Recessive dystrophic epidermolysis bullosa associated with mesangioproliferative glomerulonephritis and multifocal necrotizing leucoencephalopathy of the pons.

We report a woman with recessive dystrophic epidermolysis bullosa (RDEB) in whom there was prolonged sepsis and death at age 22 years. Autopsy revealed multiple epidermolytic skin lesions with chronic ulceration, mesangioproliferative glomerulonephritis and multifocal necrotizing leucoencephalopathy (MNL) of the pons. The latter two conditions may have been mediated by sepsis-associated cytokines. Although mesangioproliferative glomerulonephritis has previously been described in association with RDEB, to our knowledge this is the first report of MNL in a patient with RDEB.

Holoprosencephaly associated with an apparent isolated 2q37.1-->2q37.3 deletion.

A female infant survived 5(1/2) hours after delivery at 33 weeks gestation. Autopsy showed a lobar variant of holoprosencephaly (HPE). Cytogenetic analysis revealed a 2q37.1-->2q37.3 deletion. This case represents the fourth reported case of HPE associated with partial monosomy 2q37 and the first with an apparent isolated 2q37 deletion. Chromosome segment 2q37.1-->2q37.3 may harbor yet another locus important in forebrain development, which, when disrupted, can lead to brain malformations within the HPE spectrum.

Modulation of RTX cytotoxicity by thymidine and dipyridamole in vitro: implications for chemotherapy.

PURPOSE: The cytotoxicity of the thymidylate synthase (TS) inhibitor raltitrexed (RTX) is reversed by supraphysiologic thymidine concentrations. Dipyridamole (DP) blocks thymidine salvage (uptake) and potentiates several chemotherapeutic agents in vitro. The purpose of this study was to determine whether DP is capable of potentiating RTX cytotoxicity in the presence of physiologic thymidine concentrations. METHODS: We examined the effect of DP on RTX cytotoxicity in the presence of various physiologic thymidine concentrations in eight colorectal adenocarcinoma cell lines by colony formation assay, and in p53-defective HL60 S and p53-competent HL60 SN3 promyelocytic leukemia cells by a tetrazolium reduction-based assay. RESULTS: In WiDr colon adenocarcinoma cells the cytotoxicity of 1.0 microM RTX (4 h) varied from 0% to >99% within the reported range of human serum thymidine concentrations from 500 to <50 nM, respectively. DP potentiated RTX cytotoxicity 2- to >93-fold within this range. Free DP concentrations of 10 to 20 nM, achievable with oral dosing, potentiated RTX at thymidine concentrations up to 100 nM. Potentiation at higher physiologic thymidine concentrations of >/=200 nM required DP concentrations of at least 50 nM, or about twice the steady-state DP concentration obtainable from oral and intravenous dosing, but well within that obtainable by intraperitoneal infusion. Maximal potentiation required 72 to 120 h of DP exposure. DP also potentiated RTX in the absence of exogenous thymidine in six of eight colon cell lines and HL60 S and SN3 cells suggesting a second, nonthymidine salvage-dependent mechanism of potentiation. CONCLUSIONS: Clinically achievable free DP concentrations potentiated RTX cytotoxicity within the range of physiologic serum thymidine concentrations. RTX/DP or DP analogue-based combination therapy should, therefore, be considered for clinical trial. Serum or tumor thymidine concentration determinations may aid in identification of patients likely to respond to TS and nucleoside salvage inhibitors versus alternate, non-TS-directed therapies.

Toxic optic neuropathy after concomitant use of melatonin, zoloft, and a high-protein diet.

Melatonin is a neuromodulating hormone found in the pineal gland and retina. It is involved in light-dark circadian rhythms and mediates retinal processes in a manner antagonistic to that of dopamine. Zoloft (sertraline) is an antidepressant drug that blocks the reuptake of serotonin at the neural synapse. Serotonin is the natural precursor of melatonin. A 42-year-old woman sought treatment for visual acuity loss, dyschromatopsia, and altered light adaptation. Neuro-ophthalmologic examination was otherwise normal except for evolving bilateral cecocentral scotomas. She had taken Zoloft for 4 years and began a high-protein diet with melatonin supplementation 2 weeks before onset of visual symptoms. Visual acuity and color vision improved within 2 months after melatonin and the high-protein diet were discontinued. Combined use of melatonin, Zoloft, and a high-protein diet may have resulted in melatonin/dopamine imbalance in the retina, manifesting as a toxic optic neuropathy. Physicians and patients should be alerted to this potential drug interaction.

1H MRS in acute traumatic brain injury.

