Increase in circulating SDF-1 after treatment with sulfated glycans. The role of SDF-1 in mobilization.

SDF-1 is a potent chemoattractant for mature white blood cells and hemopoietic stem/progenitor cells (HPCs). An important role for this chemokine in mobilization has been postulated, but in vivo studies directly addressing its effects are lacking. After one injection of fucan sulfate (FucS) or dextran sulfate, plasma levels of SDF-1 are greatly increased in mice or primates. Increases are dose-dependent and correlate with mobilization of HPCs. Elevated levels of circulating SDF-1 appear to be uniquely associated with this treatment, as it was not seen with cytokine or anti-integrin antibody treatments that induce mobilization. In vitro, these sulfated glycans specifically bind to SDF-1 and inhibit SDF-1/heparin binding, suggesting a mechanism of release from sequestration on heparan sulfate proteoglycans in vivo. Although other chemokines including IL8 and cytokines like G-CSF also increase, evidence in GCSFR-deficient mice suggests that at least these two factors are unlikely participants in FucS-induced mobilization. Likewise, although the activity of the metallo-protease MMP9 increases after FucS treatment, experiments in MMP9-/- mice indicate its presence is dispensable for mobilization or SDF-1 release. However, effects of other proteases cannot be ruled out by these experiments. Finally, anti-SDF-1 antibodies partially inhibit FucS-induced mobilization, supporting a causative relationship. Our data offer a unique insight into the mechanism of sulfated glycan-induced mobilization and suggest a novel way of disturbing SDF-1 gradients between bone marrow and peripheral blood.

Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence.

Employing carbohydrate ligands, which have been extensively used to block selectin function in vitro and in vivo, we have examined the involvement of such ligands in stem/progenitor cell mobilization in mice and monkeys. We found that sulfated fucans, branched and linear, are capable of increasing mature white cells in the periphery and mobilizing stem/progenitor cells of all classes (up to 32-fold) within a few hours posttreatment in a dose-dependent manner. To elicit the effect, the presence of sulfate groups was necessary, yet not sufficient, as certain sulfated hexosamines tested (chondroitin sulfates A or B) were ineffective. Significant mobilization of stem/progenitor cells and leukocytosis was elicited in selectin-deficient mice (L(-/-), PE(-/-), or LPE(-/-)) similar to that of wild-type controls, suggesting that the mode of action of sulfated fucans is not through blockade of known selectins. Other mechanisms have been entertained, in particular, the release of chemokines/cytokines, including some previously implicated in mobilization. Significant increases were documented in the levels of seven circulating chemokines/cytokines within a few hours after fucan sulfate treatment and support such a proposition. Additionally, an increase was noted in plasma metalloproteinase (MMP) 9, which might independently contribute to the mobilization process by enzymatically facilitating chemokine/cytokine release. Mobilization by sulfated polysaccharides provides a distinct paradigm in the mobilization process and uncovers an additional novel in vivo biological role for sulfated glycans. As similarly sulfated compounds were ineffective in vivo, the data also underscore the fact that polysaccharides with similar structures may elicit diverse in vivo effects.

Increases in ceramide levels in normal human mesangial cells subjected to different cellular stresses result from changes in distinct enzyme activities and can influence cellular responses to other stimuli.

Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to in vivo disease states.

Selectivity of sphingosine-induced apoptosis. Lack of activity of DL-erythyro-dihydrosphingosine.

Sphingosine (Sph) is emerging as an intracellular regulator of cellular differentiation and apoptosis (Ohta, et al., Cancer Res., 55, 691-697, 1995). We have recently found that both Sph and its methylated derivative N,N-dimethylsphingosine (DMS) inhibit mitogen-activated protein kinase (MAPK) activity, suggesting that Sph-induced apoptosis may be mediated at least partly through inhibition of MAPK (Sakakura, et al., Int J Oncol, 11, 31-39, 1997). We report in this study that three stereoisomers, D-erythro-Sph, L-threo-Sph, and DL-erythro-dihydrosphingosine, were tested in induction of apoptosis and inhibition of MAPK activity in three different kinds of solid tumor cell lines. D-erythro-Sph was strongest in these effects among three compounds. L-threo-Sphingosine was partly active. On the other hand, DL-erythro-dihydrosphingosine was totally inactive. These results demonstrate the specificity of sphingosine action in induction of apoptosis and inhibition of MAPK, suggesting that Sph may play an important role as a physiological intracellular messenger of apoptosis in these cancer cells.

