The-Scientist.com, March 8, 2010  —  In the fall of 2009, a group of New Zealand scientists were putting the finishing touches on a new therapeutic to help cancer patients recover from chemotherapy, in preparation for a clinical trial. All they had left to do was choose a flavor.

“It was no easy task,” says Arie Geursen, general manager of LactoPharma, the entity developing the therapeutic, presented in the form of an ice cream. “Everyone has their favorites. We were already leaning toward strawberry since one of the bioactive agents was tinting the ice cream pink… But the decision really came down to who won the taste tests. Strawberry came out on top.”

Development of the ice cream, named ReCharge, began 8 years ago with the formation of LactoPharma, a collaborative research venture between the University of Auckland, the New Zealand government, and the country’s largest dairy company, Fonterra Ltd.

Various components of milk have demonstrated antimicrobial, antiviral, and anti-inflammatory properties, and in the past decade, scientists have begun to identify the molecular components driving these reactions. LactoPharma was created with the goal of trying to incorporate milk’s protective mechanisms into food, health supplements, and pharmaceuticals.

One protein in particular, lactoferrin, has been shown to inhibit tumor growth, promote intestinal cell growth, and regulate immune response in the intestine (Biochem Cell Biol, 89:95–102, 2002). The scientists reasoned it could therefore help patients receiving chemotherapy, which can damage normal cells that multiply quickly, such as infection-fighting white blood cells, known as neutrophils, and intestinal cells. A lack of neutrophils exposes cancer patients to a high risk of infection, while the destruction of intestinal cells can lead to digestive problems, such as diarrhea and poor nutrient uptake. Geoff Krissansen, a molecular biologist at the University of Auckland, and colleagues began testing whether bovine lactoferrin and other dairy components could reduce these side effects of chemotherapy.

Indeed, when fed to mice 2 weeks prior to chemotherapy, bovine lactoferrin helped increase immunoresponsive cytokines in the intestine, decreasing cell damage caused by chemo, and restored both red blood cell and neutrophil numbers (Immunol Cell Biol, 86:277–88, 2008). The researchers also found that another bioactive component present naturally in milk—a type of “lipid fraction,” according to Krissansen—demonstrated similar results in mice. The scientists expect to publish these results in 2010.

Wouldn’t it be nice if all drugs came in the form of ice cream?

“Since lactoferrin has been shown to help restore immune response, it makes sense to incorporate it into a therapy for chemo side effects, which can cause immunosuppression,” says Marian Kruzel, a biologist at the University of Texas Medical School in Houston, who was not involved with ReCharge. “But the dosing levels are very important; too much of it and its immune-regulating effects may be negated.”

To figure out how to deliver these milk ingredients to patients, Krissansen and LactoPharma looked to Kate Palmano at the Fonterra Research Center. “We needed to formulate a product that was acceptable and palatable to patients, but that was also suitable for the bioactives,” says Palmano. They had to avoid anything that would require high temperatures during production, she explains, since the heat could change the protein structure and the bioactives’ functions.

Palmano considered incorporating the bioactives into a liquid drink or yogurt, but in the end, ice cream won out. “Creating a frozen product meant we didn’t have to worry about the bioactives’ shelf life,” she says. “Plus, people going through chemotherapy typically lose their appetite. Why not give them a treat like ice cream?”

The scientists worked with New Zealand’s top ice cream manufacturers to create six tons of strawberry-flavored ReCharge. They then made a placebo ice cream with the same taste, color, and calorie count. ReCharge started its Phase II clinical trial in October 2009, in which 200 prechemotherapy cancer patients will be required to eat 100 grams of either ReCharge or the placebo ice cream each day.

“It has been a wonderful ride creating this product,” says Geursen. “We don’t know if ReCharge will work—it is always a challenge going from mice to humans—but we are keeping our fingers crossed.”

Read more: Sweet relief – The Scientist – Magazine of the Life Sciences http://www.the-scientist.com/article/display/57167/#ixzz0hKgOqyZh

An artist’s impression of “Inuk,” an ancient Eskimo whose genome has been sequenced. Credit: Nuka Godfredsen

Researchers say their subject had brown eyes, thick hair, and dry ear wax

Thanks to new sequencing technologies, and a few tufts of hair rescued from a museum basement, researchers have reconstructed the genome sequence of a man who lived in western Greenland about 4000 years ago. Researchers from the University of Copenhagen were able to read about 80 percent of his genome at a level of accuracy comparable to the genome sequences of living people. (Only eight human genome sequences have been published to date.)

That level of accuracy allowed scientists to analyze 350,000 single nucleotide polymorphisms, or SNPs–spots of common genetic variation within the genome–enabling them to draw conclusions about both the physiology of the man and his origins.

According to a commentary accompanying the paper in Nature,

“he had an A+ blood group, brown eyes, non-white skin, thick dark hair and ‘shovel-graded’ front teeth typical of Asian and Native American populations. What’s more, he had an increased susceptibility to baldness, dry earwax and a metabolism and body-mass index commonly found in those who live in cold climates.”

