Breastfeeding for Primary Prevention of Stroke.

A recent review by Zhang et al. published in the "Journal of the National Medical Association" focused on the major and emerging strategies for the prevention of stroke. We would highlight the evolving role of breastfeeding as an efficient intervention in primary prevention of such cardiovascular disease, including in black women.

"3M": Migraine, Microbiota and Melatonin.

We read with deep interest the recent article published on "Medical Hypotheses" about migraine and associated gastrointestinal disorders. Beyond the exhaustive pathophysiological explanations, we would address some relevant points to the role of melatonin as well as the high potential of intestinal microbiota in such conditions.

Ataxia in children: think about vitamin E deficiency ! (comment on: ataxia in children: early recognition and clinical evaluation).

A recent article from Pavone et al. published in the Italian Journal of Pediatrics entitled «Ataxia in children: early recognition and clinical evaluation¼ made an exhaustive overview of the large spectrum of pediatric ataxias. However, we would underline the importance of considering hereditary ataxia due to isolated vitamin E deficiency as a specific and treatable cause of child ataxia. We present a short clinical and therpeutic synopsis of this peculiar genetic etiology, frequently encountred in the mediterranean region.

Development of a blood-based molecular biomarker test for identification of schizophrenia before disease onset.

Recent research efforts have progressively shifted towards preventative psychiatry and prognostic identification of individuals before disease onset. We describe the development of a serum biomarker test for the identification of individuals at risk of developing schizophrenia based on multiplex immunoassay profiling analysis of 957 serum samples. First, we conducted a meta-analysis of five independent cohorts of 127 first-onset drug-naive schizophrenia patients and 204 controls. Using least absolute shrinkage and selection operator regression, we identified an optimal panel of 26 biomarkers that best discriminated patients and controls. Next, we successfully validated this biomarker panel using two independent validation cohorts of 93 patients and 88 controls, which yielded an area under the curve (AUC) of 0.97 (0.95-1.00) for schizophrenia detection. Finally, we tested its predictive performance for identifying patients before onset of psychosis using two cohorts of 445 pre-onset or at-risk individuals. The predictive performance achieved by the panel was excellent for identifying USA military personnel (AUC: 0.90 (0.86-0.95)) and help-seeking prodromal individuals (AUC: 0.82 (0.71-0.93)) who developed schizophrenia up to 2 years after baseline sampling. The performance increased further using the latter cohort following the incorporation of CAARMS (Comprehensive Assessment of At-Risk Mental State) positive subscale symptom scores into the model (AUC: 0.90 (0.82-0.98)). The current findings may represent the first successful step towards a test that could address the clinical need for early intervention in psychiatry. Further developments of a combined molecular/symptom-based test will aid clinicians in the identification of vulnerable patients early in the disease process, allowing more effective therapeutic intervention before overt disease onset.

Diabetic db/db mice exhibit central nervous system and peripheral molecular alterations as seen in neurological disorders.

The db/db mouse is a widely used preclinical model in diabetes research. Recent studies have shown that these mice also display aspects of psychosis and depression-like behaviors as seen in some psychiatric disorders. Here, we have performed multiplex immunoassay and liquid chromatography mass spectrometry profiling of the plasma and brain samples from db/db and control mice to identify altered pathways, which could be related to these behavioral abnormalities. This is the first study to carry out profiling of the brain proteome in this model. Plasma from the db/db mice had increased levels of leptin and insulin, decreased levels of peptide YY, glucagon and prolactin and alterations in inflammation-related proteins, compared with control mice. Frontal cortex tissue from the db/db mice showed changes in proteins involved in energy metabolism, cellular structure and neural functioning, and the hippocampus had changes in proteins involved in the same pathways, with additional effects on cellular signalling proteins. The overlap of these findings with effects seen in type 2 diabetes, schizophrenia, major depressive disorder and Alzheimer's disease might contribute to a common endophenotype seen in metabolic and neurological disorders.

Identification of proteomic signatures associated with depression and psychotic depression in post-mortem brains from major depression patients.

Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis of post-mortem dorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

Identification of a biological signature for schizophrenia in serum.

