Quantitative MRI reveals heterogeneous impacts of treatment on diseased bone marrow in a mouse model of myelofibrosis.

PURPOSE: Analyzing bone marrow in the hematologic cancer myelofibrosis requires endpoint histology in mouse models and bone marrow biopsies in patients. These methods hinder the ability to monitor therapy over time. Preclinical studies typically begin treatment before mice develop myelofibrosis, unlike patients who begin therapy only after onset of disease. Using clinically relevant, quantitative MRI metrics allowed us to evaluate treatment in mice with established myelofibrosis. METHODS: We used chemical shift-encoded fat imaging, DWI, and magnetization transfer sequences to quantify bone marrow fat, cellularity, and macromolecular components in a mouse model of myelofibrosis. We monitored spleen volume, the established imaging marker for treatment, with anatomic MRI. After confirming bone marrow disease by MRI, we randomized mice to treatment with an approved drug (ruxolitinib or fedratinib) or an investigational agent, navitoclax, for 33 days. We measured the effects of therapy over time with bone marrow and spleen MRI. RESULTS: All treatments produced heterogeneous responses with improvements in bone marrow evident in subsets of individual mice in all treatment groups. Reductions in spleen volume commonly occurred without corresponding improvement in bone marrow. MRI revealed patterns associated with effective and ineffective responses to treatment in bone marrow and identified regional variations in efficacy within a bone. CONCLUSIONS: Quantitative MRI revealed modest, heterogeneous improvements in bone marrow disease when treating mice with established myelofibrosis. These results emphasize the value of bone marrow MRI to assess treatment in preclinical models and the potential to advance clinical trials for patients.

Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor-Positive Breast Cancer.

Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor-positive (ER+) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER+ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER+ breast cancer and overcome resistance to standard clinical therapies. IMPLICATIONS: This study reveals how MSCs reprogram metabolism of ER+ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.

Multiparametric MRI to quantify disease and treatment response in mice with myeloproliferative neoplasms.

Histopathology, the standard method to assess BM in hematologic malignancies such as myeloproliferative neoplasms (MPNs), suffers from notable limitations in both research and clinical settings. BM biopsies in patients fail to detect disease heterogeneity, may yield a nondiagnostic sample, and cannot be repeated frequently in clinical oncology. Endpoint histopathology precludes monitoring disease progression and response to therapy in the same mouse over time, missing likely variations among mice. To overcome these shortcomings, we used MRI to measure changes in cellularity, macromolecular constituents, and fat versus hematopoietic cells in BM using diffusion-weighted imaging (DWI), magnetization transfer, and chemical shift-encoded fat imaging. Combining metrics from these imaging parameters revealed dynamic alterations in BM following myeloablative radiation and transplantation. In a mouse MPLW515L BM transplant model of MPN, MRI detected effects of a JAK2 inhibitor, ruxolitinib, within 5 days of initiating treatment and identified differing kinetics of treatment responses in subregions of the tibia. Histopathology validated the MRI results for BM composition and heterogeneity. Anatomic MRI scans also showed reductions in spleen volume during treatment. These findings establish an innovative, clinically translatable MRI approach to quantify spatial and temporal changes in BM in MPN.

Targeting disseminated estrogen-receptor-positive breast cancer cells in bone marrow.

Estrogen receptor-positive (ER+) breast cancer can recur up to 20 years after initial diagnosis. Delayed recurrences arise from disseminated tumors cells (DTCs) in sites such as bone marrow that remain quiescent during endocrine therapy and subsequently proliferate to produce clinically detectable metastases. Identifying therapies that eliminate DTCs and/or effectively target cells transitioning to proliferation promises to reduce risk of recurrence. To tackle this problem, we utilized a 3D co-culture model incorporating ER+ breast cancer cells and bone marrow mesenchymal stem cells to represent DTCs in a bone marrow niche. 3D co-cultures maintained cancer cells in a quiescent, viable state as measured by both single-cell and population-scale imaging. Single-cell imaging methods for metabolism by fluorescence lifetime (FLIM) of NADH and signaling by kinases Akt and ERK revealed that breast cancer cells utilized oxidative phosphorylation and signaling by Akt to a greater extent both in 3D co-cultures and a mouse model of ER+ breast cancer cells in bone marrow. Using our 3D co-culture model, we discovered that combination therapies targeting oxidative phosphorylation via the thioredoxin reductase (TrxR) inhibitor, D9, and the Akt inhibitor, MK-2206, preferentially eliminated breast cancer cells without altering viability of bone marrow stromal cells. Treatment of mice with disseminated ER+ human breast cancer showed that D9 plus MK-2206 blocked formation of new metastases more effectively than tamoxifen. These data establish an integrated experimental system to investigate DTCs in bone marrow and identify combination therapy against metabolic and kinase targets as a promising approach to effectively target these cells and reduce risk of recurrence in breast cancer.

