Ibudilast attenuates doxorubicin-induced cytotoxicity by suppressing formation of TRPC3 channel and NADPH oxidase 2 protein complexes.

BACKGROUND AND PURPOSE: Doxorubicin is a highly effective anticancer agent but eventually induces cardiotoxicity associated with increased production of ROS. We previously reported that a pathological protein interaction between TRPC3 channels and NADPH oxidase 2 (Nox2) contributed to doxorubicin-induced cardiac atrophy in mice. Here we have investigated the effects of ibudilast, a drug already approved for clinical use and known to block doxorubicin-induced cytotoxicity, on the TRPC3-Nox2 complex. We specifically sought evidence that this drug attenuated doxorubicin-induced systemic tissue wasting in mice. EXPERIMENTAL APPROACH: We used the RAW264.7 macrophage cell line to screen 1,271 clinically approved chemical compounds, evaluating functional interactions between TRPC3 channels and Nox2, by measuring Nox2 protein stability and ROS production, with and without exposure to doxorubicin. In male C57BL/6 mice, samples of cardiac and gastrocnemius muscle were taken and analysed with morphometric, immunohistochemical, RT-PCR and western blot methods. In the passive smoking model, cells were exposed to DMEM containing cigarette sidestream smoke. KEY RESULTS: Ibudilast, an anti-asthmatic drug, attenuated ROS-mediated muscle toxicity induced by doxorubicin treatment or passive smoking, by inhibiting the functional interactions between TRPC3 channels and Nox2, without reducing TRPC3 channel activity. CONCLUSIONS AND IMPLICATIONS: These results indicate a common mechanism underlying induction of systemic tissue wasting by doxorubicin. They also suggest that ibudilast could be repurposed to prevent muscle toxicity caused by anticancer drugs or passive smoking.

Hepatocyte-Specific Deletion of TIPARP, a Negative Regulator of the Aryl Hydrocarbon Receptor, Is Sufficient to Increase Sensitivity to Dioxin-Induced Wasting Syndrome.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3-/-) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice given a single injection of 10 µg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3-/- mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

Changes in body composition and muscle attenuation during taxane-based chemotherapy in patients with metastatic breast cancer.

PURPOSE: Body composition parameters including low muscle mass, muscle attenuation (which reflects muscle quality) and adipose tissue measurements have emerged as prognostic factors in cancer patients. However, knowledge regarding the possibility of excessive muscle loss during specific systemic therapies is unknown. We describe the changes in body composition and muscle attenuation (MA) during taxane- and anthracycline-based regimens and its association with overall survival (OS) in metastatic breast cancer patients. METHODS: The lumbar skeletal muscle index (LSMI) was used as marker of muscle mass. LSMI, MA, subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and intramuscular adipose tissue (IMAT) were measured before and after first-line treatment with paclitaxel (n = 73) or 5-fluorouracil-doxorubicin-cyclophosphamide (FAC) (n = 25) using CT-images. Determinants of the change of LSMI and MA were analyzed using multiple linear regression. OS was assessed using Cox proportional hazard models. RESULTS: MA significantly decreased during paclitaxel treatment (- 0.9 HU, p = 0.03). LSMI (p = 0.40), SAT (p = 0.75), VAT (p = 0.84) and IMAT (p = 0.10) remained stable. No significant alterations in body composition parameters during FAC-treatment were observed. Previous (neo-)adjuvant chemotherapy contributed to larger loss of MA during the current treatment. Body composition changes during chemotherapy were not associated with OS. CONCLUSIONS: MA decreased during treatment with paclitaxel, while muscle mass was stable. Body composition changes are not associated with survival in the absence of progressive disease.

Mitochondria: Inadvertent targets in chemotherapy-induced skeletal muscle toxicity and wasting?

Chemotherapy has been associated with increased mitochondrial reactive oxygen species production, mitochondrial dysfunction and skeletal muscle atrophy leading to severe patient clinical complications including skeletal muscle fatigue, insulin resistance and wasting. The exact mechanisms behind this skeletal muscle toxicity are largely unknown, and as such co-therapies to attenuate chemotherapy-induced side effects are lacking. Here, we review the current literature describing the clinical manifestations and molecular origins of chemotherapy-induced myopathy with a focus on the mitochondria as the target organelle via which chemotherapeutic agents establish toxicity. We explore the likely mechanisms through which myopathy is induced, using the anthracycline doxorubicin, and the platinum-based alkylating agent oxaliplatin, as examples. Finally, we recommend directions for future research and outline the potential significance of these proposed directions.