Biomarkers are now used in many areas of medicine but are still lacking for psychiatric conditions such as schizophrenia (SCZ). We have used a multiplex molecular profiling approach to measure serum concentrations of 181 proteins and small molecules in 250 first and recent onset SCZ, 35 major depressive disorder (MDD), 32 euthymic bipolar disorder (BPD), 45 Asperger syndrome and 280 control subjects. Preliminary analysis resulted in identification of a signature comprised of 34 analytes in a cohort of closely matched SCZ (n=71) and control (n=59) subjects. Partial least squares discriminant analysis using this signature gave a separation of 60-75% of SCZ subjects from controls across five independent cohorts. The same analysis also gave a separation of ~50% of MDD patients and 10-20% of BPD and Asperger syndrome subjects from controls. These results demonstrate for the first time that a biological signature for SCZ can be identified in blood serum. This study lays the groundwork for development of a diagnostic test that can be used as an aid for distinguishing SCZ subjects from healthy controls and from those affected by related psychiatric illnesses with overlapping symptoms.

Sex-specific serum biomarker patterns in adults with Asperger's syndrome.

Autism spectrum conditions have been hypothesized to be an exaggeration of normal male low-empathizing and high-systemizing behaviors. We tested this hypothesis at the molecular level by performing comprehensive multi-analyte profiling of blood serum from adult subjects with Asperger's syndrome (AS) compared with controls. This led to identification of distinct sex-specific biomarker fingerprints for male and female subjects. Males with AS showed altered levels of 24 biomarkers including increased levels of cytokines and other inflammatory molecules. Multivariate statistical classification of males using this panel of 24 biomarkers revealed a marked separation between AS and controls with a sensitivity of 0.86 and specificity of 0.88. Testing this same panel in females did not result in a separation between the AS and control groups. In contrast, AS females showed altered levels of 17 biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. Classification of females using this biomarker panel resulted in a separation between AS and controls with sensitivities and specificities of 0.96 and 0.83, respectively, and testing this same panel in the male group did not result in a separation between the AS and control groups. The finding of elevated testosterone in AS females confirmed predictions from the 'extreme male brain' and androgen theories of autism spectrum conditions. We conclude that to understand the etiology and development of autism spectrum conditions, stratification by sex is essential.

Impaired glycolytic response in peripheral blood mononuclear cells of first-onset antipsychotic-naive schizophrenia patients.

Little is known about the biological mechanisms underpinning the pathology of schizophrenia. We have analysed the proteome of stimulated and unstimulated peripheral blood mononuclear cells (PBMCs) from schizophrenia patients and controls as a potential model of altered cellular signaling using liquid-chromatography mass spectrometry proteomic profiling. PBMCs from patients and controls were stimulated for 72 h in vitro using staphylococcal enterotoxin B. In total, 18 differentially expressed proteins between first-onset, antipsychotic-naive patients and controls in the unstimulated and stimulated conditions were identified. Remarkably, eight of these proteins were associated with the glycolytic pathway and patient-control differences were more prominent in stimulated compared with unstimulated PBMCs. None of these proteins were altered in chronically ill antipsychotic-treated patients. Non-linear multivariate statistical analysis showed that small subsets of these proteins could be used as a signal for distinguishing first-onset patients from controls with high precision. Functional analysis of PBMCs did not reveal any difference in the glycolytic rate between patients and controls despite increased levels of lactate and the glucose transporter-1, and decreased levels of the insulin receptor in patients. In addition, subjects showed increased serum levels of insulin, consistent with the idea that some schizophrenia patients are insulin resistant. These results show that schizophrenia patients respond differently to PBMC activation and this is manifested at disease onset and may be modulated by antipsychotic treatment. The glycolytic protein signature associated with this effect could therefore be of diagnostic and prognostic value. Moreover, these results highlight the importance of using cells for functional discovery and show that it may not be sufficient to measure protein expression levels in static states.

Expression profiling of fibroblasts identifies cell cycle abnormalities in schizophrenia.