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Short-Term Environmental Conditioning Enhances Tumorigenic Potential of Triple-Negative Breast Cancer Cells.

Tumor microenvironments expose cancer cells to heterogeneous, dynamic environments by shifting availability of nutrients, growth factors, and metabolites. Cells integrate various inputs to generate cellular memory that determines trajectories of subsequent phenotypes. Here we report that short-term exposure of triple-negative breast cancer cells to growth factors or targeted inhibitors regulates subsequent tumor initiation. Using breast cancer cells with different driver mutations, we conditioned cells lines with various stimuli for 4 hours before implanting these cells as tumor xenografts and quantifying tumor progression by means of bioluminescence imaging. In the orthotopic model, conditioning a low number of cancer cells with fetal bovine serum led to enhancement of tumor-initiating potential, tumor volume, and liver metastases. Epidermal growth factor and the mTORC1 inhibitor ridaforolimus produced similar but relatively reduced effects on tumorigenic potential. These data show that a short-term stimulus increases tumorigenic phenotypes based on cellular memory. Conditioning regimens failed to alter proliferation or adhesion of cancer cells in vitro or kinase signaling through Akt and ERK measured by multiphoton microscopy in vivo, suggesting that other mechanisms enhanced tumorigenesis. Given the dynamic nature of the tumor environment and time-varying concentrations of small-molecule drugs, this work highlights how variable conditions in tumor environments shape tumor formation, metastasis, and response to therapy.

Verification of the Cepheid Xpert Carba-R assay for the detection of carbapenemase genes in bacterial isolates cultured on alternative solid culture media.

The Xpert Carba-R (Cepheid) is a polymerase chain reaction (PCR) assay for detection and differentiation of five common carbapenemase genes. If used according to manufacturer's instructions, bacterial isolates tested must be cultured on blood or MacConkey agar. This study confirms that the assay performs well against a diverse panel of bacterial isolates with known carbapenemase genes. It also demonstrates that the assay performs well using three solid agar culture media that have not been recommended by the PCR assay's manufacturer: CLED (cystine-, lactose-, and electrolyte-deficient), ChromID CARBA-SMART, and a nutrient slope. By testing isolates directly from any of these media, delays in laboratory reporting can be avoided.

External prolonged electrocardiogram monitoring in unexplained syncope and palpitations: results of the SYNARR-Flash study.

AIMS: SYNARR-Flash study (Monitoring of SYNcopes and/or sustained palpitations of suspected ARRhythmic origin) is an international, multicentre, observational, prospective trial designed to evaluate the role of external 4-week electrocardiogram (ECG) monitoring in clinical work-up of unexplained syncope and/or sustained palpitations of suspected arrhythmic origin. METHODS AND RESULTS: Consecutive patients were enrolled within 1 month after unexplained syncope or palpitations (index event) after being discharged from emergency room or hospitalization without a conclusive diagnosis. A 4-week ECG monitoring was obtained by external high-capacity loop recorder (SpiderFlash-T(®), Sorin) storing patient-activated and auto-triggered tracings. Diagnostic monitorings included (i) conclusive events with reoccurrence of syncope or palpitation with concomitant ECG recording (with/without arrhythmias) and (ii) events with asymptomatic predefined significant arrhythmias (sustained supraventricular or ventricular tachycardia, advanced atrio-ventricular block, sinus bradycardia <30 b.p.m., pauses >6 s). SYNARR-Flash study enrolled 395 patients (57.7% females, 56.9 ± 18.7 years, 28.1% with syncope, and 71.9% with palpitations) from 10 European centres. For syncope, the 4-week diagnostic yield was 24.5%, and predictors of diagnostic events were early start of recording (0-15 vs. >15 days after index event) (OR 6.2, 95% CI 1.3-29.6, P = 0.021) and previous history of supraventricular arrhythmias (OR 3.6, 95% CI 1.4-9.7, P = 0.018). For palpitations, the 4-week diagnostic yield was 71.6% and predictors of diagnostic events were history of recurrent palpitations (P < 0.001) and early start of recording (P = 0.001). CONCLUSION: The 4-week external ECG monitoring can be considered as first-line tool in the diagnostic work-up of syncope and palpitation. Early recorder use, history of supraventricular arrhythmia, and frequent previous events increased the likelihood of diagnostic events during the 4-week external ECG monitoring.