By comparing the SNP data with that of several surrounding populations, researchers were able to pinpoint his geographical origin.

Surprisingly, the ancient eskimo proved to be most closely related to three Old World Arctic populations: the Nganasans, Koryaks and Chukchis of the Siberian far east. This suggests that there was a substantial and relatively recent migration across the Bering Strait and over North America to Greenland. The authors’ analysis indicates that the Saqqaqs diverged from the Chukchis about 200 generations (5,400 years) ago, implying that the ancestral Saqqaqs separated from their Old World relatives almost immediately before their migration to the New World.

Scientists had previously analyzed DNA from Neandertal bone and tooth samples, but not to this level of accuracy. Sequencing ancient DNA samples is notoriously difficult, thanks to degraded DNA and contamination from bacteria, fungus and humans handling the samples. In this case, researchers say only Europeans handled the DNA, and the sample itself was found to have no European ancestry. They used sequencing technology from the company Illumina.

According to the New York Times, the hair, originally dug out of the permafrost at Qeqertasussuk on the west coast of Greenland in 1986, was kept in a plastic bag in the National Museum of Denmark. It was found with other waste, and the scientists speculate that it was the result of a haircut.

“There it moldered, unfrozen, until discovered by Dr. Willerslev, an expert on ancient DNA. Having spent two months digging for ancient human DNA in Greenland without finding any human remains, he concluded that ancient Greenlanders must have disposed of their dead by laying them on the sea ice. Only on complaining of his bad luck to a friend did he learn that the friend’s father had found the hair sample 20 years earlier.”

NORTHBROOK, Ill., March 8, 2010 (GLOBE NEWSWIRE) — Nanosphere, Inc., (Nasdaq:NSPH), announced today it was selected one of the world’s most innovative companies by Massachusetts Institute of Technology’s Technology Review with its inclusion on the 2010 TR50 list. 

The 2010 TR50 is Technology Review’s first annual list identifying companies that demonstrate the most impressive and noteworthy innovations occurring in technology. 

“In choosing the TR50, we picked companies with this year’s most important inventions and breakthroughs. But we also selected companies that are successfully growing businesses and markets around innovative new products,” said Jason Pontin, editor in chief and publisher of Technology Review. “The TR50 list is our selection of companies that show the most impressive innovation in commercializing new technologies.”

“Nanosphere is a leader in commercializing microfluidic technology for use in clinical diagnostic tests, helping to pave the way for personalized medicine. The FDA’s recent approval of its instrument for pharmacogenomic testing is a significant milestone,” said David Rotman, editor, Technology Review. 

Nanosphere joins such notable companies chosen by MIT’s Technology Review as Amazon, GlaxoSmithKline, Google, Tesla Motors, and Twitter. It is one of six public bio-medical companies listed.

“To be recognized as an innovative global leader furthering healthcare and personalized medicine is gratifying,” said William P. Moffitt, president and chief executive officer of Nanosphere, Inc. “Our focus on achieving excellence will continue to generate new solutions and breakthroughs that will improve diagnostic testing and ultimately the diagnosis and treatment of disease and the health of people worldwide.”  

The additional five selected public biomedical companies include Alnylam, AthenaHealth, GlaxoSmithKline, Illumina, and Medtronic. The list also acknowledged five private biomedicine companies including BIND Biosciences, Complete Genomics, Fate Therapeutics, Fluidigm, and Pacific Biosciences. 

Nanosphere was also featured in the March/April edition of Technology Review, with a focus on the company’s Verigene® System, troponin testing and overall approach to personalized medicine. The link to the online version is: http://www.technologyreview.com/biomedicine/24581/

About 2010 TR50

The first annual list compiled by MIT’s Technology Review, Inc. recognizes companies of all sizes, both public and private, within the energy, computing, web, biomedicine, and materials sectors that have demonstrated the most promising technologies and remarkable potential to transform the world. Companies were evaluated on their business model, strategies for deploying and scaling up their technologies, and the likelihood of success. For more information and full access to the list, please visit: http://www.technologyreview.com/companywatch/tr50/

About Technology Review, Inc.

Technology Review, Inc., an independent media company owned by MIT, is the authority on the future of technology, identifying emerging technologies and analyzing their impact for leaders. Technology Review’s media properties include Technology Review magazine, the oldest technology magazine in the world (founded in 1899); the daily news website TechnologyReview.com; and events such as the annual EmTech@MIT Conference.

About Nanosphere, Inc.

Nanosphere develops, manufactures, and markets an advanced molecular diagnostics platform, the Verigene® System, for direct genomic and ultra-sensitive protein detection. This easy to use and cost effective platform enables simple, low cost and highly sensitive genomic and protein testing on a single platform. Nanosphere is based in Northbrook, IL. Additional information is available at http://www.nanosphere.us.