Central Diabetes Insipidus and Cisplatin-Induced Renal Salt Wasting Syndrome: A Challenging Combination.

We describe a 2-year-old female with a suprasellar primitive neuroectodermal tumor and central diabetes insipidus (DI) who developed polyuria with natriuresis and subsequent hyponatremia 36 hr after cisplatin administration. The marked urinary losses of sodium in combination with a negative sodium balance led to the diagnosis of cisplatin-induced renal salt wasting syndrome (RSWS). The subsequent clinical management is very challenging. Four weeks later she was discharged from ICU without neurological sequela. The combination of cisplatin-induced RSWS with DI can be confusing and needs careful clinical assessment as inaccurate diagnosis and management can result in increased neurological injury.

Cushing syndrome and giant sterile abscess induced by self intramuscular injection of supra-therapeutic doses of triamcinolone.

OBJECTIVE: Corticosteroid are among the most commonly used medications for a wide range of inflammatory, autoimmune and neoplastic disorders. Therefore, it is important for the physician to be aware of their side effects, in relation to different forms and delivery, that occur more often with oral therapy. METHODS: A 33-year old professional drummer had been suffering from bilateral tenosynovitis of the wrists for three months. In order to accelerate the recovery and play drums again, he self-injected supra-therapeutic doses of triamcinolone acetonide (a total of injected drug > 2,400 mg was calculated) in his right gluteus. RESULTS: The intramuscular overuse of triamcinolone acetonide caused Cushing syndrome associated with a giant sterile abscess that involved the right gluteal muscles and the adjacent subcutaneous tissue. CONCLUSION: A large intramuscular sterile abscess can be a side effect of supra-therapeutic intramuscular triamcinolone dose and this should be explained to the patient prior to initiation of treatment with triamcinolone.

Selenium-binding protein 1: its physiological function, dependence on aryl hydrocarbon receptors, and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

BACKGROUND: Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1. METHODS: To address this issue, we generated Selenbp1-null [Selenbp1 (-/-)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model. RESULTS: Selenbp1 (-/-) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (-/-) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (-/-) and (+/+) mice. CONCLUSIONS: The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity. GENERAL SIGNIFICANCE: Selenbp1 (-/-) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.

Dietary zinc mediates inflammation and protects against wasting and metabolic derangement caused by sustained cigarette smoke exposure in mice.

In mouse asthma models, inflammation can be modulated by zinc (Zn). Given that appetite loss, muscle wasting and poor nutrition are features of chronic obstructive pulmonary disease (COPD) and that poor dietary Zn intake is in itself accompanied by growth retardation and appetite loss, we hypothesised that dietary Zn limitation would not only worsen airway inflammation but also exaggerate metabolic effects of cigarette smoke (CS) exposure in mice. Conversely, Zn supplementation would lessen inflammation. Mice were exposed to CS [2× 2RF, 3×/day; 15 min/cigarette] and fed diets containing 2, 20 or 140 mg/kg Zn ad libitum. Airway cells were collected by bronchoalveolar lavage (BAL). Plasma Zn was measured by fluorometric assay. Inflammatory, metabolic and Zn transport markers were measured by real-time RT-PCR. Mice fed low Zn diets had less plasma labile zinc (0-0.18 µM) than mice fed moderate (0.61-0.98 µM) or high (0.77-1.1 µM) Zn diets (SDs 0.1-0.4, n = 8-10). Smoke exposure increased plasma and BAL labile Zn (1.5-2.5 fold, P < 0.001), bronchoalveolar macrophages (2.0 fold, P < 0.0001) and MT-1 (1.5 fold), MIP-2 (2.3 fold) and MMP-12 (3.5 fold) mRNA. Zn supplementation reduced alveolar macrophage numbers by 62 and 52% in sham and smoke-exposed mice, respectively (Zn effect: P = 0.011). Gastrocnemius, soleus and tibialis anterior muscle mass were affected by both smoke and dietary Zn in the order of 3-7%. The 50-60% reduction in alveolar macrophages in Zn-supplemented mice supports our evolving hypothesis that Zn is an important anti-inflammatory mediator of airway inflammation. Restoring airway Zn levels through dietary supplementation may lessen the severity of lung inflammation when Zn intake is low.