The objective of this study was to demonstrate 1H MR spectroscopy (MRS) changes in cerebral metabolites after acute head trauma. Twenty-five patients (12 children, 13 adults) were examined with quantitative 1H MRS after closed head injury. Clinical grade (Glasgow Coma Scale [GCS]) and outcome (Rancho Los Amigos Medical Center Outcome Score [ROS]) were correlated with quantitative neurochemical findings. N-acetylaspartate (NAA), a neuronal and axonal marker, was reduced (P < .03-.001). In children, a reduced NAA/creatine plus phosphocreatine (Cr) level and the presence of detectable lipid/lactate predicted bad outcome (sensitivity, 89%; specificity, 89%). The first MRS examination of all patients correlated with ROS versus NAA (r = .65, P < .0001). Although most patients showed MRS abnormalities, striking heterogeneity of 1H MRS characterized the individual patients. 1H MRS identifies multiple patterns of diffuse brain injury after blunt head trauma. There was a strong correlation between MRS and outcome. Future prospective studies will be needed to determine the clinical usefulness of MRS in predicting outcome from closed head injury.

Non-lysosomal cycling pathway for atrial natriuretic peptide activated by protein kinase C in human NPE cells.

Atrial natriuretic peptide (ANP) stimulates aqueous humor formation in primates, but the membrane-bound receptors which mediate this effect have not been well studied in the eye. Endocytosis of [125I]ANP bound to natriuretic peptide C receptors was characterized in fetal human nonpigmented ciliary epithelial (NPE) cells. [125I]ANP which bound to cells at 4 degreesC was detected in the cell interior after a temperature shift to 37 degreesC. Appearance of ligand within the cell peaked at 5 min, and then declined towards zero over 20 min. The endocytosis inhibitor phenylarsine oxide blocked the appearance of internalized ligand, whereas the lysosomotropic drug chloroquine had no effect on internalization but blocked subsequent loss of internalized ligand. Chloroquine also blocked the accumulation of degraded ligand in the extracellular medium. Treatment with phorbol 12-myristate, 13-acetate accelerated the loss of internalized ligand from cells and increased the accumulation of ligand in the extracellular medium. Ligand in the medium was also increased by dioctanoylglycerol but not by 4alpha phorbol didecanoate, an isomer which does not activate protein kinase C. The protein kinase inhibitors staurosporine and bisindolylymaleimide blocked the increase in ligand. Phorbol ester-stimulated loss of internalized ligand occurred in the presence of chloroquine. TCA precipitation of ligand in the extracellular medium showed that both degraded and undegraded [125I]ANP were present. However, in the presence of chloroquine only, undegraded ANP was detected in the medium, and phorbol esters stimulated its rate of appearance by approximately 2 fold. A similar stimulation occurred when cells containing internalized ligand, but stripped of membrane-bound ligand, were exposed to phorbol esters. The data suggest that ANP bound to natriuretic peptide C receptors on NPE cells is endocytosed, and that protein kinase C activates a non-lysosomal pathway for ANP retroendocytosis in these cells.

The stereospecific cytotoxic potency of (6R) and (6S)-5,10- dideazatetrahydrofolate correlates with cellular folylpolyglutamate synthetase levels.

The de novo purine synthesis inhibitor 5,10-dideazatetrahydrofolate (DDATHF) has previously been shown to inhibit the growth of mouse L1210 and human CCRF-CEM leukemia cells. The present study demonstrates that both the 6R and 6S diastereomers of DDATHF are also cytotoxic to mammalian cells in a stereospecific manner. The cytotoxic potency of (6R)-DDATHF (also known as Lometrexol) towards different cell lines varied by approximately 14-fold and that of (6S)-DDATHF by as much as 156-fold. Compared to (6R)-DDATHF, (6S)-DDATHF was 6.0- and 7.2-fold more cytotoxic to human WiDr colon adenocarcinoma and Chinese hamster ovary (CHO) cells, respectively, and only 1.5- and 2.0-fold more cytotoxic to human T24 bladder carcinoma and mouse L1210 leukemia cells, respectively. However, compared to (6S)-DDATHF, (6R)-DDATHF was 8.7- and 6.9-fold more cytotoxic to C3H/10T1/2 clone 8 and clone 16 mouse fibroblasts, respectively. Weak inhibition of aminoimidazolecarboximide ribonucleotide formyltransferase (AICARFT, EC 2.1.2.3) appeared to have little role in the cytotoxicity of DDATHF diastereomers to WiDr cells during a 24-h exposure. Although glycinamide ribonucleotide formyltransferase (GARFT, EC 2.1.21) is the main biochemical target of DDATHF, DDATHF stereoisomers' cytotoxic potency showed no clear negative correlation with cellular GARFT levels. However, cellular folylpolyglutamate synthetase (FPGS, EC 6.3.2.17) levels correlated with cytotoxic potency in a positive manner. Surprisingly, two enzyme-dose/DDATHF LD90-response curves were observed for FPGS corresponding to differences in (6R) and (6S)-DDATHF cytotoxic potency among the six cell lines studied.

