Archivi del mese: febbraio 2014

MIMIC: metodi alternativi per la valutazione dei vaccini e mancata predittività dei modelli animali

[Byers AM, Tapia TM, Sassano ER, Wittman V. In vitro antibody response to tetanus in the MIMIC system is a representative measure of vaccine immunogenicity. Biologicals. 2009 Jun;37(3):148-51.]

human variety

Abstract:

In response to the recurrent failure of animal vaccine protection studies to accurately predict human trial results, we have developed a fully human modular immune in vitro construct (MIMIC) to serve as a preliminary screen for efficacy testing of potential vaccine formulations. To validate the potential of this approach, we monitored the in vitro-generated tetanus (TT)-specific antibody levels in a cohort of donors before and after receiving tetanus vaccination. Purified CD4_T cell and B cell populations were combined with autologous tetanus vaccine-pulsed dendritic cells to generate specific antibody. Enumeration of TT-specific IgG antibody-secreting cells by ELISPOT displayed a significant increase in the magnitude of this population after vaccination. The relative magnitudes of the in vitro-generated TT-specific antibody response before and after vaccination largely recapitulated the TT-specific IgG serum titer profiles measured in the same individuals. These findings provide evidence that the MIMIC system can be a rapid and representative in vitro method for measuring vaccine immunogenicity via induction of the memory B cell response.

Generalizzabilità delle scoperte sui modelli animali nello studio del cervello ed evoluzione del cervelletto nei primati

[Rilling JK, Insel TR. Evolution of the cerebellum in primates: differences in relative volume among monkeys, apes and humans. Brain Behav Evol. 1998;52(6):308-14.]

Abstract:

According to the ‘developmental constraint hypothesis’ of comparative mammalian neuroanatomy, brain structures enlarge predictably as the entire brain grows both ontogenetically and phylogenetically. In this study, brain and cerebellum volumes are measured from in vivo magnetic resonance scans of 44 primates from 11 haplorhine species. After controlling for overall brain volume, the cerebellum in both pongid and hylobatid apes is, on average, 45% larger than in monkeys. These results demonstrate that all primate brains are not similarly organized and that developmental constraints are not tight enough to preclude selection for increased cerebellar volume independent of selection on overall brain size.

Uomo e animale: differenze negli studi di neuroscienze cognitive

[Premack D. Human and animal cognition: continuity and discontinuity. Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):13861-7. Epub 2007 Aug 23.]

Full Text: http://www.pnas.org/content/104/35/13861.full

Abstract:

Microscopic study of the human brain has revealed neural structures, enhanced wiring, and forms of connectivity among nerve cells not found in any animal, challenging the view that the human brain is simply an enlarged chimpanzee brain. On the other hand, cognitive studies have found animals to have abilities once thought unique to the human. This suggests a disparity between brain and mind. The suggestion is misleading. Cognitive research has not kept pace with neural research. Neural findings are based on microscopic study of the brain and are primarily cellular. Because cognition cannot be studied microscopically, we need to refine the study of cognition by using a different approach. In examining claims of similarity between animals and humans, one must ask: What are the dissimilarities? This approach prevents confusing similarity with equivalence. We follow this approach in examining eight cognitive cases—teaching, short-term memory, causal reasoning, planning, deception, transitive inference, theory of mind, and language—and find, in all cases, that similarities between animal and human abilities are small, dissimilarities large. There is no disparity between brain and mind.

Conclusioni:

Animal competencies are mainly adaptations restricted to a single goal. Human competencies are domain-general and serve numerous goals. For instance “planning” may be tied to episodic memory, suggesting a broad competence. However, if episodic memory is confined to one (or only a few) activities, planning itself will be a narrow competence. Differences in the evolutionary origins of animal and human abilities help explain why the one is tied to a single goal, and the other to indeterminately many goals.

In humans, teaching did not evolve in the context of food seeking (by stalking or coping with toxic food) as it apparently did in animals; but in a far broader context arguably involving TOM, language, and aesthetics. In causal reasoning, animal limitations are of a different kind: The sense of cause may originate in personal actions that result in a desirable or undesirable outcome. In the human, the sense graduates to impersonal actions: a rock that falls on a plant crushing it, a wind that blows out a flame, etc., to events that humans recognize as causal. Does the animal make the human transition, or does its sense of cause remain tied to personal action? This question awaits decisive evidence. Further, because animals have no concept of monotonic order, although “transitive inference” is widely found in animals, it cannot be based on logic or reasoning.