Inhibition of sphingolipid induced apoptosis by caspase inhibitors indicates that sphingosine acts in an earlier part of the apoptotic pathway than ceramide.

Caspases are specific proteases involved in apoptosis, and their inhibition by specific peptide inhibitors can inhibit apoptosis. With these inhibitors we examined the relationship of caspases and sphingolipids involved in the induction of apoptosis of human leukemic HL60 cells. We have previously shown that sphingosine (Sph) and its methylated derivative dimethylsphingosine (DMS) effectively induce apoptosis in HL60 cells. Using these lipids as well as ceramide analogues we found both similarities and differences in the caspase involvement in apoptosis induced by the two distinct lipid types. The wide-spectrum caspase inhibitor Z-VAD-FMK and Z-DEVD-FMK, an inhibitor of the downstream caspases 3 (CPP32, Yama) and 7, both inhibited apoptosis induced by all the lipids tested. Z-AAD-FMK which inhibits the serine protease Granzyme B, inhibited Sph/DMS induced apoptosis, but little or no effect on ceramide induced apoptosis. Granzyme B shares a substrate sequence preference with upstream caspases capable of activating themselves and other caspases downstream. Z-IETD-FMK, which inhibits caspase 8/FLICE also inhibited Sph/DMS induced apoptosis with no inhibition of apoptosis induced by either ceramide. Together, these data indicate that Sph/DMS act independently from ceramide in the apoptosis pathway and further suggest that Sph/DMS act earlier in the pathway than ceramide and are involved upstream of even the early proteases, whereas the point of action for ceramide is downstream of the early proteases but upstream from the late caspases.

Altered sphingomyelinase and ceramide expression in the setting of ischemic and nephrotoxic acute renal failure.

Diverse physical and chemical stimuli can activate sphingomyelinases (SMases), resulting in sphingomyelin (SM) hydrolysis with ceramide release. Since ceramide can profoundly impact a host of homeostatic mechanisms, the concept of a "SM (or SMase) signaling pathway" has emerged. We recently documented that ceramide levels fall abruptly during renal ischemia, and then rebound to twice normal values during early reperfusion (30 to 90 min) Therefore, the present study assessed whether these ceramide changes are paralleled, and hence potentially mediated, by comparable changes in SMase activity. Mice were subjected to 45 minutes of renal ischemia +/- 30 minutes, 90 minutes, or 24 hours of reperfusion. Renal cortices (or isolated proximal tubules) were then assayed for SMase activity (acidic, neutral forms). To characterize whether early post-ischemic ceramide increments are a relatively persistent event, ceramide was assayed following a 24-hour reperfusion period. Finally, to assess whether the observed perturbations were unique to post-ischemic injury, SMase and ceramide were quantified in the setting of glycerol-induced myohemoglobinuria and anti-glomerular basement membrane (alpha GBM) antibody-induced acute renal failure (ARF). Ischemia induced abrupt declines (approximately 50%) in both acidic and neutral SMase activities, and these persisted in an unremitting fashion throughout 24 hours of reperfusion. Nevertheless, increased ceramide expression (2x normal) resulted. Myohemoglobinuria also suppressed acidic/neutral SMases, and again, "paradoxical" ceramide increments were observed. Finally, alpha GBM nephritis increased ceramide levels, but in this instance, a correlate was increased SMase activity. These results suggest that: (1) ceramide is an acute renal "stress rectant" increasing in response to diverse renal insults; (2) this response may occur independently of the classic SM pathway, since the ceramide increments can seemingly be dissociated from increased SMase activity; and (3) given the well documented impact of ceramide and the SM(ase) pathway on apoptosis, cell proliferation, differentiation, and tissue inflammation, the present results have potentially broad ranging implications for the induction and evolution of diverse forms of ARF.

Sphingosine 1-phosphate regulates melanoma cell motility through a receptor-coupled extracellular action and in a pertussis toxin-insensitive manner.