Many previous studies have attempted to gain insight into the underlying pathophysiology of schizophrenia by studying postmortem brain tissues of schizophrenia patients. However, such analyses can be confounded by artifactual features of this approach such as lengthy agonal state and postmortem interval times. As several aspects of schizophrenia are also manifested at the peripheral level in proliferating cell types, we have studied the disorder through systematic transcriptomic and proteomic analyses of skin fibroblasts biopsied from living patients. We performed comparative transcriptomic and proteomic profiling to characterize skin fibroblasts from schizophrenia patients compared to healthy controls. Transcriptomic profiling using cDNA array technology showed that pathways associated with cell cycle regulation and RNA processing were altered in the schizophrenia subjects (n = 12) relative to controls (n = 12). LC-MS(E) proteomic profiling led to identification of 16 proteins that showed significant differences in expression between schizophrenia (n = 11) and control (n = 11) subjects. Analysis in silico revealed that these proteins were also associated with proliferation and cell growth pathways. To validate these findings at the protein level, fibroblast protein extracts were analyzed by Western blotting which confirmed the differential expression of three key proteins associated with these pathways. At the functional level, we confirmed the decreased proliferation phenotype by showing that cultured fibroblasts from schizophrenia subjects (n = 5) incorporated less (3)H-thymidine into their nuclei compared to those from controls (n = 6) by day 4 over an 8 day time course study. Similar abnormalities in cell cycle and growth pathways have been reported to occur in the central nervous system in schizophrenia. These studies demonstrate that fibroblasts obtained from living schizophrenia subjects show alterations in cellular proliferation and growth pathways. Future studies aimed at characterizing such pathways in fibroblasts and other proliferating cell types from schizophrenia patients could elucidate the molecular mechanisms associated with the pathophysiology of schizophrenia and provide a useful model to support drug discovery efforts.

Proteoglycans: pericellular and cell surface multireceptors that integrate external stimuli in the mammary gland.

Proteoglycans consist of a core protein and an associated glycosaminoglycan (GAG) chain of heparan sulfate, chondroitin sulfate, dermatan sulfate or keratan sulfate, which are attached to a serine residue. The core proteins of cell surface proteoglycans may be transmembrane, e.g., syndecan, or GPI-anchored, e.g., glypican. Many different cell surface and matrix proteoglycan core proteins are expressed in the mammary gland and in mammary cells in culture. The level of expression of these core proteins, the structure of their GAG chains, and their degradation are regulated by many of the effectors that control the development and function of the mammary gland. Regulatory proteins of the mammary gland that bind GAG include many growth factors and morphogens (fibroblast growth factors, hepatocyte growth factor/scatter factor, members of the midkine family, wnts), matrix proteins (collagen, fibronectin, and laminin), enzymes (lipoprotein lipase) and microbial surface proteins. Structural diversity within GAG chains ensures that each protein-GAG interaction is as specific as necessary and a number of sequences of saccharides that recognize individual proteins have been elucidated. The GAG-protein interactions serve to regulate the signal output of growth factor receptor tyrosine kinase and hence cell fate as well as the storage and diffusion of extracellular protein effectors. In addition, GAGs clearly coordinate stromal and epithelial development, and they are active participants in mediating cell-cell and cell-matrix interactions. Since a single proteoglycan, even if it carries a single GAG chain, can bind multiple proteins, proteoglycans are also likely to act as multireceptors which promote the integration of cellular signals.

Reversal of type 2 diabetes in mice by products of malaria parasites. II. Role of inositol phosphoglycans (IPGs).

We have previously shown that infection with Plasmodium yoelii malaria or injection of extracts from malaria-parasitized red cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in mice made moderately diabetic with streptozotocin. Inositol phosphoglycans (IPGs) are released outside cells by hydrolysis of membrane-bound glycosylphosphatidylinositols (GPIs), and act as second messengers mediating insulin action. The C57BL/Ks-db/db and C57BL/6J-ob/ob mice offer good models for studies on human obesity and Type 2 diabetes. In the present study, we show that a single iv injection of IPG-A or IPG-P extracted from P. yoelii significantly (P < 0.02) lowers the blood glucose in STZ-diabetic, db/db, and in ob/ob mice for at least 4--6 h. Using rat white adipocytes, IPG-P increased lipogenesis by 20--30% in the presence and absence of maximal concentrations of insulin (10(-8) M) (P < 0.01) and stimulated pyruvate dehydrogenase (PDH) phosphatase in a dose-related manner. Both IPG-A and IPG-P inhibited c-AMP-dependent protein kinase (PKA) in a dose-related manner. Compositional analysis of IPGs after 24 h hydrolysis revealed the presence of myo-inositol, phosphorus, galactosamine, glucosamine, and glucose in both IPG-A and IPG-P. However, hydrolysis of IPGs for 4 h highlighted differences between IPG-A and IPG-P. There are some functional similarities between P. yoelii IPGs and those previously described for mammalian liver. However, this is the first report of the hypoglycemic effect of IPGs in murine models of Type 2 diabetes. We suggest that IPGs isolated from P. yoelii, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes mellitus.

