High risk and low prevalence diseases: Metformin toxicities.

INTRODUCTION: Metformin toxicity is a rare but serious condition that carries with it a high rate of morbidity and mortality. OBJECTIVE: This review highlights the pearls and pitfalls of metformin toxicity, including diagnosis, initial resuscitation, and management in the emergency department (ED) based on current evidence. DISCUSSION: Metformin is a common medication used for treatment of diabetes mellitus. Metformin toxicity is a spectrum of conditions that may be differentiated into three subgroups: metformin-associated lactic acidosis (MALA), metformin-induced lactic acidosis (MILA), and metformin-unrelated lactic acidosis (MULA). MILA is a condition found predominantly in patients chronically taking metformin or those with large acute overdoses. Conversely, MULA occurs in patients on metformin but with a critical illness stemming from a separate cause. MALA is rare but the most severe form, with mortality rates that reach 50%. Differentiating these entities is difficult in the ED setting without obtaining metformin levels. Patients with metformin toxicity present with nonspecific gastrointestinal symptoms and vital sign abnormalities. Laboratory analysis will reveal a high lactate with anion gap metabolic acidosis. Patients presenting with elevated lactate levels in the setting of metformin use should be considered at risk for the most severe form, MALA. Patients with MALA require aggressive treatment with intravenous fluids, treatment of any concomitant condition, and early consideration of hemodialysis, along with specialist consultation such as nephrology and toxicology. CONCLUSIONS: An understanding of metformin toxicity can assist emergency clinicians in diagnosing and managing this potentially deadly disease.

Tetanus: A Rare Complication of Black Tar Heroin Use.

Tetanus is associated with high morbidity and mortality, although this is rarely encountered in high-income countries. We present a case of tetanus in an unvaccinated patient secondary to black tar heroin use that highlights the importance of considering tetanus in appropriate clinical contexts, harm reduction interventions, and universal tetanus vaccination campaigns.

Substrate Reduction Therapy for Sandhoff Disease through Inhibition of Glucosylceramide Synthase Activity.

Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of disease-associated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivo imaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay (∼28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity (∼17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.

In Vivo Optical Imaging of Myelination Events in a Myelin Basic Protein Promoter-Driven Luciferase Transgenic Mouse Model.

The compact myelin sheath is important for axonal function, and its loss can lead to neuronal cell death and irreversible functional deficits. Myelin is vulnerable to a variety of metabolic, toxic, and autoimmune insults. In diseases like multiple sclerosis, there is currently no therapy to stop myelin loss, underscoring the need for neuroprotective and remyelinating therapies. Noninvasive, robust techniques are also needed to confirm the effect of such therapies in animal models. This article describes the generation, characterization, and potential uses for a myelin basic protein-luciferase (MBP-luci) transgenic mouse model, in which the firefly luciferase reporter gene is selectively controlled by the MBP promoter. In vivo bioluminescence imaging can be used to visualize and quantify demyelination and remyelination at the transcriptional level, noninvasively, and in real time. Transgenic mice were assessed in the cuprizone-induced model of demyelination, and luciferase activity highly correlated with demyelination and remyelination events as confirmed by both magnetic resonance imaging and postmortem histological analysis. Furthermore, MBP-luci mice demonstrated enhanced luciferase signal and remyelination in the cuprizone model after treatment with a peroxisome proliferator activated receptor-delta selective agonist and quetiapine. Imaging sensitivity was further enhanced by using CycLuc 1, a luciferase substrate, which has greater blood-brain barrier penetration. We demonstrated the utility of MBP-luci model in tracking myelin changes in real time and supporting target and therapeutic validation efforts.

Multifunctional, High Molecular Weight, Post-Translationally Modified Proteins through Oxidative Cysteine Coupling and Tyrosine Modification.

Glycoproteins and their mimics are challenging to produce via chemical or biological methods because of their long protein backbones and large number of polysaccharide side chains that form a densely grafted protein-polysaccharide brush architecture. Herein, we demonstrate a new approach to protein bioconjugate synthesis that can approach the molar mass and functionalization densities of natural glycoproteins such as mucins and aggrecans. In this method, a tyrosine-enriched protein sequence is engineered and synthesized in E. coli, and sugars or other functional moieties can be efficiently and polyvalently grafted to the backbone through tyrosine modification chemistry. Cysteine residues on the chain ends are used for oxidative chain polymerization into high molar mass chains larger than can be easily expressed in the host. The effects of tyrosine-enrichment and cysteine-incorporation on the physical and expression properties on a model protein are explored. Elastin-like peptides (ELPs) are chosen because of their high expression yields, repetitive sequence, substitutable amino acids, and well-studied physical properties. The sequence modifications to mimic glycoproteins are shown to affect the maximum length of expressible sequence but not yield. The tyrosine modification chemistry is shown to functionalize up to 73% of all tyrosines on the peptide, and the scope of functional groups that can be mass conjugated to proteins is expanded through multistep conjugation strategies involving copper(I)-catalyzed alkyne-azide cycloaddition showing up to 97% alkyne functionalization. All of the functionalization chemistries preserve the ability to polymerize the backbone.