The Nanosphere, Inc. logo is available at http://www.globenewswire.com/newsroom/prs/?pkgid=4344

CONTACT:  Nanosphere, Inc.
          William P. Moffitt, President and CEO
          847-400-9021
          wmoffitt@nanosphere.us
          The Torrenzano Group
          Media:
          Ed Orgon
          212-681-1700
          ed@torrenzano.com

From Structure and Function of Ribosomes to New Antibiotics

Sterling Professor of Molecular Biophysics and Biochemistry, HHMI Investigator, and recipient of the 2009 Nobel Prize for Chemistry Thomas A. Steitz, Ph.D., gives his Nobel Lecture

Biofilm Research – Paul Webster, Ph.D.

Dr. Webster discusses the nature of biofilms – what they are and what they do in the human body. He shares some of the new research taking place on biofilms

March 9, 2010, by Gabe Mirkin MD  —  A report in the medical journal, Cancer Research, shows that colon cancer may be prevented by eating curry powder, a spice you add to flavor food.

A chief ingredient in curry powder is curcumin (sir’qumin), the pigment that gives turmeric spice its bright yellow-orange color. Nutritional biochemist Bandaru Reddy, of the American Health Foundation in Valhalla, N.Y., noted that people who live in certain parts of Asia have a very low incidence of colon cancer. Her team wanted to see if they could find something in their diets that helps to prevent colon cancer. They know that aspirin and other anti-inflammatory drugs may help to prevent colon cancer in America, so they checked to see what drugs or herbals were used by people who live in areas where there is a very low incidence of colon cancer. They found that people in India use turmeric, found in curry powder, to treat traumatic aches and sprains and also the pain of arthritis. They then fed huge amounts of a chemical that is known to cause colon cancer to rats. Half of them also ate large amounts of circumin, and they had half the rate of colon cancer of the rats who did not receive the curry powder extract.

Now entrepreneurs are promoting expensive supplements that contain tumeric. Virtually all spices, herbs and vegetables contain phytochemicals that help to prevent cancers. You will get maximum protection from cancer by eating a healthful diet with large amounts of fruits and vegetables, seasoned with your favorite of herbs and spices, rather than by spending your money to supplement an unhealthful diet with pills.

By Gabe Mirkin, M.D., for CBS Radio News

Patrizia Stoitzner, Kristian Pfaller*, Hella Stössel and Nikolaus Romani

  1. Department of Dermatology, University of Innsbruck, Innsbruck, Austria
  2. *Department of Anatomy and Histology, University of Innsbruck, Innsbruck, Austria

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Langerhans cells of the epidermis and dermal dendritic cells screen the skin for invading antigens. They initiate primary immune responses after migrating from sites of antigen uptake to lymphoid organs. The skin is a feasible model to study the morphology and regulation of dendritic cell migration. We therefore used murine skin explant cultures for tracking the pathways of dendritic cell migration by electron microscopy. Several novel observations are reported. (i) In 48 h cultures of epidermal sheets numerous Langerhans cells migrated out between keratinocytes extending long and thin cytoplasmic processes (“veils”). (ii) Langerhans cells in transition from epidermis to dermis were observed by transmission electron microscopy. Where Langerhans cells penetrated the basement membrane, the lamina densa was focally absent. (iii) This was highlighted by scanning electron microscopy, which presented the basement membrane as a tightly packed and dense network of fibrils. (iv) Scanning electron microscopy of the dermis revealed dendritic cells extending their cytoplasmic processes and clinging to collagen fibrils. (v) Entry of dendritic cells into dermal lymphatics was observed by transmission electron microscopy. It occurred by transmigration through intercellular spaces of adjacent endothelial cells. Entry through wide gaps between endothelial cells also seemed to take place. (vi) Dendritic cells inside the afferent lymphatics frequently carried material such as melanosomes and apoptotic bodies. These observations visualize the cumbersome pathway that dendritic cells have to take when they generate immunity.

Dendritic cells are highly motile antigen-presenting cells. They have optimized the migratory capacity to fulfill their prime task, i.e., to initiate primary immune responses . Like sentinels they scan peripheral compartments for invading foreign particles and phagocytose or macropinocytose these antigens very effectively. After uptake of antigens they start to mature and migrate to draining lymph nodes to stimulate antigen-specific T cells there. The gap between the sites of antigen uptake and the sites of clonal T cell activation is efficiently bridged by these migratory dendritic cells. They carry immunogenic complexes of major histocompatibility complex and antigenic peptides on their cell surface and possibly also antigenic proteins in antigen retention organelles into the T cell areas of lymphoid organs .