Association of skeletal muscle wasting with treatment with sorafenib in patients with advanced renal cell carcinoma: results from a placebo-controlled study.

PURPOSE Effects of specific antineoplastic therapies on progression of cancer-associated wasting remain uncharacterized. We selected a targeted therapy, sorafenib, because of its reported association with weight loss. PATIENTS AND METHODS Patients with metastatic renal cell cancer (RCC) who were resistant to standard therapy (N = 80) received sorafenib 400 mg twice daily or placebo in a randomized, double-blinded clinical trial. Computed tomography image analysis, which has high precision and specificity for evaluation of specific muscles and adipose tissues, was used to define change in total skeletal muscle and adipose tissue. Results At inclusion, 51% of patients were overweight or obese (ie, body mass index [BMI] > 25 kg/m(2)). Only 5% were underweight. Advanced muscle wasting (ie, sarcopenia) was present in 72% of patients with BMI less than 25 and in 34% of those with a BMI greater than 25. Patients received placebo for an average of 6 months and received sorafenib for 1 year. Patients in the placebo group had stable body weight during 6 months (0.8 kg +/- 0.7 kg), with no significant alteration of muscle or fat. Patients who received sorafenib lost 2.1 kg +/- 0.6 kg (P < .01) in 6 months and lost 4.2 kg +/- 0.7 kg (P < .01) by 1 year. Sorafenib-treated patients lost skeletal muscle progressively at 6 months (decrease of 4.9%; P < .01) and 12 months (decrease of 8.0%; P < .01). CONCLUSION Sarcopenia is prevalent in patients with metastatic RCC and is an occult condition in patients with normal or high BMI. Muscle loss is specifically exacerbated by sorafenib, consistent with the evidence for a role of kinases in regulating muscle mass. Muscle loss is a sorafenib adverse effect that may relate to asthenia, fatigue, and physical disability.

UCP3 in muscle wasting, a role in modulating lipotoxicity?

UCP3 has been postulated to function in the defense against lipid-induced oxidative muscle damage (lipotoxicity). We explored this hypothesis during cachexia in rats (zymosan-induced sepsis), a condition characterized by increased oxidative stress and supply of fatty acids to the muscle. Muscle UCP3 protein content was increased 2, 6 and 11 days after zymosan injection. Plasma FFA levels were increased at day 2, but dropped below control levels on days 6 and 11. Muscular levels of the lipid peroxidation byproduct 4-hydroxy-2-nonenal (4-HNE) were increased at days 6 and 11 in zymosan-treated rats, supporting a role for UCP3 in modulating lipotoxicity during cachexia.

Muscle-specific expression of IGF-1 blocks angiotensin II-induced skeletal muscle wasting.

Advanced congestive heart failure is associated with activation of the renin-angiotensin system and skeletal muscle wasting. We previously showed that angiotensin II infusion in rats produces cachexia secondarily to increased muscle proteolysis and also decreases levels of circulating and skeletal muscle IGF-1. Here we show that angiotensin II markedly downregulates phospho-Akt and activates caspase-3 in skeletal muscle, leading to actin cleavage, an important component of muscle proteolysis, and to increased apoptosis. These changes are blocked by muscle-specific expression of IGF-1, likely via the Akt/mTOR/p70S6K signaling pathway. We also demonstrate that mRNA levels of the ubiquitin ligases atrogin-1 and muscle ring finger-1 are upregulated in angiotensin II-infused WT, but not in IGF-1-transgenic, mice. These findings strongly suggest that angiotensin II downregulation of IGF-1 in skeletal muscle is causally related to angiotensin II-induced wasting. Because the renin-angiotensin system is activated in many catabolic conditions, our findings have broad implications for understanding mechanisms of skeletal muscle wasting and provide a rationale for new therapeutic approaches.

Alterations of plasma ghrelin levels in rats with lipopolysaccharide-induced wasting syndrome and effects of ghrelin treatment on the syndrome.