Differential regulation of FGF-1 and -2 mitogenic activity is related to their kinetics of binding to heparan sulfate in MDA-MB-231 human breast cancer cells.

The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth.

Hepatocyte growth factor/scatter factor has distinct classes of binding site in heparan sulfate from mammary cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan sulfate (HS)-binding growth factor and morphogen for mammary epithelial cells that is produced by mammary stromal fibroblasts. HS chains, purified as peptidoglycans from a panel of cell lines representative of the ductal epithelial cell (Huma 123), the myoepithelial cell (Huma 109), the stromal fibroblast (Rama 27), and malignant mammary epithelial cells (MCF-7 and ZR-75), were used in a biosensor-based assay to identify the classes of HGF/SF-binding sites in the polysaccharide chains. At least three distinct binding sites were identified. One site exhibits fast association and fast dissociation kinetics [kass (1.4-7.7) x 10(6) M-1 s-1; kdiss 0. 0032-0.0096 s-1] and is present on the HS from benign Huma 123 epithelial cells, Huma 109 myoepithelial-like cells, and ZR-75 malignant cells. The second binding site, found on HS from the malignant MCF-7 cells, has slower HGF/SF-binding kinetics (kass 0.20 x 10(6) M-1 s-1; kdiss 0.00055 s-1). The third binding site possesses fast association and slow dissociation kinetics (kass 1.1 x 10(6) M-1 s-1; kdiss 0.00020 s-1) and was found on the HS isolated from the culture medium of the Huma 123 benign epithelial cells. The first and second binding sites have a similar Kd, 1-3 nM, while the third binding site has a considerably higher affinity for HGF/SF (Kd 200 pM). The three binding sites seem to be mutually exclusive, since each sample of HS possessed just one of the sites.

Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate.

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.

Hepatocyte growth factor/scatter factor binds with high affinity to dermatan sulfate.

We have demonstrated by affinity chromatography that hepatocyte growth factor/scatter factor (HGF/SF) binds strongly to dermatan sulfate (DS), with a similar ionic strength dependence to that previously seen with heparan sulfate (HS). Analysis of binding kinetics on a biosensor yields an equilibrium dissociation constant, KD, of 19.7 nM. This corresponds to a 10-100-fold weaker interaction than that with HS, primarily due to a faster dissociation rate of the complex. The smallest DS oligosaccharide with significant affinity for HGF/SF by affinity chromatography appears to be an octasaccharide. A sequence comprising unsulfated iduronate residues in combination with 4-O-sulfated N-acetylgalactosamine is sufficient for high affinity binding. The presence of 2-O-sulfation on the iduronate residues does not appear to be inhibitory. These observations concur with our previous suggestions, from analyses of HS binding (Lyon, M., Deakin, J. A., Mizuno, K., Nakamura, T., and Gallagher, J.T. (1994) J. Biol. Chem. 269, 11216-11223), that N-sulfation of hexosamines and 2-O-sulfation of iduronates are not absolute requirements for glycosaminoglycan binding to HGF/SF. This is the first described example of a high affinity interaction between a growth factor and DS, and is likely to have significant implications for the biological activity of this paracrine-acting factor.

State of airway surface liquid on guinea pig trachea.

Two preparations of the guinea pig trachea have been examined: an isolated preparation and a preparation in vivo, both exposed to air on the mucosal surface. Ion-selective microelectrodes have been used to measure Na (alpha Na) and K activities (alpha K) in airway surface liquid (ASL) while airflows tending either to evaporate or to condense water were applied. Other variables measured included ASL depth and transepithelial potential difference (TEPD). In isolated preparations, condensation did not progressively alter depth, alpha Na, or TEPD but caused a slight increase in alpha K. Evaporation decreased depth; increased alpha Na, alpha K, and osmotic pressure; and changed TEPD. Measurements on preparations in vivo broadly supported these observations. In addition, the depth of ASL developed on isolated preparations was related to the humidity of the air to which animals had been previously exposed. We conclude that condensation and evaporation at the ASL-air interface in isolated preparations and in preparations in vivo do significantly modify key ASL variables as does the relative humidity of the air to which animals are exposed before experimentation.