Inflammatory stimuli, such as bacterial lipopolysaccharide, tumor necrosis factor , and interleukin-1 , and chemotactic cytokines, like macrophage inflammatory protein 3 (MIP-3 /CCL19), secondary lymphoid tissue chemokine (SLC/CCL21), and interleukin-16 , trigger and guide the migration of dendritic cells from peripheral tissues towards the draining lymphoid organs. As a first step E-cadherin, which mediates Langerhans cell-keratinocyte adhesion, is downregulated by inflammatory cytokines so that Langerhans cells are able to leave the epidermis . By secretion of matrix metalloproteinases (MMP) dendritic cells can digest extracellular matrices , which facilitates their crossing of the basement membrane and migration through the dermis. When they encounter lymphatic vessels they enter the lumen and migration proceeds towards the lymph nodes. This was demonstrated by immunohistochemistry and transmission electron microscopy (TEM) in murine and human skin explant organ culture models where dendritic-cell-filled lymph vessels were originally described as “cords” . Skin in general and the skin explant model in particular represent suitable tools to investigate morphologic and regulatory aspects of dendritic cell migration. Contact hypersensitivity is another widely used experimental model . In order to learn more about the requirements for efficient dendritic cell migration we were interested in morphologic aspects of emigrating dendritic cells in situ. Therefore, we analyzed in detail cultured murine skin explants, primarily by scanning electron microscopy (SEM) but also by TEM.

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Mice

Mice of inbred strains BALB/c and C57BL/6 were purchased from Charles River Germany (Sulzfeld, Germany) and used at 8–12 wk of age.

Media and reagents

Culture medium was RPMI 1640 supplemented with 10% fetal bovine serum, L-glutamine (Sebac, Stuben, Austria), gentamycin (all from PAA, Linz, Austria), and 2-mercaptoethanol (Sigma Chemical, St. Louis, MO).

Skin explant culture

Mice were sacrificed and ears were cut off at the base. Ear skin was split in dorsal and ventral halves by means of strong forceps and the dorsal halves (i.e., cartilage-free, thinner halves) were cultured in 24-well tissue culture plates (one ear per well) as described previously . Alternatively, epidermis and dermis were separated from each other by means of the bacterial enzyme dispase , and the epidermal sheets were placed in culture. In most experiments whole skin or epidermis was cultured continuously for 48 h. At the end of the cultures skin explants were further processed for electron microscopy. All observations described are based on the ultrastructural inspection of several explants from three to four separate experiments.

SEM

Tissue was fixed immediately after termination of cultures with half-strength Karnovsky’s formaldehyde-glutaraldehyde fixative , followed by three washes with 0.1 M cacodylate buffer and postfixation in 3% OsO4 in water. After a short rinse in 0.1 M sodium cacodylate buffer specimens were dehydrated in ascending concentrations of ethanol (50%-100%). Samples were then critical-point dried (CP Dryer, Balzers, Liechtenstein), mounted on aluminum stubs with collodial silver, and subsequently coated with a layer of 5–10 nm of gold-palladium in a sputtering device (Balzers). Specimens were viewed on a Zeiss Gemini 985 scanning electron microscope (Zeiss, Oberkochen, Germany) at 5–8 kV.

TEM

Skin organ cultures were minced into small blocks and fixed by submersion for 5 h in half-strength Karnovsky’s formaldehyde-glutaraldehyde reagent . Further processing was as described previously . Briefly, specimens were postfixed in 3% aqueous OsO4, en bloc contrasted with veronal-buffered uranyl acetate, and dehydrated in a graded series of ethanols. After dehydration specimens were infiltrated in mixtures consisting of varying proportions of propylene oxide as an apolar solvent and epoxy resin (Epon 812; Serva Feinchemikalien, Heidelberg, Germany). The resin was polymerized at 60°C for 24 h. Ultrathin sections were contrasted with lead citrate and viewed with a Philips EM 400 electron microscope (Fei Company, Eindhoven, The Netherlands) at a voltage of 80 kV.

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Langerhans cells emigrate from epidermal sheets in a time-dependent manner

Langerhans cell emigration from the epidermis has been repeatedly shown to occur in vivo as well as in skin explant cultures . Here we extend these observations in a morphologic way. Emigration of Langerhans cells from the epidermis occurs irrespective of the presence of the dermis (Figure 1a). When the epidermis was separated from the dermis by means of dispase before the onset of culture, Langerhans cells emigrated into the culture medium cells equally well as in whole skin cultures. SEM of epidermal sheets cultured for 0, 5, 10, or 48 h revealed Langerhans cells coming out of the epidermis in a time-dependent manner (data not shown). Already 5 h after the onset of the explant cultures the first Langerhans cells emigrating from the epidermis could be observed. The numbers of emigrating Langerhans cells increased with duration of the organ culture. Langerhans cells detached from the surrounding keratinocytes. Gaps and holes formed in the keratinocyte layer where the emigrating Langerhans cells moved through (Figure 1b, d). The emigrating Langerhans cells extended very thin and long pseudopodia with which they seemed to be attached to surrounding keratinocytes (Figure 1c). Langerhans cells displayed pronounced cytoplasmic veils and may be considered – at least partially – mature by this criterion. It should be noted that the dermo-epidermal separation by means of dispase leaves the collagenous part of the basement membrane (lamina densa) on the dermal side. SEM of cultured epidermal sheets also emphasizes that migration is an active process. Langerhans cells do not simply fall out of a disintegrating epidermis. This point is underscored by the fact that in populations of emigrant cells retrieved from the culture medium Langerhans cells are highly enriched up to 70%.

