Arg, SCN8a), and a patient with autism (p.Thr155Ala, SCN2a). After predicting the 3D-structure of mutated proteins by homology modeling, time dependent molecular dynamic simulations were performed, using the Schrödinger algorithm. The opening of the sodium channel, including the detachment of the sodium ion to the DEKA motif and pore diameter were assessed. Results were compared to the existent patch clamp analysis in four patients, and consistency with clinical phenotype was noted. RESULTS: The Na+ ion remained attached to DEKA filter longer when compared to wild type in the p.Gly177Ala, p.Ser259Arg,SCN1a, and p.Thr155Ala, SCN2a variants, consistent with loss-of-function. In contrast, it detached quicker from DEKA than wild type in the p.Ala263Val,SCN2a variant, consistent with gain-of-function. In the p.Met1267Ile,SCN1a variant, detachment from DEKA was quicker, but pore diameter decreased, suggesting partial loss-of-function. In the p.Leu251Arg,SCN8a variant, the pore remained opened longer when compared to wild type, consistent with a gain-of-function. The molecular dynamic simulation results were consistent with the existing patch-clamp analysis studies, as well as the clinical phenotype. SIGNIFICANCE: Molecular dynamic simulation can be useful in predicting pathogenicity of variants and the disease phenotype, and selecting targeted treatment based on channel dysfunction. Further development of these bioinformatic tools may lead to "virtual patch-clamp analysis".

Novel antibodies as anticancer agents.

In recent years antibodies, whether generated by traditional hybridoma technology or by recombinant DNA strategies, have evolved from Paul Ehrlich's 'magic bullets' to a modern age 'guided missile'. In the recent years of immunologic research, we are witnessing development in the fields of antigen screening and protein engineering in order to create specific anticancer remedies. The developments in the field of recombinant DNA, protein engineering and cancer biology have let us gain insight into many cancer-related mechanisms. Moreover, novel techniques have facilitated tools allowing unique distinction between malignantly transformed cells, and regular ones. This understanding has paved the way for the rational design of a new age of pharmaceuticals: monoclonal antibodies and their fragments. Antibodies can select antigens on both a specific and a high-affinity account, and further implementation of these qualities is used to target cancer cells by specifically identifying exogenous antigens of cancer cell populations. The structure of the antibody provides plasticity resonating from its functional sites. This review will screen some of the many novel antibodies and antibody-based approaches that are being currently developed for clinical applications as the new generation of anticancer agents.

Pulp response to ferric sulfate, diluted formocresol and IRM in pulpotomized primary baboon teeth.

This study investigated the pulp response to a 15.5 percent ferric sulfate solution (FS) and a 20 percent dilution of formocresol (DFC) in pulpotomized primary teeth of baboons, after four and eight weeks. Pulpotomies were performed in seventy-nine primary teeth of 4 baboons. After coronal pulp resection, the pulp stumps were painted with ferric sulfate for fifteen seconds, in thirty-two teeth (group 1); in another thirty-two teeth, a cotton pellet moistened with dilution of formocresol was placed over the pulp stumps for five minutes, and removed (group 2). In fifteen teeth, IRM was placed directly over the pulp stumps after hemostasis (group 3--control). The teeth of all groups were sealed with IRM, and examined for inflammatory changes under a microscope by two blinded examiners. Seventy-seven teeth were assessed. Mild or no inflammation was found in 58 percent (18/31) of the teeth of group 1, in 48 percent (15/31) of those of group 2, and in 73 percent (11/15) of those of group 3. Severe inflammation was found in 35 percent (11/31) of group 1, 29 percent (9/31) of group 2, and in 7 percent (1/15) of group 3. No statistically significant difference between the three groups was observed for degree of inflammation, periradicular or interradicular abscess or inflammatory root resorption (chi-square p > 0.05). Dentin bridges were observed in 52 percent (16/31) of the teeth in group 1, 52 percent (16/31) of those of group 2, and in 73 percent (11/15) of those of group 3. No difference was found between the experimental and control groups for the presence of dentin bridge, (p > 0.05). Ferric sulfate produced pulp responses that compared favorably to those of diluted formocresol.

Effect of loading on the migration of periodontal fibroblasts in the rat incisor.