The broad range of cognitive cases, which includes teaching, causal reasoning, short-term memory, planning, TOM, etc., consistently shows fundamental limitations in the animal version of the human competence. There is no anomaly in the disparity—the disparity between human and animal cognition is compatible with the disparity between human and animal brain. The coming challenge is to understand the function of the cellular-level differences between human and animal brain. Work linking these neural changes to cognitive processes can now move forward.

Nel testo:

The fact that adaptations have a single target distinguishes teaching by animals from teaching by humans. Human teaching is not an adaptation. It is a domain-general competence with indeterminately many targets. Further, the targets of teaching differ in every culture. Toilet training and table manners are widely taught in the western countries, whereas among the Kalahari San, walking and sitting are the key activities taught to the young (12).

Human teaching consists of three distinct actions: observation, judgement, and modification. A teacher observes the novice, judges his actions or products, and modifies them when they fall short of her standards. The human recognizes that the young are incompetent and therefore need to be taught; has the technology with which to teach; and is motivated to teach by deeply rooted aesthetic standards. Each of these actions has a distinct cognitive source.

The recognition that competence develops with age humans owe to their TOM: It enables them to both differentiate the mental conditions of other individuals, and to analyze the factors, such as age, intelligence, experience, etc., that cause the differences (1318).

Humans can teach or modify the other one because they are both language-competent and expert in passive guidance (placing other’s body in desired positions).

The human motivation to teach is largely aesthetic (1920). A parent has a conception of a proper act or product and dislikes the appearance of an improper one. The evidence for such standards is twofold. First, humans “practice,” e.g., swing a golf club repeatedly, flip an omelet, sing a song, write a poem, etc., trying to improve their performance of a chosen activity. Second, humans seek to improve their appearance. The mirror is where they begin their day, combing their hair, applying makeup, etc. That humans have mental representations of preferred actions or appearances is suggested not only by the demands they make on themselves but by the corrections they make of children when teaching them. Teaching, the attempt to correct others, is the social side of the attempt to correct self.

It is no coincidence that humans both practice and teach, whereas other species do neither. A species that practices but does not teach—that corrects itself but does not correct others—will probably never be found. Nor will a species of the opposite kind, one that teaches but does not practice—corrects others but not itself. […]

Although short-term memory limits the number of units one can remember, it does not define the content of a unit. A language-trained chimpanzee exposed to the numbers 1–9 might remember, for example: 2, 6, 4, 3, 7, whereas humans might remember, for example, 21, 43, 96…; 214, 618, 109… ; 1012, 6680, 3456, etc. In language, content of the unit is even more open-ended. A chimpanzee may remember five words; a human may remember five phrases, five sentences, five stories, etc. Humans are able to make these expansions because they are capable of both recursive language and numbers. Thus, despite comparable limitations in short-term memory, animals and humans may differ dramatically in the amount of information they can remember. In addition, humans can, and often do, circumvent short-term memory with written language. Similar limitations in different species may have entirely different consequences depending on the other cognitive resources of the species (22). […]

An animal may recognize that a large rock is more likely to break a branch than a small one. But if the animal observes a large rock lying by a crushed plant, will it infer that the rock crushed the plant? There is no evidence that it will. The understanding of physical action is not the equivalent of
causal reasoning. […]

Although further research may reveal more development than is presently recognized, the cognitive elaboration leading to causal reasoning appears to be lacking in animals. […]

Complex planning differs from simple planning in these respects. It is social: two or more individuals form the plan, and the beneficiary of the plan is likely to be yet another individual, different from those who form the plan; the plan is not one-shot, but a series of plans; the plan extends not for hours but over years. Neither social nor sequential planning, nor planning that extends over long durations, is likely to be found in animals. […]

Whether the plover’s act is goal-directed could be determined by arranging two cases, one in which its display leads intruders away and another in which its displays do not succeed in leading intruders away. If, when the displays fail, the bird ceases to make them, the act is intentional. For intentional acts that fail to realize their goal extinguish. However, neither the potential intentionality of the plover’s display nor the fact that the plover can discriminate real intruders from fake ones changes the status of the display. It is an adaptation that serves only one goal. It is not comparable with human deception, a domain-general competence that can serve indeterminately many goals. […]