The basement membrane is a major physical obstacle for migrating Langerhans cells

After leaving the epidermis Langerhans cells encounter a complex barrier, the basement membrane, which they have to passage on their way to the dermis. By TEM we occasionally found the rare event of a Langerhans cell in transit through the basement membrane (Figure 2). We observed that the Langerhans cell extended a pseudopod through the basement membrane. The electron-dense part of the basement membrane (lamina densa) was absent only in the area of physical contact with the emigrating Langerhans cells (Figure 2b–d). The cells displayed ultrastructural features of mature dendritic cells, i.e., few small and runted Birbeck granules or no Birbeck granules at all and a large size. Unfortunately, this event is too rare on ultrathin sections so that it is not possible to completely reconstruct the movement of Langerhans cells through this obstacle, even though this is attempted in Figure 2.

The lamina densa stays on the dermis after dermo-epidermal separation by dispase . We studied this border structure between the skin compartments by SEM. As can be appreciated from Figure 3a the collagen fibrils in the basement membrane are very tightly packed. They form a dense network that would not leave enough space for a transmigrating Langerhans cell.

The dermis does not allow unimpeded movement of migrating Langerhans cells

In the dermis the collagen meshwork appears dense but less compact than in the lamina densa. Yet, in relation to the size of migrating dendritic cells this connective tissue appears to render movement of dendritic cells difficult. Dendritic cells squeezing through between bundles of collagen fibrils are readily visible . Migrating dendritic cells sometimes attached to individual bundles of collagen fibrils with their pseudopodia . These observations suggest that the ability of dendritic cells to flexibly change their shape may be essential for their successful movement through the dermis until they encounter a lymphatic vessel.

Migrating dendritic cells enter lymphatic vessels

As reported previously for human and mouse skin explant cultures dendritic cells accumulated in wide clefts that were lined by a thin endothelium, did not possess a continuous basement membrane, and thus qualified as lymphatic vessels. In immunohistochemistry these structures were originally termed “cords” . In semithin sections no differences were noted in the numbers of lymphatic vessels in cultured and uncultured skin. By TEM of a large number of sections of 48 h explant cultures we did observe entry of dendritic cells into vessels. Dendritic cells moved through the intercellular space of two neighboring endothelial cells . As cell-cell contacts between adjacent endothelial cells were often loose, entry of cells may be relatively easy. A tight “sealing” of the pore through which the migrating cell entered the vessel was described for dendritic cells acquiring antigens through the intestinal wall . Such tight contacts were not observed between migrating dendritic cells and skin lymphatics. In addition, we repeatedly noted distinct gaps in the endothelial layer. The endothelial cells were intact but they were not adjacent to each other, thus forming an interruption that was sometimes very wide . Clearly, these gaps appeared large enough to allow the entry of a dendritic cell and, indeed, dendritic cells in the vessel lumen were occasionally found close to these gaps, although never really in transit . A quantitative comparison of dendritic cells entering the vessel by transmigration between endothelial cells and dendritic cells that had possibly entered via the gaps was not possible. Both events were too rare.

Dendritic cells inside lymph vessels carry antigens

The vessels contained cells with the morphology of dendritic cells such as an electron-lucent cytoplasm, veils, and an irregularly shaped nucleus. Frequently we could find Birbeck granules , i.e., the Langerin (CD207)-containing cell organelles typical for Langerhans cells and possibly important for antigen processing. This had been described previously . Granules with an electron-dense core, typical for dendritic epidermal T cells , were not found in the cells evaluated. Moreover, as BALB/c mice, which contain only very few dendritic epidermal T cells, were used for most experiments, it is highly unlikely that the cells described here might be dendritic epidermal T cells rather than dendritic cells. Frequently these lymph-borne cells contained substantial amounts of material that they must have taken up earlier. Some dendritic cells had ingested melanosomes . Other cells carried various forms of cellular material including apoptotic bodies .

It has been shown before that Langerhans cells emigrate out of the epidermis after receiving an inflammatory stimulus such as the application of contact allergen or the onset of skin explant culture . After passage through the basement membrane the migrating dendritic cells enter lymphatic vessels in the dermis and travel to the draining lymph nodes to initiate primary immune responses . Here we present for the first time a three-dimensional view of migrating Langerhans cells with the help of SEM. We were able to follow Langerhans cells on their way out of the epidermis, through the basement membrane and the dermis, and into the dermal lymphatics. This view allows some novel insights to be gained into the mechanism of dendritic cell migration.