The influence of occlusal loading on periodontal fibroblasts was investigated in hypoloaded (shortened out of occlusion), functionally loaded and hyperloaded (constant linguointrusive mechanical loads of 9.4 +/- 0.06 g) lower left rat incisors. One hour following injection of 3H-thymidine, half of the animals in each group were killed, while the remaining rats were killed 2 weeks later. The decalcified incisors were embedded in glycolmethacrylate and sectioned (2 microns) serially, perpendicularly to the long tooth axis. Labeled and unlabeled fibroblasts in the tooth-related periodontal ligament were counted in 8 x 80 microns consecutive layers. Cell density (CD) and labeling index (LI) were plotted according to their location on the apico-incisal and cementum-bone axes. Loading caused a decrease in CD and a shift of cells from the cementum towards the middle of the ligament, proportionally to load intensity and duration. The average tooth-to-bone movement of the cells was 2 microns/day in the hypoloaded and 4 microns/day in the two loaded groups. The mean daily tooth eruption rate was 975 +/- 60 microns, 499 +/- 18 microns and 103 +/- 27 microns in the hypo-, functionally- and hyperloaded teeth, respectively. The respective concomitant average daily cell migration rates in the incisal direction were 786 microns, 500 microns, and 500 microns, i.e. 80%, 100% and 485% of the tooth eruption rates. The gross disparity between cell velocity and tooth movement under conditions of restrained eruption indicates active motility of the fibroblasts, rather than their passive tooth-eruption dependent translation.

Dynamics of tissue changes found after mechanical loading of the rat incisor. II. A three-dimensional longitudinal study of the histopathologic aspects.

The study comprised 60 lower left rat incisors subjected to 2 weeks of loading (19.7 +/- 1.6 gm) and 5 control teeth. After the loading period, 10 rats were killed with the springs in situ, and the remainder were killed in groups of 5 at intervals of 1 through 10 weeks after the springs were removed. The teeth were cross-sectioned serially (2 microns). The distance of each section from the apex and the time of its tissue formation in relation to load removal were calculated. Bone remodeling and dental and periodontal trauma were plotted according to their location on the tooth axis and circumference. Vigorous bone remodeling continued throughout the observation period, apposition and resorption sites intermingling around the tooth. Directionality, as expressed by the resorption/apposition ratio, for the formerly tensed and stressed areas was 0.61 +/- 0.07 and 1.79 +/- 0.52, respectively. However, in some of the groups directionality was negligible or reversed. New lesions of dentin (folding and resorption) were formed for 6 weeks after loading, whereas new damage to the enamel organ and enamel matrix appeared until week 9. The integrity of the PDL deteriorated for the first 8 weeks, as evidenced by edema, cell loss, scarring, and collagen fiber disorientation. Necrosis occurred throughout the recovery period. Fiber to bone attachment was disrupted by formation of big sinusoids adjacent to the bone. The frequency of most lesions decreased toward the end of the observation period. It is suggested that tissue recovery proceeds in subsiding cycles of tissue injury/tissue repair, a process that is much more prolonged than is generally believed.

Histomorphometric study of the periodontal vasculature of the rat incisor.

This study assessed quantitatively the vascular system in the cementum-related periodontal ligament (PDL) along the rat incisor. The lower left incisors of six rats (+/- 200 g) were subjected to routine histological procedures and cross-sectioned serially (2 microns), and the distance between each section and the apex was computed. The PDL of five sections at different levels along the tooth was divided into mesial, lingual, and lateral parts. The number and area of small and terminal arterioles, capillaries (C), sinusoids (S), post-capillary venules (PCV), and connecting venules, as well as the area of the PDL, were established. Blood vessels (BV) occupied 47 +/- 2% of the PDL area in the apical half and 4 +/- 2% at the incisal end. Of the total BV area, 41%, 32%, and 27% were located on the lingual, mesial, and lateral tooth sides, respectively. The majority of BV belonged to the venous system (98.5 +/- 0.6% and 82.5 +/- 3.0% in the apical and incisal parts, respectively). The apical venous system comprised 95.4 +/- 1.6% S and 3.2 +/- 1.0% PCV, reversing to 27.2 +/- 14.2% S and 55.2 +/- 11.3% PCV in the incisal half. The number of arterial profiles increased gradually from 6.8 +/- 1.5 at the apex to 25.3 +/- 2.4 in the incisal part and that of C from 9.0 +/- 1.18 to 25.0 +/- 4.3. The extensive vascularization in the apical half of the PDL is consistent with the high metabolic demands and with the need for protective cushioning of the constantly growing dental and periodontal tissues.2+_

Histopathology of the pulp in primary incisors with deep dentinal caries.