Ghrelin not only strongly stimulates GH secretion, but is also involved in energy homeostasis by stimulating food intake and promoting adiposity through a GH-independent mechanism. These effects of ghrelin may play an important role in the pathophysiology of inflammatory wasting syndrome, in which both the somatotropic axis and energy balance are altered. In this study we investigated plasma ghrelin concentrations after lipopolysaccharide (LPS) administration to rats, a model of the wasting syndrome and critical illness. In addition, the therapeutic potential of the antiwasting effects of ghrelin was explored using LPS-injected rats. A single LPS injection suppressed plasma ghrelin levels 6 and 12 h later. Maximal reduction was observed 12 h after LPS injection, in a dose-dependent manner. In contrast, plasma ghrelin levels were elevated after repeated LPS injections on d 2 and 5. Peripheral administration of ghrelin twice daily (10 nmol/rat) for 5 d increased body weight gain in repeated LPS-injected rats. Furthermore, both adipose tissue weight and plasma leptin concentrations were increased after ghrelin administration in these rats. In conclusion, plasma ghrelin levels are altered in LPS-injected rats, and ghrelin treatment may provide a new therapeutic approach to the wasting syndrome and critical illness.

Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn's disease.

BACKGROUND & AIMS: Crohn's disease (CD) is a chronic debilitating disease of unknown etiology that is characterized by severe inflammation of the colon. Vasoactive intestinal peptide (VIP) has recently emerged as a promising candidate for treatment of inflammatory Th1-driven diseases. We studied the effect of VIP in trinitrobenzene sulfonic acid (TNBS)-induced colitis, which has clinical and molecular features in common with CD. METHODS: A 3-mg enema of TNBS was given to BALB/c mice, and VIP (1 nmol) was given either as a single dose at 12 hours or every other day. Weight loss, histopathology, and chemokine and cytokine levels in serum and colon extracts were assessed. VIP was also tested given 5 days after the onset of TNBS-induced colitis, and its effect was analyzed given a second dose of TNBS. RESULTS: Treatment with VIP reduced the clinical and histopathologic severity of TNBS-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of VIP were associated with down-regulation of both inflammatory and Th1-driven autoimmune responses, including tumor necrosis factor alpha, interleukin 1, and interleukin 6 in colon extracts and serum as well as interferon gamma by splenic and lamina propria CD4(+) T cells. VIP reduced disease severity when given after disease onset and dramatically reduced disease recurrence given a second dose of TNBS. CONCLUSIONS: Our data suggest that VIP has beneficial prophylactic and therapeutic effects in TNBS-induced colitis and is a promising candidate to test for potential benefits in CD.

The use of c-src knockout mice for the identification of the main toxic signaling pathway of TCDD to induce wasting syndrome.

The effect of single intraperitoneal injection of 115 microg/kg of TCDD (i.e., approximately 1/2 of LD50) to male C57BL/6 mice on the liver mRNA expression changes of several growth factor related genes was assessed at 3 h, 24 h, 10 days, and 30 days posttreatment. The results revealed that the most consistently elevated mRNAs during the entire test period were those of c-Src, TGFalpha, and PDGFa. In contrast, those observed to be consistently suppressed were mRNAs for EGF receptor (EGFR), Ki-Ras, SAPKK, Sp-1, C/EBPbeta, and NFkB. Elevation of mRNAs for TGFbeta and STAT3 was observed only on day 10 and day 30. To assess the role of c-Src in the above action of TCDD, we conducted a parallel study with congenic C57BL/6 male c-src -/- mice. The results showed that in scr -/- mice the effect of TCDD was less in the case of mRNA expression of PDGF(AA), STAT3, C/EPBbeta, NMT-1, and AP-2gamma in addition to c-src as compared to scr +/+ mice. Those affected least by the absence of c-Src were SAPKK, and surprisingly, EGF receptor mRNAs, both of which were consistently downregulated in both strains. In most of the other cases, the extent of TCDD-induced changes were generally less pronounced in src -/- mice as compared to +/+ mice. These observations support the notion that c-Src is an important mediator of the effects of TCDD on TGFalpha, PDGF(AA), and C/EBPalpha, beta.

Interaction of estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature male chickens (Gallus domesticus).

The interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and estrogen was studied in chickens to more clearly define this relationship in an avian species and its role in the enhanced sensitivity of female chickens to TCDD-induced wasting syndrome. Twenty male chickens (7-9 weeks old) were divided evenly into four groups: control (CTL, received the same volume of vehicle); estrogen-treated (E2, 1 mg/kg estradiol cypionate injections on days 1, 2 and 3); TCDD-treated (TCDD, single 50 microg/kg injection on day 4); and estrogen plus TCDD (E2+TCDD, as above), with measurements taken on day 14. The E2 group compared with the CTL group had decreased comb height (24%), comb length (26%) and adipose tissue (AT) lipoprotein lipase (LPL) activity relative to AT mass (51%), while liver mass and body weight gain were each increased by 28%. The TCDD group had increased liver mass (62%), reduced comb length (17%), and reduced AT LPL activity indexed to AT mass (70%) compared with the CTL group. Finally, the E2+TCDD group had 37% lower body weight gain and 30% larger livers relative to body mass compared with the E2 group, but were not significantly different from the TCDD group. These data show that TCDD antagonized several effects of exogenous estrogen in male chickens, while estrogen enhanced TCDD toxicity in a tissue-specific manner.