Langerhans cell migration appears to be a highly active process

After an inflammatory stimulus, here given by the onset of the organ culture, Langerhans cells start to emigrate from the epidermis in a time-dependent way. They squeeze out between the keratinocytes. This may happen in an active way in that they extend long thin pseudopodia, attach them to neighboring keratinocytes, and so seem to pull themselves out of the epidermis. Also in the dermal meshwork of collagen fibrils the migrating dendritic cells appear far from being passive. Again, they frequently stretch out processes and cling onto bundles of collagen. Although we are aware that the study of the dynamics of a process requires serial or real-time analysis – something that is not possible with electron microscopy – it is tempting to speculate that they were actively pulling. Such active dendritic cell movement has been shown in artificial collagen lattices by time-lapse video microscopy . Our data suggest that it may also occur in vivo.

Migrating Langerhans cells need to “create a path” with the help of enzymes and adhesion molecules

Our observations suggest critical roles for enzymes and adhesion molecules at several levels.

First, for the emigration of Langerhans cells from the epidermis it is necessary that the intercellular bonds between keratinocytes (e.g., desmosomes) and between keratinocytes and Langerhans cells be loosened. A role for E-cadherin has been proposed . Another perhaps additional way is by digestion of cell-adhesion-mediating molecules with special proteases such as the MMPs . We have evidence that a broad-spectrum inhibitor of MMPs and, more specifically, monoclonal antibodies against MMP-9 and MMP-2 strongly impaired the emigration of Langerhans cells out of epidermal sheets.1 As epidermal sheets procured by the use of the enzyme dispase possess no more lamina densa these data might suggest a role for the MMPs in helping to loosen contacts between epidermal keratinocytes. Such a function for MMP-9 on epidermal cells has been demonstrated in the context of carcinogenesis .

Second, the scanning electron micrographs strongly suggest that for the transit through the basement membrane Langerhans cells need to be equipped with enzymatic tools. We2 and others have shown that migrating Langerhans cells express MMP-9 and MMP-2. Kobayashi et al additionally demonstrated that Langerhans cells produce MMP-9, which is able to digest collagen IV . TEM highlighted an important qualitative aspect. The basement membrane was absent (presumably “digested away”) only in the very focused area of cell-basement membrane contact. This mode of action is typical for the MMPs .

Third, the scanning electron microscopic view of the dermis suggests that similar cellular tools may also be needed for the second leg of the Langerhans cell journey within the skin, namely from the basement membrane through the collagen thicket until they gain access to draining lymphatic vessels. The pictures suggest that migrating cells contact and temporarily adhere to collagen fibrils. One might speculate that the cells are actively pulling themselves along the fibrils.

Migrating dendritic cells appear to enter dermal lymphatic vessels by endothelial transmigration and through gaps in the endothelium

Langerhans cells and dermal dendritic cells en route are chemotactically attracted towards the dermal lymphatic vessels by the chemokine SLC/CCL21, which is expressed in lymph endothelial cells in situ . How exactly they get into the lumen of the vessels is not clear. Our observations would suggest two possibilities: entry by transmigration through intercellular spaces and entry through gaps in the endothelial lining.

Transmigration of dendritic cells through intact layers of endothelium occurs. The experiments by Randolph et al in vitro and in vivo suggest an additional important role for this type of entry into lymphatic vessels. Inflammatory tissue monocytes may transform into dendritic cells upon ablumenal-to-lumenal transmigration through lymphatic endothelium. Even though the lymph vessel endothelial cells are very thin we noted a close apposition of transmigrating dendritic cells and endothelial cells that may suffice to deliver the necessary signals for cell transformation as described by Randolph et al. Transmigration through the endothelium of dermal lymphatics may also be facilitated by the fact that endothelial cells are only loosely connected to each other; sometimes the most distal parts of their elongated cell bodies overlap, forming some sort of flap . This is clearly in contrast to transmigration through the tightly structured walls of blood vessels.

We have previously noted that in human and murine skin explant cultures the lymphatic endothelium shows interruptions that appear large enough for a dendritic cell to go through. In these studies, however, we have not investigated the frequency of the gaps in detail. In this study we gained the impression that such gaps were frequent. We saw dendritic cells close to the gaps and even in contact with the gaps ; we did not see dendritic cells going across these gaps, however. Nevertheless, it seems likely that dendritic cells also enter via the gaps. It should be noted that there was always a well-delineated separation of ablumenal connective tissue and the lumen of the vessel, indicating that also the gaps had some sort of a vessel wall, perhaps consisting of electron-lucent lamina lucida materials such as integrins, laminins, etc.