Cytotoxicity of antifolate inhibitors of thymidylate and purine synthesis to WiDr colonic carcinoma cells.

We have studied the cytotoxicity of 5,10-dideazatetrahydrofolate (DDATHF) and of D-1694 to human WiDr colonic carcinoma cells as a model system for the effects of pure inhibitors of either the de novo purine synthesis pathway or thymidylate synthesis. The growth of this cell line was inhibited by very low concentrations of either agent and the lethality of DDATHF and D-1694 was completely prevented by continuous exposure to either hypoxanthine or thymidine, respectively, indicating that these compounds were very potent metabolic inhibitors, each specific for one of these pathways. D-1694 was highly cytotoxic (> 3 logs of kill) after a 4-h exposure to 1 microM drug, or a 24-h exposure to very low concentrations (0.04 microM). On the other hand, the cytotoxicity of DDATHF was substantially lower, with 2 logs of cell kill requiring >> 100 microM with 4 h of exposure or approximately 40 microM for 72 h of exposure. Maximal cell kill induced by D-1694 was 5-6 logs, consistent with elimination of all viable cells except preexisting mutants. A maximum of 2-3 logs of cell kill was observed with DDATHF. Exposure of WiDr cells to either D-1694 or DDATHF caused striking cellular changes, but the morphologies of cells treated with the two drugs were remarkably different. D-1694-treated cells detached from the dish within 1-2 days after a megaloblastosis, whereas DDATHF-treated cells remained adherent to the dishes for at least 10 days after treatment. The addition of thymidine to D-1694-treated cultures or hypoxanthine to DDATHF-treated cells after up to 20 h of drug exposure completely prevented cytotoxicity of either drug. With longer exposures, cytotoxicity of both drugs progressively increased in spite of such rescue. Our results indicate that substantial (99-99.9%) tumor cell kill can be induced by a pure inhibitor of purine synthesis, but that the rate of commitment to cell death and the extent of cell kill is greater with a pure inhibitor of thymidylate synthesis.

Study of the ability of phenacetin, acetaminophen, and aspirin to induce cytotoxicity, mutation, and morphological transformation in C3H/10T1/2 clone 8 mouse embryo cells.

Use of the analgesic compounds acetylsalicylic acid (aspirin), phenacetin, and acetaminophen has been correlated with increased risk of renal cancer in humans. Hence, we studied these compounds for ability to induce cytotoxicity, mutation to ouabain resistance, and morphological transformation in cultured C3H/10T1/2 clone 8 (10T1/2) mouse embryo cells. All three compounds were cytotoxic from 0.5-mg/ml to 2-mg/ml concentrations as evidenced by decreased plating efficiency. None of the compounds induced detectable base substitution mutations to ouabain resistance even at cytotoxic concentrations. Aspirin did not induce morphological transformation. Both phenacetin and acetaminophen induced low but concentration-dependent numbers of atypical, weak type II morphologically transformed foci; at equimolar concentrations, phenacetin was 1.1- to 3.0-fold more active in inducing these foci. Neither phenacetin nor acetaminophen was cotransforming with 3-methylcholanthrene, and neither compound promoted cell transformation when added to 3-methylcholanthrene-initiated 10T1/2 cells. The focus-inducing potency of both compounds was increased by addition of an Arochlor-induced hamster liver S9 fraction as an exogenous metabolizing system. However, seven putative metabolites of phenacetin and acetaminophen that were tested--N-hydroxyphenacetin, p-phenetidine, p-aminophenol, p-nitrosophenol, benzoquinone, acetamide, and N-acetyl-p-benzoquinoneimine--were inactive in transformation assays at the concentrations reducing plating efficiency of treated cells to 50% of the plating efficiency of nontreated (control) cells. Several acetaminophen- and phenacetin-induced foci were cloned, expanded into cell lines, and characterized. These cell lines stably formed type II foci when maintained at confluence for 2 to 4 wk in reconstruction experiments with nontransformed 10T1/2 cells; however, they did not exhibit significantly increased saturation density compared to 10T1/2 cells, and they did not grow in soft agarose. These results suggest that metabolic intermediates of high concentrations of phenacetin and acetaminophen induce a low frequency of nonneoplastic morphological transformation of 10T1/2 mouse embryo cells.