Respiratory viral infections during the 2009-2010 winter season in Central England, UK: incidence and patterns of multiple virus co-infections.

Acute viral respiratory infections are the most common infections in humans. Co-infection with different respiratory viruses is well documented but not necessarily well understood. The aim of this study was to utilise laboratory data from the winter season following the 2009 influenza A(H1N1) outbreak to investigate rates of respiratory virus co-infections, virus prevalence in different age groups and temporal variations in virus detection. The Health Protection Agency Public Health Laboratory (HPA PHL) Birmingham, UK, routinely uses polymerase chain reaction (PCR) to detect common respiratory viruses. The results from specimens received for respiratory virus investigations from late September 2009 to April 2010 were analysed. A total of 4,821 specimen results were analysed. Of these, 323 (13.2 %) had co-detections of two viruses, 22 (0.9 %) had three viruses and four (0.2 %) had four viruses. Reciprocal patterns of positive or negative associations between different virus pairs were found. Statistical analysis confirmed the significance of negative associations between influenza A and human metapneumovirus (HMPV), and influenza A and rhinovirus. Positive associations between parainfluenza with rhinovirus, rhinovirus with respiratory syncytial virus (RSV) and adenovirus with rhinovirus, parainfluenza and RSV were also significant. Age and temporal distributions of the different viruses were typical. This study found that the co-detection of different respiratory viruses is not random and most associations are reciprocal, either positively or negatively. The pandemic strain of influenza A(H1N1) was notable in that it was the least likely to be co-detected with another respiratory virus.

Viral respiratory infections during the 2009 influenza A(H1N1) outbreak in the West Midlands Region, UK.

In spring 2009 a new strain of influenza A(H1N1) emerged and caused a worldwide pandemic. This study utilized a large collection of respiratory specimens from suspected cases of influenza A(H1N1) in the UK West Midlands during the pandemic in order to investigate which other respiratory viruses were circulating and whether they played any role in the increased hospitalization rates seen during that period. Study specimens were selected from community and hospitalized patients positive and negative for influenza A(H1N1) and tested by PCR for other respiratory viruses. A number of infections diagnosed as influenza during the summer influenza outbreak were found to be due to other virus infections (most commonly rhinovirus). No statistically significant difference was found between the rates of respiratory virus co-infection with H1N1 in patients from community or hospital locations suggesting underlying factors were likely to be more significant than viral co-infections in determining severity of influenza A(H1N1) disease.

Extended multilocus variable-number tandem-repeat analysis of Clostridium difficile correlates exactly with ribotyping and enables identification of hospital transmission.

PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.

Cytokine activation and disease progression in patients with stable moderate chronic heart failure.

BACKGROUND: Activation of the cytokine and the complement system is associated with disease progression in severe congestive heart failure (CHF). Magnitude and prognostic relevance of cytokine and complement activation remain uncertain in patients with moderate CHF. OBJECTIVES: Measurement of cytokine and complement activation in patients with moderate CHF and testing whether C-reactive protein (CRP) can serve as a surrogate marker of their activation, adding independent prognostic information when co-measured with B-type natriuretic peptide (BNP). METHODS: The 118 study participants were separated into three groups based on pre-determined CRP and BNP levels: Group I (n = 27; CRP > 5 mg/liter, BNP > or = 200 pg/ml); Group II (n = 46; CRP < or = 5 mg/liter, BNP > or = 200 pg/ml); and Group III (n = 45; CRP < or = 5 mg/liter, BNP < 200 pg/ml). RESULTS: Mortality was high in Group I (30%; log-rank p < 0.001) but low in Groups II and III (2% and 4%, respectively; log rank, p = 0.7). No differences were observed for left ventricular ejection fraction (LVEF) and left ventricular end-diastolic diameter (LVEDD) between Groups I and II (31 +/- 16 vs 32 +/- 14% and 66 +/- 16 vs 65 +/- 11 mm, respectively), whereas in Group III LVEF was higher (42 +/- 17%, p = 0.002) with smaller LVEDD (57 +/- 13 mm, p = 0.012). Cytokine sCD14 and tumor necrosis factor (TNF)-alpha levels were not different between the three groups. However, interleukin-6 levels (9.75 +/- 8.17 pg/ml, p = 0.001) and the terminal complement complex C5b-9 (109.9 +/- 68 ng/ml; p = 0.04) were elevated in Group I, both correlating with CRP (interleukin-6: r = 0.5, p < 0.001; C5b-9: r = 0.41, p = 0.001). CONCLUSIONS: CRP may be used as a surrogate parameter for interleukin-6 and complement activation in moderate CHF. CRP in combination with BNP identifies a high-risk group with a tendency for poor outcome not discriminated by cardiac function.