Migrating dendritic cells carry antigens

The prime function of dendritic cells in peripheral organs is to take up, process, and transport antigens to lymphoid organs. Observations with dendritic cells from the intestine have suggested that immature dendritic cells may also carry self antigens to the lymph nodes, thereby maintaining tolerance . We show here that this may happen also in the skin: dendritic cells transport self antigens such as melanosomes that they have probably collected from dying keratinocytes in the epidermis. They also carry apoptotic bodies. This would suggest that the described efficient pathway of cross-presentation, which uses apoptotic cells as the preferred form of antigen , may also be operative in the skin. In contrast to, however, melanosome- and apoptotic-body-containing dendritic cells in lymph vessels looked morphologically mature. Whether this would ultimately result in the generation of immunity of tolerance cannot be judged from ultrastructural observations only. Additionally, our observations complement and extend the work by, who showed recently that melanosomes are transported to draining nodes in two ways. First, we noted melanosome transport under conditions of normal melanogenesis; Hemmi et al used experimentally increased melanogenesis. Second, we show uptake and melanosome transport directly in the dermal lymphatics; Hemmi et al describe the end result of melanosome transport, i.e., accumulation in the lymph node.

Relevance

Dendritic cells are crucial for the generation of immunity to epicutaneously or intracutaneously arriving pathogens or vaccines. The use of in vitro generated, tumor-antigen-loaded dendritic cells has come to the stage of clinical evaluation  When dendritic cells are injected into the skin most of them have been shown to remain at the injection site Migration to the lymph nodes is ineffective, and it therefore appears desirable to improve this process and, as a consequence, improve immunogenicity. Better knowledge about the pathways and the regulation of dendritic cell migration in model systems will help to achieve this goal.

The first dendritic cells (DCs) to be discovered, in 1868 were the Langerhans cells of human epidermis. It took however until the 1970s to demonstrate that these cells belong to the immune system. Simultaneously, in 1973, the pioneering work of Steinman and Cohn permitted the identification of DCs in lymphoid tissue and their functional relationship with Langerhans cells. Realization of the extraordinary capacity of DCs for antigen -presentation set the stage for an exponentially rising interest in their biology. Major advances in the early 1990s subsequently led to the ability to generate DCs in vitro from myeloid hematopoietic progenitors or from monocytes, and greatly facilitated their study. The initial unified model of DC life history held that immature DCs patrol peripheral tissues and upon encounter with microbial products or other danger signals undergo maturation as they migrate to lymphoid tissue where they present antigen and activate naive T cells (16).

While most elements of this model still hold true, in particular the unique capacity of DCs to initiate adaptive immunity, many different and contrasting facets of DCs have since been discovered (17). One aspect that has become clearly appreciated is the great diversity of DC Subtypes with considerable functional differences. Part of this heterogeneity is intrinsic (eg “conventional” versus plasmacytoid DCs), but a high degree of plasticity is also characteristic of the DC system. For instance, DCs can be instructed by the nature of the early signals they receive, with greatly divergent consequences on the immune response. Thus, in addition to their classic function to drive strong Th1-type adaptive responses, DCs can be polarized by microbial products towards a Th2- type response, or towards peripheral immune tolerance via the induction of regulatory T cells (18, 19).

Today, DCs are thus positioned as the master regulators of immunity. Pharmacological intervention to exploit the full range of DC regulatory potential will undoubtedly lead to a variety of therapeutic applications to either boost, suppress or repolarize the immune system (20, 21). Another recently recognized important function of DCs is to link the innate and adaptive immune response. This is illustrated by antiviral responses of plasmacytoid DCs (22), and by crosstalk between DCs and NK cells (23). A major breakthrough in DC  biology has been the recent unraveling of the mechanisms responsible for their

Dendritic Cells: regulators of the immune response

regulatory functions, an advance made possible by the molecular cloning of genes expressed by DCs. Thus, it was realized that DCs are remarkably equipped with Pattern -Recognition Receptors (PRRs), the innate sensors that recognize conserved molecular patterns on microbes and self- tissue. Outstanding PRRs are the C- type Lectin Receptors and the Toll – Like Receptors. The key role played by Chemokines and their receptors in the migration patterns of DCs is now well established. Finally, an array of Cytokines and corresponding receptors are known to be responsible for the crosstalk between DCs and a host of other cell types that will determine the net outcome of the immune response. Collectively, this rapidly-evolving knowledge allows for drug-discovery programs to design pharmacological compounds to agonize or antagonize DC molecules in a number of clinical settings.

Dendritic Cell Migration

The ability to migrate is a central feature of DCs and indispensable for their functions within the immune system (64). Studies on the migratory properties of DCs provide highly valuable information about their biology. DCs reside in an immature state in peripheral tissues where they act as sentinels to induce protective immunity against incoming danger or immune tolerance to maintain homeostasis to self antigens. In the steady-state, heterogeneous populations of DCs (see section 3) are localized in distinct anatomic sites (eg skin) according to their unique homing properties. In the absence of danger, tissue- resident DCs emigrate via afferent lymph towards draining lymph nodes, presumably to instruct T cell tolerance (21).  Inflammatory conditions result in an important mobilization of blood DC precursors into sites of tissue injury. Illustrating the plasticity of the DC system, conventional DCs are recruited in part from inflammatory monocytes (65).