Rapamycin Inhibition of mTOR Reduces Levels of the Na+/H+ Exchanger 3 in Intestines of Mice and Humans, Leading to Diarrhea.

BACKGROUND & AIMS: The immunosuppressant rapamycin frequently causes noninfectious diarrhea in organ transplant recipients. We investigated the mechanisms of this process. METHODS: We performed a retrospective analysis of renal transplant recipients treated with rapamycin from 2003 through 2010 at Albany Medical College, collecting data on serum levels of rapamycin. Levels of the Na+/H+ exchanger 3 (NHE3) were measured in human ileal biopsy specimens from patients who did and did not receive rapamycin (controls), in ileum tissues from rats or mice given rapamycin, and in mice with intestine-specific disruption of mammalian target of rapamycin (Mtor) (mTOR(f/f):Villin-cre mice) or Atg7 (Atg7(flox/flox); Villin-Cre). Exchange activity and intestinal water absorption were measured using a pH-sensitive dye and small intestine perfusion, respectively. RESULTS: Episodes of noninfectious diarrhea occurred in organ recipients after increases in serum levels of rapamycin. The expression of NHE3 was reduced in the ileal brush border of patients with diarrhea. In rats and mice, continuous administration of low doses of rapamycin reduced levels of NHE3 in intestinal tissues; this effect was not observed in mice with intestinal deletion of ATG7, indicating that autophagy is required for the reduction. Administration of single high doses of rapamycin to mice, to model the spikes in rapamycin levels that occur in patients with severe diarrheal episodes, resulted in reduced phosphorylation of S6 and AKT in ileal tissues, indicating inhibition of the mTOR complex (mTORC1 and mTORC2). The intestines of mice with intestine-specific deletion of mTOR were dilated and contained large amounts of liquid stools; they also had reduced levels of total NHE3 and NHERF1 compared with control mice. We observed a significant reduction in Na(+)/H(+) exchange activity in ileum tissues from these mice. CONCLUSIONS: Rapamycin inhibition of mTOR reduces levels of NHE3 and Na(+)/H(+) exchange activity in intestinal tissues of patients and rodents. This process appears to require the autophagic activity mediated by ATG7. Loss of mTOR regulation of NHE3 could mediate the development of diarrhea in patients undergoing rapamycin therapy.

The use of optical imaging to assess the potential for embryo-fetal exposure to an exogenous material after intravaginal administration.

A ß-actin-luc transgenic mouse model was used to evaluate whether embryo-fetal exposure could occur after intravaginal administration of a compound. A bioluminescent substrate, d-luciferin, was delivered intravaginally to mimic compound exposure to the female reproductive track and the embryo-fetus. Bioluminescence was observed throughout the reproductive tract during diestrus, but not during estrus, 2-5min after intravaginal d-luciferin administration to female ß-actin-luc mice. Intravaginal administration of d-luciferin to wild-type females mated with male ß-actin-luc mice indicated that the substrate reached the developing embryo-fetus, with bioluminescence corresponding to transgene expression in the embryo-fetus. d-Luciferin substrate rapidly reached the embryo-fetus regardless of the administration route (intravaginal, intraperitoneal, subcutaneous, or intravenous). Vaginal ligation appeared to block at least some direct exposure to the embryo-fetus, but did not prevent d-luciferin from eventually reaching the embryo-fetus. Additional work will be necessary to form the basis for a reliable assessment of the human risk for male-mediated teratogenicity.

Mephedrone, a new designer drug of abuse, produces acute hemodynamic effects in the rat.