The purpose of this study was to assess the histological appearance of the pulp of human primary incisors extracted because of deep, unrestorable caries, and to determine how clinical pulp exposures affected the histological status of the pulp compared to nonexposures. Caries was removed carefully from all teeth after fixation; 24 incisors had pulp exposures, and 29 teeth had no pulp exposures. Histological examination showed normal pulps in 69% of the teeth without pulp exposures, compared to 33% of teeth with exposed pulps (P < 0.05). Microabscesses were observed in 33% of cases with pulp exposures, compared to 10% of cases without pulp exposures. In this study, 46 of 53 pulps remained vital in spite of the multiple and deep carious lesions. Teeth without pulp exposures were diagnosed in the treatable category in 20 of 26 cases.

Compensatory hyperplasia of the rat submandibular gland following unilateral extirpation.

This study investigated the morphological changes in rat submandibular glands undergoing compensatory hyperplasia. Fifteen adult male rats underwent left submandibulectomy, after which they were killed in groups of five (at days 3, 7, and 14), and their right submandibular glands (SMG) were excised. Fifteen control rats were killed in groups of five (at days 0, 7, and 14), and their right SMG were removed. Sections of 3 microns were cut, and the parenchymal and stromal cells were counted in 50 microscopic fields and sorted according to their morphological features and "class". Class is equivalent to the number of nuclei in an acinar or tubular cross-section. No change in glandular weight was noted post-surgery. Total cell count/field rose to 138.5 +/- 7.1% of control values on day 3 after gland extirpation, remaining almost constant thereafter until the end of the experiment. Acinar cell count and class showed a 154.1% peak on day 3, followed by a 30% drop in cell count by day 7 and an equivalent decline in class by day 14. Tubular cell count increased gradually to 146.5% by day 14, without a change in class. In the first week, the increase in tubular cells was mainly due to intercalated duct (ID) cells, while in the second week, there was a sharp rise in granular duct (GD) cells. This diverging cellular behavior indicates that the GD cell stems from the ID cell. The cellular changes in the hyperplastic SMG indicate death of newly generated acinar cells and expansion of the glandular progenitor compartment, as expressed in elongation of the ID.

Dynamics of tissue changes found after mechanical loading of the rat incisor. I. A three-dimensional longitudinal study of the morphologic aspects.

A three-dimensional morphometric method was used to evaluate progressive changes in shape and size of recovering dental and periodontal tissues after orthodontic loading. In 35 female rats weighing 212 +/- 4 gm, loads of 19.7 +/- 1.6 gm generated by closed-coil springs were applied for 2 weeks to the shortened lower left incisor. The rats were killed in groups of five at 0, 1, 3, 5, 7, 9, and 10 weeks (groups 0-w to 10-w) after the springs were removed. A group of rats with normal incisors (group C-1) and one with five incisors that had been continually shortened for 10 weeks (group C-2) served as controls for groups 0-w and 10-w, respectively. Width, area, and volume of the tooth and enamel-bordering periodontal ligament (e-PDL) and cementum-bordering PDL (c-PDL) were measured. After 2 weeks of loading (group 0-w), the volume of the compressed e-PDL had decreased by 22%, and the volume of the stretched c-PDL had increased by 72%, suggesting that bone apposition lags behind the rate of tooth movement. During the recovery period, the dental and periodontal parameters tended toward a gradual return to control (C-2) values, although at the end of 10 weeks many still lagged significantly behind the controls. Recovery was slowed by repeated reversals at different sites in the various groups. The ability of the preloaded incisor to adjust to changes in occlusal function was lastingly impaired.

Pulp response to collagen and glutaraldehyde in pulpotomized primary teeth of baboons.

This investigation assessed histologically the pulp tissue reaction to glutaraldehyde (GA) and to a commercial collagen preparation in pulpotomized primary teeth of baboons. One hundred and eighty-eight primary teeth were pulpotomized; in half of them inflammation was induced prior to the treatment. The teeth were divided into five groups: in three of them GA was used as a pulp dressing and applied for 1 min (group 1), 5 min (group 2), or mixed into the paste (group 3); collagen was used in group 4 and in group 5 (control) IRM was placed directly over the pulp stumps. Follow-up times were two, eight, and 24 weeks. Total necrosis was observed only in the collagen group. Partial necrosis and severe inflammation also were seen mainly in this group, and when the GA was incorporated into the paste. Slight to moderate inflammation was evident in all groups two and eight weeks postoperatively; however, 78% of the teeth of group 2 (GA 5 min) were inflammation-free after 24 weeks. Partial dentin bridges were seen in 92% of the teeth of the control group, in 82% of group 2, and 50% each of groups 1 and 3 eight weeks postoperatively. Dentin bridges were present in only 4% of the collagen group. After 24 weeks, all the teeth in group 2 and 83% of group 1 had dentin bridges. We conclude that Zyderm (Colagen Corp. Palo Alto, CA) led to unacceptable results, 5 min application of GA presented the best healing response, and GA 1 min and IRM also were satisfactory.