Trafficking directly from blood to lymph nodes via high endothelial venules, plasmacytoid DCs (pDCs) are normally absent from peripheraltissues (66).  However, pDCs may be recruited to inflammatory sites under pathological conditions.

It was realized early on that activation (eg by LPS or pro-inflammatory cytokines) of resident or newlyrecruited conventional DCs at inflammatory sites results in their rapid egress into afferent lymphatics. After crosstalk with the

Tuning of Dendritic cell function

lymphatic vessels (67) such DCs reach the draining lymph nodes in a fully-mature state with optimal capacity for both antigen -presentation and T cell activation. Several mechanisms are responsible for the distinct migration patterns of DCs observed in the steady- state and in perturbed tissues. First, DCs respond to a variety of chemokines, a family of secreted chemotactic factors that guide the navigation of various types of leukocytes (68).

Immature DCs express many receptors for “ inflammatory ” chemokines such as MIP-3a/CCL20 (mAb DENDRITICS) produced by epithelial cells (eg epidermal keratinocytes) (11). MIP-3a/CCL20 (mAb DENDRITICS) and its receptor CCR6 are important components for the recruitment of Langerhans cells, and consistently are strongly up-regulated in inflammatory skin disorders, eg in psoriasis (69). Activation of DCs under pro-inflammatory conditions induces a change in their profile of chemokine receptors. This modification allows for enhanced trafficking of DCs from inflamed tissues towards draining lymph nodes, a process in which the chemokine receptor CCR7 plays a critical role (70).  

In addition to the chemokines, several other mechanisms control the migration of DCs. These include non-chemokine chemotactic factors such as formylated peptides (fMLP) or the antimicrobial defensins. In addition, several non – chemotactic molecules promote or inhibit DC migration. Finally, physical barriers must be crossed by migrating DCs. Matrix metalloproteinases (MMPs) play an essential role in the degradation of such obstacles. MMP12 (mAb DENDRITICS) is expressed by Langerhans cells, and strikingily abundant in Langerhans cell histiocytosis, a malignancy in which this protease may play an important role (71). Further understanding the patterns and mechanisms of DC migration will undoubtedly open new avenues for therapeutic translation.

Cytokines

As other leukocytes, DCs rely on a network of soluble cytokines to communicate with a variety of cell types to orchestrate the nature and intensity of the immune response. A number of immunoregulatory cytokines are thus produced by DCs to directly imprint the functions of neighboring cells. In response to maturation-inducing stimuli, conventional DCs produce IL-12, IL-23, and IL-27, three related cytokines that promote Th1-type responses (72), and IL- 18, a member of the IL-1 superfamily, which shares many activities with IL-12, including stimulation of IFN-(mAb DENDRITICS) (73) production by activated T cells. The pro-inflammatory cytokines IL- 1, IL-6, and TNF-, likewise produced by conventional DCs in response to activation/maturation stimuli, display highly pleiotropic effects on both innate and adaptive immunity. In contrast to the above cytokines, IL – 10 (mAb DENDRITICS) which can be induced in DCs, plays an important role in the induction of regulatory T cells that mediate immune tolerance (74). Likewise, TGF-b displays immunosuppressive properties mediated via several mechanisms. Type- I interferons (IFN-ƒÑ/ƒÒ) play a crucial role in innate immunity against virus infection. Plasmacytoid DCs (pDCs) are specialized to produce high amounts of IFN-a /b (IFN-2b subtype mAb DENDRITICS) in response to TLR ligands or engagement of the IL – 3 receptor alpha chain (CD123) (mAb DENDRITICS) (see sections 3 and 5).

Of note, several of the DC – derived cytokines also function in an autocrine fashion resulting in signal amplification. In addition to their capacity to secrete cytokines, DCs and their precursors are well -equipped with receptors for cytokines produced by other cells present in their micro- milieu.

This property allows for a cytokine dialogue between DCs and other cell types during DC development, at sites of inflammation, and in lymphoid tissue. 

Besides its capacity to favor the generation of conventional DCs in combination with GM-CSF, a widely – exploited asset to generate DCs in vitro from monocytes, IL-4 (mAb DENDRITICS) is a regulatory cytokine that governs IL- 12 production by DCs (75). A minor proportion of conventional DCs also express receptors for IL- 17 (mAb DENDRITICS), a T- cell pro-inflammatory cytokine which promotes DC maturation (76). DCs can be generated both from myeloid or lymphoid hematopoietic progenitors. The cytokine Flt3 – ligand is a key early – acting factor for the development of both lineages (77). IL -7 (mAb DENDRITICS) and its receptor (IL -7R alpha chain) (CD127) (mAb DENDRITICS) (78) are involved in the generation of DCs from lymphoid-committed precursors. Through their functions exerted at every key decision point in the life-cycle of DCs, cytokines thus represent central players in tuning the immune response. A wide range of diseases are targets for intervention via the cytokine network.