Mephedrone (4-methylmethcathinone) is a new and popular drug of abuse widely available on the Internet and still legal in some parts of the world. Clinical reports are now emerging suggesting that the drug displays sympathomimetic toxicity on the cardiovascular system but no studies have yet explored its cardiovascular effects. Therefore we examined the effects of mephedrone on the cardiovascular system using a combination of in vitro electrophysiology and in vivo hemodynamic and echocardiographic measurements. Patch clamp studies revealed that mephedrone, up to 30 µM, had little effect on the major voltage-dependent ion channels of the heart or on action potentials recorded in guinea pig myocytes. Subcutaneous administration of mephedrone (3 and 15 mg/kg) to conscious telemetry-implanted rats produced dose-dependent increases in heart rate and blood pressure which persisted after pre-treatment with reserpine. Echocardiographic analysis demonstrated that intravenous injection of mephedrone (0.3 and 1mg/kg) increased cardiac function, including cardiac output, ejection fraction, and stroke volume, similar to methamphetamine (0.3mg/kg). We conclude that mephedrone is not directly pro-arrhythmic, but induces substantial increases in heart rate, blood pressure and cardiac contractility and this activity contributes to the cardiovascular toxicity in people who abuse the drug.

Crosstalk between integrin αvß3 and estrogen receptor-α is involved in thyroid hormone-induced proliferation in human lung carcinoma cells.

A cell surface receptor for thyroid hormone that activates extracellular regulated kinase (ERK) 1/2 has been identified on integrin αvß3. We have examined the actions of thyroid hormone initiated at the integrin on human NCI-H522 non-small cell lung carcinoma and NCI-H510A small cell lung cancer cells. At a physiologic total hormone concentration (10(-7) M), T(4) significantly increased proliferating cell nuclear antigen (PCNA) abundance in these cell lines, as did 3, 5, 3'-triiodo-L-thyronine (T(3)) at a supraphysiologic concentration. Neutralizing antibody to integrin αvß3 and an integrin-binding Arg-Gly-Asp (RGD) peptide blocked thyroid hormone-induced PCNA expression. Tetraiodothyroacetic acid (tetrac) lacks thyroid hormone function but inhibits binding of T(4) and T(3) to the integrin receptor; tetrac eliminated thyroid hormone-induced lung cancer cell proliferation and ERK1/2 activation. In these estrogen receptor-α (ERα)-positive lung cancer cells, thyroid hormone (T(4)>T(3)) caused phosphorylation of ERα; the specific ERα antagonist ICI 182,780 blocked T(4)-induced, but not T(3)-induced ERK1/2 activation, as well as ERα phosphorylation, proliferating-cell nuclear antigen (PCNA) expression and hormone-dependent thymidine uptake by tumor cells. Thus, in ERα-positive human lung cancer cells, the proliferative action of thyroid hormone initiated at the plasma membrane is at least in part mediated by ERα. In summary, thyroid hormone may be one of several endogenous factors capable of supporting proliferation of lung cancer cells. Activity as an inhibitor of lung cancer cell proliferation induced at the integrin receptor makes tetrac a novel anti-proliferative agent.

Molecular basis for certain neuroprotective effects of thyroid hormone.

The pathophysiology of brain damage that is common to ischemia-reperfusion injury and brain trauma include disodered neuronal and glial cell energetics, intracellular acidosis, calcium toxicity, extracellular excitotoxic glutamate accumulation, and dysfunction of the cytoskeleton and endoplasmic reticulum. The principal thyroid hormones, 3,5,3'-triiodo-l-thyronine (T(3)) and l-thyroxine (T(4)), have non-genomic and genomic actions that are relevant to repair of certain features of the pathophysiology of brain damage. The hormone can non-genomically repair intracellular H(+) accumulation by stimulation of the Na(+)/H(+) exchanger and can support desirably low [Ca(2+)](i.c.) by activation of plasma membrane Ca(2+)-ATPase. Thyroid hormone non-genomically stimulates astrocyte glutamate uptake, an action that protects both glial cells and neurons. The hormone supports the integrity of the microfilament cytoskeleton by its effect on actin. Several proteins linked to thyroid hormone action are also neuroprotective. For example, the hormone stimulates expression of the seladin-1 gene whose gene product is anti-apoptotic and is potentially protective in the setting of neurodegeneration. Transthyretin (TTR) is a serum transport protein for T(4) that is important to blood-brain barrier transfer of the hormone and TTR also has been found to be neuroprotective in the setting of ischemia. Finally, the interesting thyronamine derivatives of T(4) have been shown to protect against ischemic brain damage through their ability to induce hypothermia in the intact organism. Thus, thyroid hormone or hormone derivatives have experimental promise as neuroprotective agents.