How is it possible for species that lack the concept of monotonicity to do transitive inference? They probably use a hard-wired mechanism and do not do it as humans do. Earlier, we suggested that animals are not capable of causal reasoning. How can they have a hard-wired mechanism for one kind of reasoning but not the other? Causal reasoning is more complex than transitive inference, involving not one simple inference, like transitive inference, but many inferences. Evolving a simple hard-wired mechanism is therefore less likely. […]

The advanced function most clearly associated with the reorganization of the human brain is complex social cognition. No less than language, it distinguishes humans from animals. […]

Chimpanzee mothers do not recognize that their infants lack knowledge and cannot therefore, for example, crack nuts with rocks. Therefore, they do not teach them. Chimpanzees do not have the concept of knowledge, do not distinguish a knowing individual from an ignorant one, and do not attribute the mental state of knowing, perception, and intention being the only mental states they attribute (22).

Humans attribute embedded mental states, such as, John thinks that Bill thinks that Henry believes that John should put his kids in Sunday school. Women think that men think that they think that men think that women’s orgasm is different. There is a behavioral counterpart to embedded mental states in human social behavior. One individual watches another individual watch yet another individual engage in some act. In the classroom, for instance, we may observe child A watch child B watch child C look at the teacher. In animals there is nothing comparable. In the wild, we sometimes see one chimpanzee infant watch its mother, another infant watch its mother, etc., but this is a string of independent acts, not a sequence in which each act is embedded in the preceding one. Animals neither attribute embedded mental states nor have embedded social behavior (22). […]

The grammar of a recursive language permits an endless compacting of information limited only by human memory.

The hierarchical organization of information is a related case. Humans divide biological objects into plants and animals, plants into fruit and vegetables, fruit into…, etc. Whereas chimpanzees sort, for example, apple, grape, etc., into one bin, bread, cupcake, etc., into another, thus recognizing categories (24), and category is a precursor of hierarchical information, there is no evidence that they recognize class-inclusion, which is another precursor of the hierarchical organization of information. Class-inclusion requires that the chimpanzee recognize, for instance, that although apple is included in fruit, fruit is not included in apple. Children apparently do not acquire class-inclusion until ≈5 years of age, suggesting that chimpanzees will not acquire it. A good rule of thumb is this: Concepts acquired by children after 3 years of age are never acquired by chimpanzees (49).

Is recursion an automatic part of human language, number, and organization of information, such that these systems have only a recursive form? Or do they occur in both recursive and nonrecursive forms, being recursive only under appropriate cultural pressure? Perhaps the answer is different for the three systems. Number is likely to differ from the other two cases because humans have an innate system (located in the left and right intraparietal sulci) for representing analogue quantities, a system they share with animals, as well as a second system for representing digital quantities, which they do not share with animals (50). […]

Because animals lack recursion (and human language is recursive), the animals’ lack of language is attributed to this factor. But recursion is not the only factor animals lack. If a species lacked language, even a nonrecursive language would be an enormous boon. Yet, chimpanzees have no language of any kind, recursive or nonrecursive.

A number of factors stand between animals and language. For instance, chimpanzees lack voluntary control of their voice. When a chimpanzee wants the attention of its trainer, it does not call; instead, it pounds on a resonant surface. Chimpanzees, therefore, could not have speech. But sign language is a possibility, for they do have voluntary control of their hands.

Chimpanzee sign language, however, could not be comparable with human sign language, because chimpanzees also lack voluntary control of their face, and in human sign language, facial expression plays grammatical roles, such as denoting the boundary of clauses (51).

A weaker form of sign that dropped facial expression and relied exclusively on hand signs would still pose a problem for the chimpanzee. The young animal could not imitate the hand signs of its mother. Most species can imitate the object or location that a model chooses, but there is a second level of imitation in which the novice must copy the motor act of the model (this would be the requirement in the case of sign language). Motor acts are more difficult to copy than objects or locations, because motor acts are ephemeral, and one must form a mental representation of the motor act and then copy the representation (52). Only humans imitate motor acts, although chimpanzees, when taught by humans, can do so. But the untrained chimpanzee cannot, so if a mutant chimpanzee with a simplified sign language were to appear, the other chimpanzees could not copy it.