Our previous work showed that sphingosine 1-phosphate (Sph-1-P) inhibits the cell motility of mouse melanoma B16/F10, and other types of cells at 10-100 nM concentrations. In the present paper, we have identified and characterized specific cell surface binding sites for Sph-1-P in F10 cells. Sph-1-P immobilized on controlled pore glass beads inhibited the motility of F10 cells, suggesting that Sph-1-P acts on the cells from the outside. Binding assays with [3H]Sph-1-P revealed the presence of specific cell surface binding sites for Sph-1-P in F10 cells. Scatchard analysis demonstrated a single class of binding sites for Sph-1-P. The binding of [3H]Sph-1-P to F10 cells was inhibited by the addition of excess unlabeled Sph-1-P but not other natural sphingolipids. The specific binding was also sensitive to treatment with a protease. Using Sph-1-P-immobilized affinity chromatography, we, for the first time, identified 41-kDa and 79-kDa Sph-1-P binding proteins on the melanoma cell surface, although the 41-kDa protein was less specific to Sph-1-P. We demonstrated that pertussis toxin (PTX) treatment did not abolish the motility inhibition by Sph-1-P, suggesting that no PTX-sensitive G-protein is involved in the signaling. Furthermore, Sph-1-P was found to be specifically released from mouse BALB/3T3 clone A31 cells and F10 cells. Collectively, these results strongly suggest that Sph-1-P regulates melanoma cell motility through an extracellular action by specific binding to cell surface receptor protein(s), which is independent of PTX-sensitive G-protein.

Sphingosine induces apoptosis in androgen-independent human prostatic carcinoma DU-145 cells by suppression of bcl-X(L) gene expression.

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL-60 cells [Ohta et al. Cancer Res. 1995;55:691-697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down-regulation of bcl-2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177-180]. In this study, we examined the sphingosine-induced apoptosis of the androgen-independent human prostatic carcinoma cell line DU-145, which expresses bcl-X(L) and Bax but not bcl-2, and found that treatment of DU-145 cells with sphingosine suppressed bcl-X(L) in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl-X(L) or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1-phosphate, failed to induce apoptosis. These results indicate that, in DU-145 cells, sphingosine, but not its metabolites, induces apoptosis through down-regulation of bcl-X(L), independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl-2 or bcl-X(L) activity in these cells.

In vitro and in vivo induction of apoptosis by sphingosine and N, N-dimethylsphingosine in human epidermoid carcinoma KB-3-1 and its multidrug-resistant cells.

Sphingolipid breakdown products, including ceramide and sphingosine, regulate cell growth, cell differentiation, and apoptosis. We examined the effect of various agents, including sphingolipids, on apoptosis induction in human epidermoid carcinoma KB-3-1 and its multidrug-resistant (MDR) subclone KB-C2 cells which express P-glycoprotein. Adriamycin (ADM) induced apoptosis in KB-3-1 cells but not in KB-C2 MDR cells at the concentration of 50 microg/ml. On the other hand, 15 microM sphingosine or its methylated derivative N, N-dimethylsphingosine (DMS) induced apoptosis in both cell types in vitro. These results suggested that KB-C2 MDR cells were resistant to apoptosis induction by ADM but sensitive to that by sphingosine and DMS. Ceramide and sphingosine-1-phosphate, the initial metabolites of sphingosine, failed to induce apoptosis under the same experimental condition as sphingosine/DMS. The protein kinase C (PKC) inhibitors H7 and staurosporine did not induce apoptosis in either cell line, suggesting that PKC-independent signaling is involved in apoptosis induced by sphingosine and DMS, although both sphingosine and DMS have been shown to down-regulate PKC. Furthermore, DMS significantly inhibited the growth of KB-3-1 as well as KB-C2 MDR tumors in vivo, with evidence of increased apoptosis. The intracellular level of exogenously added [3H]sphingosine or [14C]DMS did not differ between the KB-3-1 parent cell line and its MDR subclone KB-C2, whereas that of [14C]ADM was reduced in KB-C2 MDR cells compared to KB-3-1 cells. These results suggest that P-glycoprotein acts as a transporter for ADM but not for sphingosine or DMS. Furthermore, DMS at the concentrations which induce apoptosis in KB-C2 cells did not affect the level of [14C]ADM. Because sphingosine and DMS induce apoptosis regardless of P-glycoprotein expression, they may provide a new strategy and a promising approach to the treatment of anticancer drug-resistant cancer.

Overexpression of bax enhances the radiation sensitivity in human breast cancer cells.

Bax-alpha, a splice variant of bax which promotes apoptosis, is expressed in many kinds of untransformed cell lines and breast tissue, whereas only weak or no expression could be detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weak bax gene expression, were stably transfected with pCX2neo bax, encoding human bax and neomycin-resistant genes, and two unique clones (MCF-7/bax-1 and MCF-7/bax-2) were thus generated which expressed different levels of bax-alpha. Sensitivity to ionizing radiation (IR) was examined and each was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax, and IR was found to induce intranucleosomal DNA fragmentation in stable transfectant but not in parent cells, thus suggesting that this sensitization is due to apoptosis. We suggest that exogenous bax-alpha expression might therefore be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may potentially have a therapeutic application by enhancing radiation sensitivity in breast cancer cells.

Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide.

Bax, one of the bcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weak bax gene expression, were stably transfected with pCX2neo bax, encoding human bax; and two unique clones, MCF-7/bax-1 and MCF-7/ bax-2, that expressed different levels of bax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level of bax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenous bax-alpha overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.

Overexpression of bax sensitizes human breast cancer MCF-7 cells to radiation-induced apoptosis.

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl-2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF-7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation-induced apoptosis. bax overexpression in MCF-7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21WAF1 about 5- to 10-fold and downregulated bcl-2 and bcl-XL by 80-90% at 6 hr in both parent and bax stably transfected MCF-7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells.

Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines.

In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylsphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway.

Suppression of bcl-2 gene expression by sphingosine in the apoptosis of human leukemic HL-60 cells during phorbol ester-induced terminal differentiation.

Our recent studies have shown that intracellular levels of sphingosine, an endogenous PKC inhibitor, increase during apoptosis resulting from phorbol ester (PMA)-induced terminal differentiation of human myeloid leukemic HL-60 cells, and have suggested that sphingosine may function as an endogenous mediator of apoptosis in these cells [Ohta, et al. (1995) Cancer Res. 55, 691-697]. We report here that apoptosis induced by PMA, sphingosine, and N,N-dimethylsphingosine (DMS) was accompanied by a concomitant decrease of bcl-2 expression in both RNA and protein levels in HL-60 cells, while expression of bcl-XL and bax mRNA did not change, and neither sphingosine nor DMS induced differentiation of HL-60 cells. In contrast, in apoptotic cells induced by pharmaceutical PKC inhibitors H7 or staurosporine, expression of bcl-2 did not change nor did the intracellular sphingosine concentration. These results suggest that sphingosine may function as an endogenous mediator of apoptotic signaling in PMA-induced terminal differentiation of HL-60 cells through bcl-2 down-regulation, probably independent from PKC inhibition.

Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation.

The present studies were undertaken to characterize the potential role of sphingosine in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. A 6-h exposure of HL-60 cells to sphingosine or its methylated derivative, N,N-dimethylsphingosine, caused internucleosomal DNA fragmentation and stereotypical morphological changes characteristic of apoptosis (i.e., cell shrinkage, nuclear condensation, and the formation of apoptotic bodies), as well as that to pharmacological inhibitors of protein kinase C such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine. Apoptosis by sphingosine and N,N-dimethylsphingosine was measured using a flow cytometric method. The percentages of apoptotic cells in cultures treated with sphingosine (10 microM) and N,N-dimethylsphingosine (10 microM) for 6 h were 55.6 +/- 7.8% and 84.2 +/- 11.6%, respectively. HL-60 cells were induced to differentiate toward macrophages by treatment with 5 nM 4 beta-phorbol 12-myristate 13-acetate (PMA). Internucleosomal DNA fragmentation, which was a hallmark of apoptosis, was first detected after 10-h exposure to PMA and increased with longer treatment. Sphingosine concentrations in the cells increased concomitantly with the increasing proportion of apoptotic cells during cell differentiation. Sphingosine level in HL-60 cells differentiated by treatment with PMA for 48 h was about 3.3-fold greater than that in untreated cells. Differentiated HL-60 cells exhibited a markedly increased conversion of exogenously added [3H]ceramide to [3H]sphingosine, indicating elevation of ceramidase activity. Moreover, exposure to sphingosine resulted in down-regulation of c-myc mRNA. These observations suggest the possible role of sphingosine in induction of apoptotic DNA fragmentation during PMA-induced differentiation in myeloid leukemia cells. Sphingosine may function as an endogenous modulator mediating the apoptotic signal.