Angiotensin II induces skeletal muscle wasting through enhanced protein degradation and down-regulates autocrine insulin-like growth factor I.

We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decreases circulating insulin-like growth factor I (IGF-I). The weight loss derives from an anorexigenic response and a catabolic effect of ang II. In these experiments we assessed potential catabolic mechanisms and the involvement of the IGF-I system in these responses to ang II. Ang II infusion caused a significant decrease in body weight compared with that of pair-fed control rats. Kidney and left ventricular weights were significantly increased by ang II, whereas fat tissue was unchanged. Skeletal muscle mass was significantly decreased in the ang II-infused rats, and a reduction in lean muscle mass was a major reason for their overall loss of body weight. In skeletal muscles, ang II did not significantly decrease protein synthesis, but overall protein breakdown was accelerated; inhibiting lysosomal and calcium-activated proteases did not reduce the ang II-induced increase in muscle proteolysis. Circulating IGF-I levels were 33% lower in ang II rats vs. control rats, and this difference was reflected in lower IGF-I messenger RNA levels in the liver. Moreover, IGF-I, IGF-binding protein-3, and IGF-binding protein-5 messenger RNAs in the gastrocnemius were significantly reduced. To investigate whether the reduced circulating IGF-I accounts for the loss in muscle mass, we increased circulating IGF-I by coinfusing ang II and IGF-I, but this did not prevent muscle loss. Our data suggest that ang II causes a loss in skeletal muscle mass by enhancing protein degradation probably via its inhibitory effect on the autocrine IGF-I system.

Strain-dependent cytokine profile and susceptibility to oleic acid anilide in a murine model of the toxic oil syndrome.

The toxic oil syndrome (TOS) was caused by the ingestion of an adulterated rapeseed oil containing oleic acid anilide (OAA). It was characterized by lethal symptoms in the acute phase and by symptoms of idiopathic autoimmune diseases in the chronic phase. The pathogenetic mechanisms remain unclear. In a murine model of TOS we demonstrate strain-dependent effects on the immune system after treatment with OAA intraperitoneally. While C57BL/6 (H-2b) mice develop a polyclonal B cell activation without disease symptoms, most A/J (H-2a) mice suffer an acute lethal wasting disease. These differences are reflected in the splenic cytokine gene expression and secretion and in the Ig production. Increased IgE serum levels and reduced TNF-beta mRNA suggest a Th2 cell response in C57BL/6 mice. In A/J mice, splenocytes express IL-1alpha, IL-10, and IFN-gamma mRNA in vivo and secrete high levels of TNF-alpha in vitro. These observations resemble the human condition in TOS with development of either an acute lethal disease or a chronic autoimmune-like disease. As in other chemical-induced reactions genetic susceptibility seems to be important.

Physiological effects of CNTF-induced wasting.

The authors have shown previously that in addition to its survival effects on neurons and glia, ciliary neurotrophic factor (CNTF) induced potent cachectic effects and acute phase proteins when present in the peripheral circulation at concentrations of < or = 10 ng/ml. These effects did not depend upon the induction of other cytokine family members. Described here are the specific physiological effects which systemic administration of CNTF can induce in somatic tissue. Mice implanted with C6 glioma cells, genetically modified to secrete CNTF, exhibited rapid catabolism of adipose tissue and skeletal muscle, depressed steady-state levels of glucose and triglycerides, elevations in red blood cell content, gall bladder hypertrophy and thymic atrophy, with a disproportionate loss of CD4+/CD8+ T cells. This cachectic wasting resulted in death over a period of 7-10 days. Implantation of the parental C6 line, or C6 cells which express a non-secreted form of CNTF, did not result in overt effects over this time period. These findings have implications both for the biology of CNTF family members, and the therapeutic use of factors such as CNTF in vivo.