Radiofrequency ablation of accessory pathways. Contemporary success rates and complications in 323 patients.

INTRODUCTION: 17 years ago the first radiofrequency catheter ablation of an accessory pathway (AP) was performed. The aim of this study was to describe the contemporary success rates and procedure related complication rates of radiofrequency (RF) ablation of accessory pathways (APs). In addition, the present study describes the anatomical distribution of APs according to the new nomenclature introduced by NASPE and ESC in 1999. METHODS: The analysis included all patients, who underwent RF ablation of an AP in the Heart Center Leipzig between January 2000 and December 2003. RESULTS: Over a 4 year period 336 APs were ablated in 323 patients. 201 APs (60%) presented with antegrade and retrograde conduction and showed preexcitation on ECG. For the remaining 135 APs (40%), only retrograde conduction over the AP was documented. According to the new nomenclature APs were classified as left-sided, right sided, septal and paraseptal APs. 188 APs (56%) were located on the left, 41 (12%) on the right, 64 (19%) in the paraseptal space and 31 APs (9%) presented with a septal or parahisian localization, respectively. Because of atypical course and/or characteristics 12 APs (4%) could not be classified. Ablation of all pathways were successful in 315 patients (98%). In 289 patients (89%) success was achieved within a single ablation session. The left-sided pathways had a re-intervention rate of 5%, which was significantly lower compared to the remaining localizations. The highest re-intervention rate was observed in the septal APs (23%). Complications were observed in less than 2% of all treated patients. CONCLUSIONS: 17 years after the first RF catheter ablation of an AP this therapy is established as a highly effective procedure. The success rate has improved to 98% and the complication rate has been minimized to less than 2%. The most frequent localization of APs is left posterior. Left sided APs also presented with the lowest re-intervention rate. The introduction of the new nomenclature in 1999 by NASPE and ESC has simplified the description of the exact anatomical localization of an AP.

Interaction of melanin-concentrating hormone (MCH), neuropeptide E-I (NEI), neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells.

Melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between alpha-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125l]alpha-MSH and [125I]NEI as radioligands and bioassays were performed with MCI-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of alpha-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, >300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]alpha-MSH displacement from mouse MC1-R were 50,000-fold and >200,000-fold higher than that of alpha-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [1251]alpha-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 microM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.

A new vascular Endostaple: a technical description.

OBJECTIVE: The purpose of this report is to describe a new vascular Endostapling system. METHODS: The vascular Endostapling system can be passed through a 13F insertion sheath that is inserted through the femoral artery. An optical fiber and overlying Endostaple will penetrate a previously inserted endoprosthesis and the aortic wall at whatever points are desired. Once the optical fiber is withdrawn, the Endostaple assumes its preformed shape and acts like a through-and-through wire suture. As tissue ingrowth proceeds, the long-term security and stabilization of the coiled coil mechanism are likely to increase. CONCLUSIONS: We think Endostaples can be useful in preventing endograft migration and in treating endoleak at the site of the aortic neck-proximal endograft interface.

Modulation of Na/K-ATPase activity by isoproterenol and propranolol in human non-pigmented ciliary epithelial cells.

PURPOSE: The present study investigates whether beta-adrenoreceptor agents such as isoproterenol and propranolol can regulate Na(+)-K(+)-ATPase activity in cultured human non-pigmented ciliary epithelial cells. METHODS: Human non-pigmented ciliary epithelial cells (ODM2) were grown to confluence. The active ion transport mediated by the Na(+)-K(+)-ATPase was evaluated by measuring ouabain-sensitive rubidium (Rb+) uptake. In a first set of experiments, cells were exposed to the beta-adrenoreceptor agonist isoproterenol (0.01-10 microM). In a second set of experiments, cells were exposed to isoproterenol (1 microM) in the presence of different concentrations of the beta-adrenoreceptor antagonist propranolol (0.01, 0.1, 1 microM). RESULTS: In a concentration-dependent manner, isoproterenol induced an increase in Na(+)-K(+)-ATPase activity. The maximal Na(+)-K(+)-ATPase activity was observed at a concentration of 1 microM of isoproterenol (283 +/- 58%, P < 0.001). The increase in Na(+)-K(+)-ATPase activity evoked by isoproterenol (1 microM) was inhibited. In a concentration dependent manner, by propranolol (maximum: 659 +/- 39 vs. 141 +/- 42 pM/mg protein/min, P < 0.01). CONCLUSION: The beta-adrenoreceptor agents isoproterenol and propranolol are apparently able to modulate Na(+)-K(+)-ATPase activity in cultured human non-pigmented ciliary epithelial cells.