EDTA-dependent pseudothrombocytopenia: a clinical study of 18 patients and a review of the literature.

EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is the phenomenon of a spurious low platelet count due to the appearance of antibodies that cause platelet agglutination in blood anticoagulated with EDTA. We review here the clinical features of 18 patients with EDTA-PTCP treated in our hospital from 1984 to 1987 as well as those of 34 patients reported in the literature. This phenomenon appears more frequently in severely ill patients, in association with autoimmune, neoplastic, atherosclerosis-related, and liver diseases. In the majority of our patients, EDTA-PTCP appeared during hospitalization, indicating that the antibody is an acquired one. Neither splenomegaly nor the presence of autoimmune markers were features of this entity. Unlike true thrombocytopenias, EDTA-PTCP is associated with a normal mean platelet volume. Awareness of this entity is essential since EDTA-PTCP is frequently misdiagnosed and therefore incorrectly treated.

Long-term effects of orthodontic forces on the morphology of the rat-incisor socket and its location in the mandible.

The effect of orthodontic force application on the rat-incisor socket and mandible was studied on roentgenograms. A mean linguo-intrusive force of 19 +/- 0.6 g was applied continuously to the shortened left lower incisor for a period of two (group A) and four weeks (group B). A third group of rats, subjected to shortening of the left mandibular incisor only, served as a hypofunctional control (group C). A fourth group of normal rats constituted the intact control (group D). After a recovery period of three months, the animals were killed, and standardized roentgenograms of the cleaned mandibles were taken. Socket and mandibular dimensions were measured on magnified tracings of the roentgenograms. Comparison of groups A and B with the control groups, on the one hand, and of group C with group D, on the other, facilitated isolation of the hypofunctional factor. In groups A and B, the orthodontic forces caused changes in the parameters affected (i.e., socket area, alveolar bone thickness, mandibular dimensions) and not affected by hypofunction (i.e., socket angulation and location, anterior-socket length). The former findings implies modification of the adaptive capability of dental structures to functional demands. It is concluded that mechanical loading of the incisor for two to four weeks causes long-lasting changes in the socket and its surrounding bone.

Effect of occlusal functional forces on incisor socket morphology and location in the rat mandible.

The effect of functional occlusal stress on dimensional alterations of the rat incisor socket and mandible were studied from roentgenograms. In 12 rats, the lower left incisor was shortened twice weekly, whereas the lower right incisor was allowed to remain in contact with both upper incisors. Thus, the right incisors were subjected to hyperfunction, and the left ones, to hypofunction. The lower incisors of 16 rats with normal occlusal contact served as control. Following an experimental period of 3 months, the animals were killed and standardized radiographs were taken of the cleaned mandibles. Socket and mandibular dimensions were measured on magnified tracings of the roentgenograms. Socket area, its posterior length, posterior mandibular length, and gonial angle changed in the same direction under both hyper- and hypofunction. The anterior socket was relocated in opposite directions: under hyperfunction, it assumed a more inferior position, whereas in hypofunction, it moved superiorly. The angulation of the socket became significantly more acute under hyperfunction, whereas in hypofunction, this parameter remained unchanged. It is concluded that altered functional demands affect the morphology of the incisor socket and its location within the mandibular borders.

The streaming of the submandibular gland. II: Parenchyma and stroma advance at the same velocity.

Thirty young male rats aged 7 weeks, weighing 200 g, were injected with 18.5 kBq g-1 (0.5 microCi g-1) body weight tritiated thymidine [3H]TdR (specific activity 185 GBq). The rats were then killed in groups of five, at the following times: 1 hour, and 14, 30, 60, 90, and 120 days. Autoradiograms of sections through the submandibular gland were prepared, and the location of labelled cells in tubular and acinar cross sections was recorded. The nuclear content of each cross section was defined as its 'class'. In this numbering system, narrow tubuli, e.g. intercalated ducts are of low class, and wider tubuli, e.g. striated ducts, of high class. One hour after labelling most labelled tubular cells were found in low class cross sections, i.e. intercalated ducts and narrow granular ducts. Striated ducts were not labelled. From then onward labelled cells entered wider tubuli, e.g. striated ducts. The advancing labelled epithelium was accompanied by labelled stroma. Both cell types traversed 0.089 classes per day. In acini, labelled cells advanced in the opposite direction, starting from acinar cross sections of high class and ending in class-1 cross sections.