A possible role of sphingosine in induction of apoptosis by tumor necrosis factor-alpha in human neutrophils.

Treatment of human neutrophils with tumor necrosis factor-alpha (TNF-alpha) resulted in an increase in concentration of ceramide and its catabolite, sphingosine. Sphingosine, a potent endogenous protein kinase C (PKC) inhibitor, as well as TNF-alpha, induced internucleosomal DNA fragmentation and morphological changes characteristic of apoptotic cells. Ceramide and sphingosine-1-phosphate, the initial product of sphingosine catabolism, did not cause apoptosis under our experimental conditions. In addition, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and N,N-dimethylsphingosine (DMS), known as PKC inhibitors, also induced apoptosis, suggesting that induction of apoptosis by sphingosine may be related to inhibition of PKC activity. These results indicate that sphingosine deacylated from ceramide may be an endogenous modulator mediating apoptotic signals by TNF-alpha in neutrophils.

Evidence for direct-acting oxidative genotoxicity by reduction products of azo dyes.

The intestinal flora forms a complex ecosystem that metabolizes dietary and endogenous nutrients under primarily anaerobic conditions. The ingestion of azo dyes has been proposed as one source of potential genotoxic agents. Many intestinal bacteria are able to reduce the azo bond (termed azofission), which liberates the substituted naphthol compounds. The standard Ames test has not demonstrated mutagenicity either by various common food colorings or by their reduced end products in Salmonella typhimurium strains TA98 and TA100. In contrast, genetic toxicity was demonstrated in the Escherichia coli differential kill assay and in S. typhimurium TA102 for the reduced dyes. The superoxide free radical was produced by the azo dyes only after reduction by the intestinal bacteria Enterococcus faecalis and Bacteroides thetaiotaomicron.

Treatment approaches for acute mania.

Drug treatment of mania is conceptually divided into mood stabilizers and tranquilizers. Indications for each are defined and a graphical decision tree for treatment of acute mania is presented. The anticonvulsants carbamazepine and valproic acid have an efficacy comparable to that of lithium and work in many lithium-refractory patients. They have not, however, been sufficiently studied in maintenance treatment. Guidelines are presented for the selection of a mood stabilizing agent as well as for combining two mood stabilizers. Lithium-refractoriness, a key concept in determining drug choice, is poorly defined in the literature and requires refinement. Among tranquilizers, neuroleptics are used most frequently, but their use should be minimized. Neuroleptic dosing of manic patients is probably too high and exposes patients to an unnecessary risk of side effects including tardive dyskinesia. In patients with no history of substance abuse, benzodiazepines can be used instead of neuroleptics or in augmentation of neuroleptics which can then be used at lower doses.

Oral complications associated with neonatal oral tracheal intubation: a critical review.

This paper summarizes and evaluates the oral complications associated with orotracheal intubation in neonates. The palatal defect resulting from orotracheal intubation is best described as palatal grooving, rather than clefting since no oral nasal communication has been demonstrated. Palatal grooving may be caused by the inhibition of the molding tongue forces on the lateral palatine shelves. The incidence of palatal grooving increases with duration of intubation and reportedly resolves following extubation. However, posterior cross-bites, high palatal vaults, and poor speech intelligibility have been reported in children who previously have been intubated. Impingement of an orotracheal tube on the alveolus rather than on the palate may cause alveolar grooving which can cause dilaceration of primary teeth. Bilateral linear enamel hypoplasia in premature neonates is caused by an interruption in amelogenesis from intrauterine disturbances. However, gross unilateral incisal enamel hypoplasia in children who have been intubated is probably due to traumatic intubation. Avoiding excessive pressure on the maxillary alveolus during intubation is suggested. An appliance is available which secures oral tubes and protects the palate and alveolus.