Expression of the melanin-concentrating hormone receptor in porcine and human ciliary epithelial cells.

PURPOSE: To evaluate whether the receptors for melanin-concentrating hormone (MCH) and its functional antagonist alpha-melanocyte-stimulating hormone (alpha-MSH) are expressed in the ciliary epithelium. Furthermore, to examine whether MCH, a neuropeptide involved in fluid and electrolyte homeostasis, may influence ion flux mediated by Na,K (adenosine triphosphatase)-ATPase in a ciliary epithelial cell line. METHODS: Expression of MCH receptors (MCH-R) and alpha-MSH receptors (MSH-R) on primary porcine ciliary pigmented epithelial (PE) cells and on a human nonpigmented ciliary epithelial (NPE) cell line, ODM-2 was investigated by radioligand binding studies and reverse transcription-polymerase chain reaction (RT-PCR). The MCH-R was further characterized by photocrosslinking. Influence of MCH on Na, K-ATPase activity was evaluated by an Rb(+) transport assay. RESULTS: MCH-R expression was observed at both the mRNA and protein levels in PE and NPE cells. In contrast, MSH-Rs were not detectable. At the mRNA level, expression of slc-1 was shown and with crosslinking, a 44-kDa protein was labeled. MCH showed no effect on Na,K-ATPase activity of NPE cells. CONCLUSIONS: The presence of MCH-R in ciliary epithelial cells of both human and porcine origin but the absence of MSH-Rs indicates that in these cells, MCH and alpha-MSH do not form a functionally antagonistic hormonal pair as they do in several other systems. Although effects of MCH on intestinal water and ion transport have been documented, a direct control of Na,K-ATPase activity was not detected in human NPE cells in vitro.

Thought disorder index of Finnish adoptees and communication deviance of their adoptive parents.

BACKGROUND: Diverse forms of thought disorder, as measured by the Thought Disorder Index (TDI), are found in many conditions other than schizophrenia. Certain thought disorder categories are primarily manifest during psychotic schizophrenic episodes. The present study examined whether forms of thought disorder qualify as trait indicators of vulnerability to schizophrenia in persons who are not clinically ill, and whether these features could be linked to genetic or environmental risk or to genotype-environment interactions. The Finnish Adoptive Study of Schizophrenia provided an opportunity to disentangle these issues. METHODS: Rorschach records of Finnish adoptees at genetic high risk but without schizophrenia-related clinical diagnoses (N = 56) and control adoptees at low genetic risk (N = 95) were blindly and reliably scored for the Thought Disorder Index (TDI). Communication deviance (CD), a measure of the rearing environment, was independently obtained from the adoptive parents. RESULTS: The differences in total TDI between high-risk and control adoptees were not statistically significant. However, TDI subscales for Fluid Thinking and Idiosyncratic Verbalization were more frequent in high-risk adoptees. When Rorschach CD of the adoptive rearing parents was introduced as a continuous predictor variable, the odds ratio for the Idiosyncratic Verbalization component of the TDI of the high-risk adoptees was significantly higher than for the control adoptees. CONCLUSIONS: Specific categories of subsyndromal thought disorder appear to qualify as vulnerability indicators for schizophrenia. Genetic risk and rearing-parent communication patterns significantly interact as a joint effect that differentiates adopted-away offspring of schizophrenic mothers from control adopted-away offspring.

(D-(p-benzoylphenylalanine)13, tyrosine19)-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking.

A photoreactive analogue of human melanin-concentrating hormone was designed, [D-Bpa13,Tyr19-MCH, containing the D-enantiomer of photolabile p-benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and PEG-PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [D-Bpa13,Tyr19]-MCH at its Tyr19 residue was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and HPLC. Saturation binding analysis of [125I]-[D-Bpa13,Tyr19]-MCH with G4F-7 mouse melanoma cells gave a K(D) of 2.2+/-0.2 x 10(-10) mol/l and a B(max) of 1047+/-50 receptors/cell. Competition binding analysis showed that MCH and rANF(1-28) displace [125I]-[D-Bpa13,Tyr19]-MCH from the MCH binding sites on G4F-7 cells whereas alpha-MSH has no effect. Receptor crosslinking by UV-irradiation of G4F-7 cells in the presence of [125I]-[D-Bpa13,Tyr19]-MCH followed by SDS-polyacrylamide gel electrophoresis and autoradiography yielded a band of 45-50 kDa. Identical crosslinked bands were also detected in B16-F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS-7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [125I]-[D-Bpa13,Tyr19]-MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein-coupled receptor, most likely with a varying degree of glycosylation.