A three-dimensional evaluation of the effects of functional occlusal forces on the morphology of dental and periodontal tissues of the rat incisor.

We examined the effect of function on tooth and periodontal ligament (PDL) morphology in 40 lower incisors of adult female rats. Ten teeth were exposed to occlusal hyperfunction for three months, ten to hypofunction for three weeks, ten to hypofunction for three months, while ten teeth in normal occlusion served as control. Transverse ground sections were cut at various levels perpendicular to the tooth long axis, and their distances from the apex were calculated. The outlines of the tissues were traced and fed into a computer. We plotted the measurements according to their location and fitted them by second-order polynomials. We calculated tissue volume for the proximal 18 mm of bone-embedded tooth. Hyperfunction affected tooth shape, in that it became more rounded. The volumes of the dental tissues remained unchanged, while width and volume of the cementum-bordering PDL increased. Hypofunction did not alter tooth shape, but influenced its size: After three weeks, tooth circumference decreased, and after three months, it expanded. Dentin width was reduced, with concomitant increase of pulp size. The amount of enamel diminished initially, but after three months returned to normal values. The PDL bordering enamel expanded proportionally to the duration of hypofunction. The changes in socket size reflected the total dimensional variations in the tooth and its PDL. The results demonstrate that the shape and size of growing teeth and their periodontium are influenced by functional occlusal forces.

The rat parotid gland--a renewing cell population.

The mode of turnover and displacement of cells in the rat parotid gland was studied by means of autoradiography. The parotid parenchyma and stroma act as a two-compartment cell renewal system. Parotid cell progenitors are formed mainly in the intercalated duct from which they progress in opposite directions: to the striated duct on one side, while feeding into the acini on the other. The stroma cells stream along the same tissue radii as the parenchymal cells.

Permanent neurological complications in patients with thrombotic thrombocytopenic purpura.

According to previous clinical studies, the neurological manifestations observed in patients with thrombotic thrombocytopenic purpura (TTP) are considered to be transient. The introduction of plasma therapy in 1977 as major treatment modality for TTP has changed the prognosis of the disease. In a clinical survey of 38 patients with TTP who received plasma therapy, we have observed five patients who developed permanent neurological deficits despite their prompt recovery from TTP. In this study, we describe the new complication of TTP and summarize the neurological manifestations observed in these patients during their first episode of TTP and during the relapses, which occurred in 12 of them.

Three-dimensional presentation of cell migration in the periodontal ligament of the rat incisor.

The progenitor compartments and cell migration were examined in the tooth-related periodontal ligament (t-PDL) of rat incisors. A pulse injection of 3H-Tdr was administered to 15 rats (200 gm each) and the animals were killed in groups of five, at 1 hr and at 1 and 2 weeks after injection. Three-dimensional analysis of cell counts and labeling index demonstrated the existence of two progenitor compartments (PC). The apical PC (responsible for 70% of synthesizing cells) was concentrated in the apical 5 mm of the t-PDL. The paracemental PC (30% of synthesizing cells) was located along and around the cementum, occupying 24 micron of the t-PDL. The cells from the apical PC migrated incisally at a rate of 6 mm/week, which is a rate similar to that of tooth eruption. The cells from the paracemental PC moved in a transverse direction toward either bone or cementum at the much slower rate of 16 micron/week.

The effect of load duration on long-term recovery of the eruptive function in the rat incisor.

The purpose of this study was to determine the long-term recovery of the normal rate of eruption of the rat incisor following application of loads of similar magnitude but of varying duration. The daily rate of eruption of the continuously growing tooth served as indicator of the functional recovery of its periodontium. The left mandibular incisor was shortened out of occlusion in 34 experimental and 16 control rats. The experimental animals were divided into groups A, B, and C, and continuous loads (19.47 g +/- 0.42 g) were applied to the shortened incisors by means of closed-coil springs for 5, 14, and 28 days, respectively. Following removal of the springs, the daily rate of eruption was measured and calculated in all 50 animals for a period of 10 weeks. The extended impairment of eruption in the experimental animals was directly proportional to the duration of loading: at 10 weeks postloading, it was present in 18%, 67%, and 100% of the rats in groups A, B, and C, respectively. The intragroup variations in the rate of eruption of individual teeth were more pronounced following short-term loading than after prolonged load application. Short-term loading proved to be least harmful regarding recovery of eruptive function.