Teaching is essential for language. Not for grammar, which arguably cannot be taught, but for words. Children are taught their initial words by their mother, and only later do they acquire words more or less on their own. Inasmuch as chimpanzees do not teach, even if they possessed all the other factors mentioned above, they could not have evolved language. In humans, the evolution of teaching evidently preceded that of language.

“Brain Chip”: Cellule del cervello coltivate su chip per studi sulle malattie neurodegenerative e test farmacologici

Da “Ansa.it – Scienza & Tecnica”:

Le cellule del cervello possono essere ‘coltivate’ all’interno di un chip per capire come impazzisce la comunicazione fra loro in caso di malattie neurodegenerative come l’Alzheimer. E’ quanto stanno facendo i ricercatori guidati da Michela Matteoli, direttrice del laboratorio di ‘Biologia cellulare della sinapsi’ dell’università di Milano, in collaborazione con Ibm e l’impresa spin-off NeuroZone.

Se si potesse ‘ascoltare’ quello che accade nel cervello, sentiremmo un continuo chiacchiericcio tra i neuroni che si scambiano informazioni lungo la fitta rete di collegamenti che li unisce. Su questa comunicazione possono intervenire anche le altre cellule del cervello, gli astrociti e le cellule della microglia, che fino a poco tempo fa si pensava fossero ‘mute’. Quando nel cervello si accumulano sostanze pericolose, come la proteina beta-amiloide che causa l’Alzheimer, questa comunicazione ‘impazzisce’: si scatena l’infiammazione e di conseguenza questo può aumentare il danno ai neuroni. Per capire il meccanismo di questo processo che porta alla malattia, l’equipe di Michela Matteoli ha creato ”un dispositivo – spiega l’esperta – che permette di separare fisicamente i diversi tipi cellulari in micro-camere grandi pochi millimetri e collegate da piccoli canali che permettono di tenere sotto controllo il flusso della comunicazione”. Per il momento sono stati creati dei ‘bilocali’ per cellule, piccoli dispositivi a due camere e un corridoio adagiati su chip fatti di polimero PDMS, ma in futuro questi potranno essere resi piu’ complessi e dotati di una componente elettronica per facilitare l”ascolto’ delle cellule, o vvero la registrazione dell’attivita’ dei neuroni.

Intanto i primi esperimenti sono già partiti. ”In una camera coltiviamo i neuroni – continua Matteoli – mentre nell’altra deponiamo a seconda dell’esperimento astrociti o microglia prelevati da varie aree del cervello. Poi applichiamo la proteina beta-amiloide prima in una camera e poi nell’altra e osserviamo cosa accade alle cellule facendo una valutazione completa con tecniche morfologiche, elettrofiosiologiche e di imaging”. 
Questi dispositivi, chiamati ‘Brain Chip’, potranno essere usati in futuro anche per testare i nuovi farmaci appena creati in laboratorio, riducendo i tempi di analisi e abbattendo i costi della ricerca

[Bianco F, Tonna N, Lovchik RD, Mastrangelo R, Morini R, Ruiz A, Delamarche E, Matteoli M. Overflow microfluidic networks: application to the biochemical analysis of brain cell interactions in complex neuroinflammatory scenarios. Anal Chem. 2012 Nov 20;84(22):9833-40. doi: 10.1021/ac302094z. Epub 2012 Nov 8.]

Abstract:

Neuroinflammation plays a central role in neurodegenerative diseases and involves a large number of interactions between different brain cell types. Unraveling the complexity of cell-cell interaction in neuroinflammation is crucial for both clarifying the molecular mechanisms involved and increasing efficacy in drug development. Here, we provide a versatile analytical method for specifically addressing cell-to-cell communication, using primary brain cells, a microfluidic device, and a multiparametric readout approach. Different cell types are plated in separate chambers of a microfluidic network so that culturing conditions can be independently controlled and single cell types can be selectively primed with different stimuli. When chambers are microfluidically connected, the specific contribution of each cell type can be finely monitored by analyzing morphology, vitality, calcium dynamics, and electrophysiology parameters. We exemplify this approach by examining the role of astrocytes derived from two different brain regions (cortex and hippocampus) on neuronal viability in two types of neuroinflammatory insults, namely, metabolic stress and exposure to amyloid β fibrils, and demonstrate regional differences in glial control of neuronal physiopathology. In particular, we show that during metabolic stress, cortical but not hippocampal astrocytes play a neuroprotective role; also, in an exacerbated inflammatory scenario consisting in the exposure to Aβ + IL-1β, hippocampal but not cortical astrocytes play a detrimental role on neurons. Aside from bringing novel insights into the glial role in neuroinflammation, the method presented here represents a promising tool for addressing a wide range of biological and biochemical phenomena, characterized by a complex interaction of multiple cell types.

[Lovchik RD, Bianco F, Tonna N, Ruiz A, Matteoli M, Delamarche E. Overflow microfluidic networks for open and closed cell cultures on chip. Anal Chem. 2010 May 1;82(9):3936-42. doi: 10.1021/ac100771r.]

Abstract:

Microfluidics have a huge potential in biomedical research, in particular for studying interactions among cell populations that are involved in complex diseases. Here, we present “overflow” microfluidic networks (oMFNs) for depositing, culturing, and studying cell populations, which are plated in a few microliters of cell suspensions in one or several open cell chambers inside the chip and subsequently cultured for several days in vitro (DIV). After the cells have developed their phenotype, the oMFN is closed with a lid bearing microfluidic connections. The salient features of the chips are (1) overflow zones around the cell chambers for drawing excess liquid by capillarity from the chamber during sealing the oMFN with the lid, (2) flow paths from peripheral pumps to cell chambers and between cell chambers for interactive flow control, (3) transparent cell chambers coated with cell adhesion molecules, and (4) the possibility to remove the lid for staining and visualizing the cells after, for example, fixation. Here, we use a two-chamber oMFN to show the activation of purinergic receptors in microglia grown in one chamber, upon release of adenosine triphosphate (ATP) from astrocytes that are grown in another chamber and challenged with glutamate. These data validate oMFNs as being particularly relevant for studying primary cells and dissecting the specific intercellular pathways involved in neurodegenerative and neuroinflammatory brain diseases.

Chip infiammatorio mimetico microfluidico per la ricerca su infiammazione, metastasi, funzione immunitaria e malattie autoimmuni

[Kim SK, Moon WK, Park JY, Jung H. Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion. Analyst. 2012 Sep 7;137(17):4062-8.] 

Graphical abstract: Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Abstract:

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5 : 4.3 : 3.9 (E-selectin : ICAM-1 : VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.

Fallimento del modello animale nell’identificazione del nesso tra cancro e fumo di sigaretta

[Coggins CR. A minireview of chronic animal inhalation studies with mainstream cigarette smoke. Inhal Toxicol. 2002 Oct;14(10):991-1002.]

Abstract:

This work was performed to verify whether or not the inhalation response to cigarette smoke in animal species for assessing carcinogenic potential in humans reflects the strong epidemiological evidence in human smokers. Significant increases in the numbers of malignant tumors of the respiratory tract were not seen in rats, mice, hamsters, dogs, or nonhuman primates exposed for long periods of time to very high concentrations of mainstream cigarette smoke. The results are clearly at variance with the epidemiological evidence in smokers, and it is difficult to reconcile this major difference between observational studies in humans and controlled laboratory studies.

Ingegneria tissutale nella creazione di un modello di cuore umano per la ricerca cardiologica

[Turnbull IC, Karakikes I, Serrao GW, Backeris P, Lee JJ, Xie C, Senyei G, Gordon RE, Li RA, Akar FG, Hajjar RJ, Hulot JS, Costa KD. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J. 2014 Feb;28(2):644-54. doi: 10.1096/fj.13-228007. Epub 2013 Oct 30.]

Abstract:

Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18±0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 μN/mm(2) at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 μM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.-Turnbull, I. C., Karakikes, I., Serrao, G. W., Backeris, P., Lee, J.-J., Xie, C., Senyei, G., Gordon, R. E., Li, R. A., Akar, F. G., Hajjar, R. J., Hulot, J.-S., Costa, K. D. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium.