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Last call for Lindane.

Lindane is losing much of any luster it once had. The pesticide, once widely used to kill lice and a variety of pests that attack agricultural products, livestock, and trees, has slowly been eliminated from many applications since the mid-1970s. As long ago as the early 1980s, three U.S. organizations petitioned the U.S. Food and Drug Administration (FDA) to ban lindane following an FDA doc-tor's advisory that the pesticide caused seizures in children and test animals. In two of the latest actions, the European Commission ruled in December 2000 that member states must withdraw all authorizations for plant protection products containing lindane, and California approved legislation in September 2000 that banned lindane-based products used to treat lice and scabies. Many groups lobbied heavily to have lindane added to the list of 12 persistent organic pollutants banned by the Stockholm Convention signed in May 2001. (It could still be added to a codicil to be developed once the convention comes into force.) Lindane, known technically by synonyms such as gamma benzene hexachloride and gamma-hexa-chlorocyclohexane, has been in use for about 50 years. Researchers have found that the long-lasting chemical can cause liver, kidney, neurolog-ic, and immune system damage; birth defects; cancer; and death. It may also be an endocrine disrupter. However, the FDA considers lindane an acceptable pesticide for killing lice and treating scabies when used according to the labeled instructions. Several companies still sell lindane-based lotions and shampoos. Numerous other pesticides , such as malathion, perme-thrin, and pyrethrins, also are used in products that target lice and sca-bies. To help reduce all pesticide exposures, alternative methods to detect and possibly remove lice, such as wet combing, are gaining favor in some circles. For broader indoor and outdoor uses such as wood fumigation and agricultural pest control, the U.S. Environmental Protection Agency is in the midst of evaluating lin-dane as part of its reregistration process for this and many other pesticides. At present, lindane is approved only for seed treatments to fight pests such as wireworms and white grubs that attack crops such as cantaloupes, lentils, and wheat. Such plant-protection uses are no longer valid in the 15 European Union countries. On 20 December 2000, the European Commission—primarily concerned with the safety of operators exposed to the pesticide and the adverse impacts the chemical has on the environment—banned lindane for those purposes, allowing 18 months for users and producers to phase …

For broader indoor and outdoor uses such as wood fumigation and agricultural pest control, the U.S. Environmental Protection Agency is in the midst of evaluating lindane as part of its reregistration process for this and many other pesticides. At present, lindane is approved only for seed treatments to fight pests such as wireworms and white grubs that attack crops such as cantaloupes, lentils, and wheat.
Such plant-protection uses are no longer valid in the 15 European Union countries. On 20 December 2000, the European Commission-primarily concerned with the safety of operators exposed to the pesticide and the adverse impacts the chemical has on the environment-banned lindane for those purposes, allowing 18 months for users and producers to phase out existing stocks. But lindane may still be used for nonagricultural purposes such as household pesticides.
The ban follows actions taken by several member countries and a 1999 Austrian risk assessment that concluded the chemical is a possible carcinogen with no safe exposure level. In 2000 lindane was also found by the French Institute for the Environment in a number of French waterways that are used as drinking water sources. European environmental activists support the announcement, though Helen Lynn, health coordinator for the London-based Women's Environmental Network, stated in a 13 July 2000 Friends of the Earth press release that "whilst we welcome today's decision, we are disappointed that the public will still be exposed to this dangerous chemical in the home." Water degradation and the ensuing impacts on human health and the environment were the primary drivers behind California's ban, scheduled to go into effect 1 January 2002. Research in the Los Angeles basin in 1999 revealed that, when lindane shampoos were rinsed down the drain, the pesticide readily contaminated the effluent and eventually the downstream waterways. Cutting down on lindane use through educational campaigns conducted prior to approval of the ban reduced contamination by an average of 50%, says Ann Heil, senior engineer for the Sanitation Districts of Los Angeles County. The continued use of lindane as a seed treatment shouldn't have much impact on water contamination, she adds.
However, lindane pervades the food supply. In the Total Diet Study, an FDA project published in September 2000, lindane was found in dozens of foods, such as evaporated milk, ground beef, pork chops, chicken, lima beans, peanuts, popcorn, and breads. The levels of contamination aren't sufficient to cause health problems, FDA officials say. But little information exists on the interactions lindane may be having with the dozen or more other chemical contaminants typically associated with food production-for instance, benzene and chlorpyrifos-that are often found in tandem with lindane. -Bob Weinhold

A Better Grade of School Bus
The California Air Resources Board has launched a $50 million initiative to reduce the harmful emissions produced by the state's 24,000 school buses. Each school day, the buses emit 13 tons of nitrogen oxides and almost half a ton of particulate matter. As part of the initiative, 375 of the state's oldest, most polluting buses will be replaced by cleaner diesel or alternative fuel buses. A portion of the funds will help build new fueling facilities and defray the added costs of low-sulfur diesel fuel. Another $12.5 million will be spent to retrofit the remaining diesel buses with filter traps to reduce particulate emissions by at least 85%. The new and retrofitted buses will be in place by the 2002-2003 school year.

Sensing Lead in the Environment
A lead-detecting sensor developed at the University of Illinois at Urbana-Champaign is less expensive and less complicated than current methods and can be used to obtain real-time, onsite data. The sensor employs small pieces of DNA that were discovered through an in vitro selection process to be sensitive to lead. Researchers manipulated the single-strand DNA to create a special site at which the metal ion would bond. A fluorescent tag has been added to the DNA to boost the sensor's detection abilities.
The sensor can be used for environmental and industrial monitoring as well as for clinical toxicology applications. Researchers believe that the principles used to develop this sensor can be applied to obtain DNA biosensors for other metals such as mercury, cadmium, calcium, and potassium.

Screening Chinese Children for Lead
On 6 January 2001 China started the first phase of an enormous children's lead screening program in Shenyang, the capital of the northeastern province of Liaoning. All of the city's children under age seven, as well as pregnant women, will have medical examinations to determine if and how they have been affected by lead poisoning. Study participants will also receive lessons on how to protect themselves from lead exposure.
The project, which will also be carried out in seven other provinces, is expected to test 8-10 million children, who are increasingly at risk for lead exposure as China's economy grows and with it transportation and industrial pollution levels. According to the Xinhua News Service, an earlier study conducted in the northern city of Taiyuan found that 64% of the 10,000 children tested had lead concentrations much higher than acceptable limits.

Green Lessons Boost Grades
Using the environment as a context for learning changes student outlook and results in better academic performance across grades K-12 compared with traditional schooling, according to Environment-Based Education: Creating High Performance Schools and Students, a report issued in September 2000 by the National Environmental Education & Training Foundation (NEETF). Environment-based education uses the environment as a thematic focus for interdisciplinary, hands-on learning. Students in environment-based programs not only raise their reading and math scores but also perform better in science and social studies and are better able to transfer knowledge from familiar to unfamiliar contexts, according to the report. Classroom discipline problems also decline.
The report examined a diverse group of schools and grade levels in Texas, North Carolina, Wisconsin, Minnesota, Kentucky, and Florida. It includes case studies at five individual schools with environment-based programs, a model school program, and the statewide Kentucky public school system. "The fact that environment-based education boosted achievement regardless of geographic location and socioeconomic status is most significant," says Mary Smith, director of environmental education with the National Audubon Society.
According to the report, students' natural interest in the environment motivates them to learn and understand the complexities of their world, which translates into higher test scores. For example, the report describes the case of Isaac Dickson Elementary School in Asheville, North Carolina, where fourthgrade students saw a 31% boost in math achievement after just one year of environment-based learning as measured by state achievement tests.
Teachers also observed increased student motivation and improved classroom behavior after introducing environment-based programs. In fact, known troublemakers often find an interest in academics through the hands-on opportunities the environment offers as a classroom, the report says. "Teacher after teacher in Kentucky reported that students previously performing at low academic levels 'came alive' when introduced to an environment-based curriculum," says the report. The NEETF report follows the seminal 1998 State Education and Environment Roundtable report Closing the Achievement Gap: Using the Environment as an Integrating Context for Education, which examined 40 schools in 12 states and detailed the success of environment-based programs in motivating student interest and improving academic achievement. "The new report provides more evidence the first study wasn't a fluke," says Andy Finch, senior director of NEETF's Education and the Environment Program.
The two studies attempt to take the case studies further by linking anecdotal evidence with test scores, Smith says. But there are limits to this approach as well. Whether test scores truly quantify academic achievement remains controversial. In addition, comparing data among states is difficult because achievement measures are increasingly state-specific, says report author Joanne Lozar Glenn.
The NEETF report recommends creating an Office of Ombudsman for the Environment to establish links among the Department of Education, the Environmental Protection Agency, the National Science Foundation, and other federal agencies concerned with U.S. education. The report also suggests conducting demonstration projects in order to improve community outreach programs and attract greater community involvement. It further recommends expanding cooperative educational ventures with resources such as public and private parks, marine sanctuaries, and nature centers.

Uncovering the Viral Mechanics of Croup
Researchers say they have solved the crystal structure of a key protein that allows the paramyxoviruses-the major cause of croup and other diseases-to attach to cells, which in turn permits the viruses to invade cells and cause respiratory illness. The new research, published in the November 2000 issue of Nature Structural Biology, may provide a basis for developing drugs to fight these diseases.
Members of the paramyxovirus family include the parainfluenza viruses (which cause respiratory infections), the mumps virus, and the Newcastle disease virus (which affects birds). "Most people who are infected with parainfluenza get symptoms similar to a bad cold," says Susan Crennell, the postdoctoral student at the University of Bath in England who used X-ray crystallographic techniques to identify the protein. "In very young children, this can develop into life-threatening respiratory disease including pneumonia." She adds, "Paramyxoviruses can be very dangerous to people-especially children-with compromised immune function." The multifunctional paramyxovirus protein hemagglutininneuraminidase (HN) is critical to the invasion process that creates illness, Crennell says. HN, a protein on the surface of the virus, binds to cells, permitting a cascade of molecular events that results in the virus fusing to the target cell and then incorporating itself into the cell. Once inside the cell, the virus then has established a base of operations from which it can infect other cells.
Crennell says the project was made difficult because HN is a flexible protein that changes shape with different periods of the cell's life cycle. Before HN binds to the cell, it has a different shape than after it attaches. Once HN has bound to the target, the structure of the cell prevents a mediator-a drug, for example-from interfering with HN's work setting the infectious stage of development into motion. Crennell says the key to preventing HN from working is to know its shape prior to binding so it can be attacked before initiating the events that lead to fusion.
The study was performed using a form of the Newcastle disease virus. Crennell says that particular virus was used because scientists found they could get a crystalline structure of the Newcastle HN protein, something that has so far evaded researchers using the viruses that cause human disease. "The HN protein on this virus we believe is very close to that found on the human virus," says Crennell. With the crystal form available, Crennell exposed the structure to X rays. Computers analyzed the subtle changes in the signals produced by the X rays in order to show the shape of the protein.
Crennell says the structure provides the basis for the structurebased design of inhibitors for a range of paramyxovirus-induced diseases. Other researchers, including principal investigator Garry Taylor of the University of St. Andrews in Fife, Scotland, are just beginning to use the crystal structure of HN to design drugs that can prevent binding and keep the virus from causing illness.
"We have no vaccines or effective drugs to stop viruses like parainfluenza," says Fran Rubin, program officer for respiratory diseases at the National Institute of Allergy and Infectious Diseases, which partially funded Crennell's study. "This study opens new possibilities for drug development." She adds, "This report is the most exciting discovery in the field in quite some time." -Ed Susman

Dirty Air Stunts Lung Growth
Long-term exposure to air pollutants such as nitrogen dioxide and particulate matter can cause reduced lung growth in children, and the effects are more pronounced in areas where air pollution is highest, according to findings from the Children's Health Study, a 10-year longitudinal study at the University of Southern California in Los Angeles.
Started in 1993, the Children's Health Study is following the respiratory development of over 3,000 fourth-, seventh-, and tenth-graders from 12 communities throughout California with different levels and types of air pollution. Each year the students undergo lung function tests at school. The researchers also measure levels of ozone, nitrogen dioxide, particulate matter smaller than 10 microns in diameter, and acid vapors at each test site (gasoline and diesel engine emissions are the major source for all four pollutants). Data collected during the first four years of the study were reported in the October 2000 issue of the American Journal of Respiratory and Critical Care Medicine by lead author W. James Gauderman, an assistant professor of preventive medicine at the university, and his colleagues.
Exposure to nitrogen dioxide, particulate matter, and acid vapors has been found to have the greatest association with impaired lung growth, with ozone having a statistically insignificant effect. Compared to students living in the least polluted communities, children living in the most polluted communities have shown a reduction in two measures of lung function: a cumulative 3.4% reduction in FEV 1 (forced expiratory volume) and a 5.0% reduction in MMEF (maximal midexpiratory flow). The FEV 1 , or amount of air expelled in the first second of blowing, measures how well the large and medium-sized airways in the lung are functioning, explains Gauderman, whereas the MMEF measures small airway function. Although children with reduced lung growth probably experience no outward symptoms of lung problems, they may be at increased risk as adults for chronic respiratory problems such as asthma and emphysema, says Gauderman. The longitudinal effect found in the first cohort has now been replicated in a second cohort, with identical results.
Children who spend the most time outdoors experience the greatest effect of air pollution on their lung function. Generally, advises Gauderman, children should minimize outdoor playing on high-pollution days. Reports on regional air pollutant levels are often available through TV weather channels and local meteorology offices.
"No one else has followed a cohort of schoolchildren of this age for this long a time," says pulmonologist David Bates of the University of British Columbia in Vancouver. He says the study's findings are "a major contribution" in understanding the long-term consequences of air pollution on children's lungs. Moreover, in 120 of the children who moved out of the study area and were tracked separately, lung function growth was greater in those who moved to cleaner environments compared to those who moved to more polluted areas. This is a "striking confirmation of the longitudinal study," Bates adds. -Carol Potera

Smog stunts lungs.
Results of a long-term study show that air pollutants decrease lung growth in children and may lead to increased risk for respiratory disease as adults.

Diazinon Use Exterminated
In December 2000 the U.S. EPA banned indoor use of diazinon, a pesticide sold under various trade names (including Ortho, Spectracide, and Real-Kill) that is widely used for commercial and household insect control. The 1996 Food Quality Protection Act requires the agency to restrict or ban a pesticide if it poses a specific threat to children's health. A May 1999 EPA draft study on diazinon concluded that the pesticide may pose greater health risks than previously thought. The chemical, which can cause nausea, headaches, diarrhea, and weakness when inhaled, is one of the leading causes of insecticide poisoning in the United States, according to the EPA. Outdoor applications of diazinon will be phased out over a four-year period.

EPA Acts for Kids
On 15 December 2000 the EPA announced it would for the first time require power plants to reduce mercury emissions, saying that these emissions pose significant human health hazards, especially to children and fetuses. The agency said it will propose regulations by 2003 and issue final rules by 2004.
Additional EPA efforts to protect children's health were announced in January 2001. On January 4, the agency set new standards for acceptable levels of environmental lead. Presently, nearly 1 million U.S. children have dangerously elevated blood lead concentrations. Under the new standards, lead is considered a hazard if there is more than 40 µg/ft 2 on floors, more than 250 µg/ft 2 on interior windowsills, and more than 400 ppm in bare soil in children's play areas.
On January 5, the agency established the Voluntary Children's Chemical Evaluation Program. This program calls for better evaluation of the risks to children from chemicals that they could be exposed to on a daily basis. Under this program, the EPA is asking manufacturers to voluntarily provide key testing data on 23 specific chemicals (including acetone, benzene, and toluene) that were selected because studies had found them in human tissue samples.

Enviro-Friendly Farm Funding
The USDA has granted The Rodale Institute funds for research on how to increase the profitability and efficiency of family farms through organic and regenerative agriculture methods, which have the added benefit of promoting environmental health. A portion of the $800,000 grant, which will target farms in the U.S. Mid-Atlantic region, will be used to study methods such as notill farming and nutrient loading through compost programs. Plus, farmers will be educated on how they can put the research findings into practice. Money will also be allocated to educate extension agents in organic practices and to develop alternative marketing strategies for use by small farmers to increase their profits.

A 257
The Beat Scientists since the time of the ancient Greeks have noted the phenomenon of disease clusters, in which diseases occur in greater-than-expected numbers over a short time in a small area. In the 1850s, pioneering epidemiologist John Snow was one of the first physicians to construct maps solely for the purpose of studying a disease (in his case, cholera). His work was important not only for discovering how cholera is transmitted but also for showing the correlation between geography and disease. Over the past few decades, the use of maps as epidemiologic tools has greatly expanded with the development of computer mapping technologies and the use of the Internet.
In April 2001, the National Cancer Institute (NCI) launched its Cancer Mortality Maps & Graphs Web site, located at http://www.nci.nih.gov/atlasplus/. The site brings online the Atlas of Cancer Mortality in the United States, 1950-94, one of a series of atlases produced by the institute since 1975 showing geographic patterns of disease mortality through color-coded maps. These reference works provide epidemiologists and public health scientists a unique resource for studying disease patterns on both spatial and temporal levels. The new Web site will be updated with additional cancer data as they become available.
Using atlas data, factors contributing to high rates of certain types of cancer have been identified. One example is the finding that elevated death rates from lung cancer in several southeastern U.S. coastal communities were linked to exposure of shipyard workers to asbestos during World War II. Another is that variations in cigarette smoking greatly influence the patterns of certain tobacco-related cancers, such as lung, larynx, esophagus, and oral cavity cancers.
The central core of the site is the atlas itself, accessible through the Atlas of Cancer Mortality in the United States: 1950-94 (Book) link. The atlas provides maps, text, tables, and figures for more than 40 cancers for the time periods 1950-1969 and 1970-1994. Clicking on the Mortality Maps and Rates by Cancer link leads to in-depth overviews of specific types of cancer, including conventional, complementary, and alternative treatment options; clinical trial information; and links to resources and support groups.
Visitors may also choose the Customize Mortality Maps option under each cancer type to create mortality charts and graphs based on any combination of parameters such as cancer type, geographic unit, time period, age group, race, and sex. These charts and graphs can be accessed by the visually impaired through text files that can be converted to speech with a screen reader. Back on the home page, the Customize Mortality Maps and Interactive Mortality Charts and Graphs links provide direct routes to these mapping options.
NCI officials hope this resource will be used not only by researchers and the general public to gain greater insight into patterns of cancer deaths but also by policy makers for allocating resources based on disease rates, availability of health care, and other demographic factors. The Centers for Disease Control and Prevention is using the Web site as a model for an injury mortality Web site it is developing, while several schools, including the Harvard School of Public Health and George Washington University, are using the site as a classroom teaching tool. The NCI plans to expand the site to include noncancer mortality mapping and graphing as well as other variables including environmental, occupational, and economic data. -Erin E. Dooley


extfieldsofstudy: Biology Medicine

text:
MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner

Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of ε-subunit–specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

Introduction
The establishment of the neuromuscular junction involves the accumulation of acetylcholine receptor (AChR)* at the postsynaptic membrane (Burden, 1998;Sanes and Lichtman, 1999), and requires nerve terminal-secreted agrin (McMahan, 1990;Gautam et al., 1996). Recent experiments using in vivo transfection suggested that neural agrin is sufficient to induce on muscle fiber membranes changes which resemble postsynaptic specializations (Cohen et al., 1997;Jones et al., 1997;Meier et al., 1997;Rimer et al., 1997). The agrininduced receptor accumulation requires the muscle-specific receptor tyrosine kinase (MuSK) (Valenzuela et al., 1995;DeChiara et al., 1996). According to the present view, agrin activates MuSK, which initiates an as yet unresolved complex signal transduction cascade and serves as a primary scaffolding element for the establishment of the postsynaptic membrane (Glass et al., 1996Apel et al., 1997). Attempts to identify distinct domains of MuSK that interact with other synaptic components have highlighted the importance of the kinase activity for the induction of AChR clusters Zhou et al., 1999;Herbst and Burden, 2000). The ectodomain of MuSK may be required for agrin responsiveness and for interactions with rapsyn (Zhou et al., 1999), a cytoplasmic protein that is associated with AChR (Froehner, 1991) and promotes AChR clustering at the synapse (Gautam et al., 1995).
The steps that lead to elevated levels of MuSK at synaptic sites, its interactions with agrin and rapsyn, and the signals that cause clustering of postsynaptic proteins remain elusive. Also, most experiments so far were done in cell culture systems where MuSK-dependent signaling has been assayed in the presence of the putative ligand, neural agrin. In such experiments it is not possible to exclude a direct contribution of the ligand itself to the observed effects attributed solely to the activation of MuSK. Therefore, we used ectopic expression in single muscle fibers (Jones et al., 1997;Sander et al., 2000) to analyze the function of MuSK in the formation of the postsynaptic apparatus in vivo and independent of agrin. The results show that ectopic expression of MuSK induces the formation of postsynaptic-like specializations, including the appearance of adult-type AChR ⑀ subunit transcripts and the aggregation of synaptic proteins such as AChR and acetylcholinesterase (AChE). AChR clusters were also induced by a truncated MuSK form lacking the ectodomain or a form where the ectodomain was replaced by GFP. To exclude any contribution of endogenous MuSK, we employed mutant mice (Hesser, 2000;unpublished data) where MuSK was inactivated conditionally upon expression of transgenic Cre recombinase by direct gene transfer. High resolution analysis revealed that rapsyn-green fluorescent proteins (GFPs), but not MuSK-GFP fusion proteins, were colocalized with AChR clusters. This suggests that a direct physical interaction between rapsyn and MuSK may not be a prerequisite for the formation of AChR clusters. The results show that MuSK activity initiates signals that lead to the formation of AChR clusters, a process which does not require ligand-dependent activation or ectodomain regions. Part of this work was presented in abstract form.

Transgenic MuSK, expressed in single muscle fibers, induces AChR clusters and aggregation of AChE
By direct gene transfer into individual muscle fibers it was shown that agrin induced the formation of ectopic AChR clusters (Cohen et al., 1997;Jones et al., 1997;Meier et al., 1997;Rimer et al., 1997). Using the same approach, it was found that injection of MuSK plasmid DNA alone results in the absence of neural agrin and also in the formation of AChR clusters (Fig. 1 A; Hesser et al., 1999;Jones et al., 1999). MuSK-induced AChR clusters differed, however, from clusters induced following overexpression of neural agrin. Agrin-induced clusters appeared larger in size and more spread out both on the injected fiber as well as on adjacent fibers (Jones et al., 1997;see Figs. 3 and 4). In the case of rapsyn, which is essential for the clustering of AChR at developing synapses (Gautam et al., 1995), we observed that ectopic injection of rapsyn plasmid DNA alone did not induce AChR clustering (unpublished data). Since rapsyn could be linked to MuSK and AChR , we asked whether MuSK-induced AChR clusters might be altered when expressed in the presence of transgenic rapsyn. Coinjection of MuSK and rapsyn DNA resulted in the formation of AChR clusters ( Fig. 1 B) which were similar to the clusters induced by MuSK alone as shown in Fig. 1 A.
Transgenic MuSK mediated not only the formation of AChR clusters (Fig. 1, A-C) but also the accumulation of AChE ( Fig. 1 D), which appeared colocalized with AChR clusters. No AChE clusters were detected in control fibers which had been injected with nGFP DNA only. These re- Plasmid DNA for nGFP and MuSK was injected ectopically in single muscle fibers of innervated soleus muscles of rats. Muscles were excised 21 d after injection. Green fluorescence of nGFP in single fibers indicated expression of transgenic proteins (unpublished data). AChR clusters induced by MuSK are detected by staining with r-bgt (7 rats were used for DNA injection; a total of 96 fibers were injected and AChR clusters were detected in 61 fibers). (B) Muscle fibers were injected with plasmid DNA for nGFP, MuSK, and rapsyn and analyzed as in A. Expression of MuSK and rapsyn induces AChR clusters comparable to clusters observed in A. 10 rats were used for DNA injection; a total of 138 fibers were injected and AChR clusters were detected in 76 fibers. A and B are projections of confocal images. (C-F) Muscle fibers were injected with plasmid DNA for nGFP, MuSK, and rapsyn. Muscles were excised 28 d after injection and analyzed as in A. (C) Fluorescence microscopic view of r-bgt-labeled AChR, induced by transgenic MuSK. (D) AChE aggregates, visualized according to Koelle and Friedenwald (1949), appear colocalized with the MuSK-induced AChR clusters (3 rats were used for DNA injection and a total of 59 fibers were injected. AChR clusters were detected together with AChE aggregates on 34 fibers). In control-injected fibers, no AChE clusters were detected (3 rats were used for nGFP DNA injection without MuSK and rapsyn DNA; a total of 66 fibers were injected and 31 fibers were nGFPpositive but without AChR or AChE clusters). (E) Cross-section of muscle region containing fiber with MuSK-induced ectopic AChR clusters. AChR clusters are located in the plasma membrane of the injected muscle fiber. (F) Cross-section, shown in E, was subjected to in situ hybridization using an ⑀ subunit-specific, 35 S-labeled antisense probe. No hybridization signals were detected using sense probes for hybridization (unpublished data). The AChR-expressing fiber contains ⑀ subunit-specific mRNA (three independent hybridization experiments were performed with thin sections of soleus muscle from two rats; AChR-positive fibers expressed in all cases ⑀ -subunit transcripts).
sults demonstrate that MuSK expressed at extrasynaptic sites in innervated muscle fibers caused the formation of AChR and AChE clusters. The AChR clusters could be newly synthesized or result from aggregation of preexisting, extrasynaptic AChR. Therefore, we prepared serial cross-sections of muscle with fibers containing MuSK-induced AChR clusters to examine whether ⑀ subunit transcript levels were elevated. Since the ⑀ subunit transcripts of normal innervated muscle are restricted to synaptic nuclei and are not expressed in extrasynaptic fiber regions (Brenner et al., 1990), their appearance was taken as evidence of the expression of new AChR. As shown in Fig. 1 E, AChR were located in the plasma membrane of the injected muscle fiber and appeared to form aggregates of varying size. In situ hybridization of the cross-section shown in Fig. 1 E revealed ectopic expression of ⑀ subunit transcripts in fibers containing MuSKinduced AChR clusters ( Fig. 1 F). Positive hybridization signals were never observed outside the AChR cluster regions of injected fibers nor in adjacent noninjected fibers ( Fig. 1  F), suggesting that MuSK-dependent signals mediate either directly or indirectly expression of ⑀ subunit gene transcripts and formation of AChR clusters at the sites of MuSK DNA injection.

MuSK kinase activity is required for induction of AChR clusters
Expression of the kinase-inactive mutant MuSK K608A  in single muscle fibers confirmed that no AChR clusters were induced (Fig. 2). To visualize and demonstrate directly the expression of recombinant MuSK in the injected muscle fibers, we fused GFP with MuSK (MuSK-GFP) and with the kinase-inactive mutant MuSK K608A (MuSK K608A -GFP), as schematically summarized in Fig. 2. Fig. 3, A and D, demonstrate that both constructs were expressed with similar efficiency. MuSK-GFP gave rise to AChR clusters which were clearly identified with rhodamine-labeled ␣ -bungarotoxin (r-bgt) (Fig. 3 B). The overlay of the green and red fluorescence confocal images (Fig. 3 C) shows that MuSK-GFP was not strictly colocalized with the AChR clusters. Statistical analysis revealed that in all transgeneexpressing fibers, as marked by GFP, AChR clusters were formed ( Fig. 3 F). No AChR clusters, however, were induced by the kinase-inactive mutant MuSK K608A -GFP ( Fig.  3, D, E, and G and Fig. 2), demonstrating that kinase activity is required for the induction of AChR clusters. Rapsyn coinjected with the MuSK mutant was unable to form AChR clusters.

Inactivation of endogenous MuSK by Cre-mediated recombination of the MuSK gene in single muscle fibers
The results so far suggest that expression of transgenic MuSK initiates signals which lead to the formation of AChR clusters. However, overexpression of transgenic MuSK could activate the expression of endogenous MuSK, as suggested by Jones et al. (1999). In this case, it would be difficult to ana-  lyze the functional role of transgenic MuSK expressed in single muscle fibers. To exclude any contribution of endogenous MuSK in live muscle fibers, endogenous MuSK should be inactivated. Since mice lacking MuSK die at birth (DeChiara et al., 1996), we made use of genetically modified mice where the exon encoding the kinase domain of MuSK was flanked by loxP sites (unpublished data). In these mice it is possible to express Cre recombinase upon DNA injection. Transgenic Cre should mediate the recombination of the loxP-containing MuSK gene and thus lead to the inactivation of endogenous MuSK. Biochemical analysis of single muscle fibers or immunochemical detection of endogenous MuSK in whole mount muscle preparations using commercially available antibodies lack the sensitivity required to unequivocally establish the presence or absence of endogenous MuSK. Therefore, we developed a bioassay based on the AChR cluster-inducing activity of agrin, which revealed that endogenous MuSK was indeed inactivated locally in fibers expressing Cre: injection of agrin DNA into the extrasynaptic regions of muscle fibers led to the appearance of AChR clusters on the injected and adjacent fibers (Fig. 4 A). In such a view, however, it was not possible to decide on the exact location of the AChR clusters. Cross-sections of single confocal images revealed that the r-bgt-labeled AChR were located in the plasma membrane of the injected and noninjected muscle fibers (unpublished data). Fig. 4 B represents a reconstructed xz projection of the complete confocal image series over the range indicated by white arrows shown in Fig. 4 A. It demonstrated that the injected fiber contained AChR clusters in the plasma membrane "surrounding" the nGFP-expressing fiber.
Since agrin-mediated AChR clustering requires MuSK (Glass et al., 1996;Zhou et al., 1999), AChR clusters should not appear in fibers lacking endogenous MuSK. Therefore, muscle fibers were injected with Cre DNA together with agrin and nGFP. The maximum projection of a confocal image series in Fig. 4 C demonstrates successful gene transfer by the appearance of agrin-induced AChR clusters. Cross-sections of single confocal images (unpublished data) as well as the reconstructed xz projection (Fig. 4 D) of the confocal image series (Fig. 4 C) demonstrated clearly that the Cre-injected fibers, identified by nGFP, were not surrounded by r-bgt fluorescence and thus had not formed AChR clusters. However, AChR clusters were located on fibers adjacent to or in the vicinity of the injected fibers. Thus, the locally restricted gene knock-out of MuSK in the Cre-injected muscle fibers inhibited agrin-mediated AChR cluster formation.
To strengthen the observation that AChR cluster formation was completely prevented in muscle fibers expressing Cre, we developed an additional bioassay using rapsyn-GFP. In cell culture experiments it has been shown previously that rapsyn-GFP forms fluorescently labeled complexes with AChR (Ramarao and Cohen, 1998). If such complexes were also formed in rapsyn-GFP-injected muscle fibers, one could directly and reliably monitor the appearance of AChR clusters. In fact, coinjecting rapsyn-GFP with agrin DNA resulted in the appearance of AChR clusters that were exactly colocalized with rapsyn-GFP (Fig. 5 A) and demonstrated that induction and aggregation of AChR can be readily detected and assigned to the injected muscle fiber. Transgenically expressed rapsyn-GFP is therefore a specific and sensitive marker to monitor the appearance of newly formed AChR clusters in vivo. We never observed rapsyn-GFP-AChR clusters in Cre-expressing fibers (Fig. 5 B), demonstrating that local, Cre-mediated DNA recombination in single muscle fibers efficiently inactivated endogenous MuSK and thus prevented agrin-induced activation of AChR expression.
Finally, if MuSK signaling activates AChR expression it should be possible to rescue the Cre-mediated MuSK knock-out by expressing MuSK transgenically. MuSK DNA was therefore coinjected together with Cre and agrin DNA. Cross-sections of the confocal images allow to assign AChR clusters to individual muscle fibers. Reconstructed xz projection of the confocal image series (A) over the range (y) marked by white arrowheads. AChR clusters, located in the plasma membrane, surround the nGFP, transgene-expressing muscle fiber (marked by asterisk). AChR clusters are also located on neighboring noninjected fibers. (Bottom) Location of muscle fibers is indicated schematically and AChR cluster-expressing fibers are labeled red. Transgene-expressing fiber is marked by asterisk. (C) Maximum projection of a confocal image series. Two nGFP-expressing fibers (green) had been injected with agrin and nGFP DNA plus Cre DNA to conditionally inactivate endogenous MuSK. Agrin-induced AChR clusters (red) are apparently distributed as in A. Expression of nGFP and AChR clusters shows successful expression of the transgenic proteins. (D) Reconstructed xz projection of the confocal image series (C) over the range (y) marked by white arrowheads. AChR clusters are not found on the nGFP, Cre-expressing muscle fibers (marked by asterisks), but only on neighboring noninjected fibers. Cre-mediated inactivation of endogenous MuSK prevents formation of AChR clusters. (Bottom) Location of muscle fibers is indicated schematically. AChR cluster-expressing fibers are labeled red. Transgene-expressing fibers are marked by asterisks. 7 mice were used for DNA injection; a total of 39 GFP-expressing fibers were analyzed but contained no ectopic AChR clusters.
Fig. 5 C reveals that AChR/rapsyn-GFP complexes were clustered in the injected muscle fiber and successful expression of transgenic proteins was again confirmed by the agrin-induced AChR clusters on the neighboring fibers. These results, summarized in Fig. 5 D, demonstrate that endogenous MuSK can be inactivated conditionally in single muscle fibers and transgenic MuSK mediates the appearance of AChR clusters in the absence of endogenous MuSK.

Ectodomain of transgenic MuSK is not required for induction of AChR clustering
There is evidence that MuSK signaling requires the ectodomain of MuSK Glass et al., 1997). To determine whether MuSK-induced AChR clustering requires the ectodomain, we constructed a mutant lacking the complete extracellular region ( ⌬ ectoMuSK; see Fig. 2). Fig. 6 A shows several representative AChR cluster patterns induced by transgenic MuSK. The number and size of the AChR clusters vary to some extent and is probably due to the variable amounts of MuSK expressed upon injection. The expression of ⌬ ectoMuSK led to the formation of AChR clusters ( Fig. 6 B), which were comparable to the clusters induced by full length MuSK. Again, to prove successful expression of the transgene in the injected muscle fibers, the ⌬ ectoMuSK construct was fused with GFP where GFP replaced the ectodomain ( ⌬ ectoMuSK-GFP; see Fig. 2). Injection of this DNA led to efficient expression of ⌬ ecto-MuSK-GFP, which induced AChR clusters (Fig. 6 C). The kinase-inactive mutant ⌬ ectoMuSK K608A -GFP (Fig. 2) was also expressed efficiently, but was unable to induce AChR clusters (Fig. 6 D). Thus, deletion of the ectodomain does not interfere with MuSK's ability to induce aggregation of AChR.
To exclude any contribution of endogenous MuSK, we performed the same experiments in MuSK loxP/ Ϫ mice where endogenous MuSK was inactivated locally in the injected muscle fiber by transgenic Cre. The results demonstrate that ⌬ ectoMuSK-GFP was expressed efficiently (Fig. 7 A) and induced the appearance of AChR clusters (Fig. 7 B). Since endogenous MuSK had been inactivated by Cre, this observation confirms that the ectodomain of MuSK is not an integral component of the mechanisms underlying MuSKinduced AChR expression. The overlay of green and red fluorescence images (Fig. 7 C) suggests that ⌬ ectoMuSK-GFP is not colocalized with r-bgt-labeled AChR.

Distribution of AChR clusters, MuSK-GFP, and rapsyn-GFP
When comparing the distribution of r-bgt-labeled AChR clusters and MuSK-GFP (Fig. 3 C) or ⌬ ectoMuSK-GFP (Fig. 7  C), it appeared that the two proteins were not strictly colocalized on the surface of the muscle fibers and MuSK-GFP fusion proteins were observed outside of AChR clusters and vice versa. This was surprising in view of the current model, in which the ectodomain of MuSK has to be structurally linked to AChR. Therefore, one would expect that a significant portion of the AChR clusters overlaps with the GFP-labeled MuSK molecules. Rapsyn is thought to interconnect AChR and MuSK via the hypothetical linker RATL . Therefore, we analyzed at higher resolution whether rapsyn was distributed like MuSK or was associated more strictly with AChR (as Figure 5. Rapsyn-GFP identifies ectopically induced AChR clusters. Single muscle fibers of MuSK loxP/Ϫ mice were injected with DNA, as indicated by asterisks, and expression of transgenic products was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images of a confocal image series are shown as maximum projections. (A) Expression of agrin and rapsyn-GFP as indicated. Agrin induces ectopic AChR clusters. AChR in the injected muscle fiber form complexes with rapsyn-GFP and AChR clusters appear yellow in the overlay. The r-bgt labeled AChR clusters on noninjected fibers are red. (2 MuSK loxP/Ϫ mice were used for injection; a total of 43 fibers were injected and 26 fibers expressed agrin and rapsyn-GFP. In all injected transgene-expressing fibers AChRs were colocalized with rapsyn-GFP and appeared yellow). (B) Expression of agrin, Cre, and rapsyn-GFP as indicated. Transgenic Cre inactivates endogenous MuSK. The absence of AChR/rapsyn-GFP clusters demonstrates that the formation of AChR clusters was prevented in injected muscle fibers. Agrin-induced AChR clusters are found only on neighboring noninjected fibers. 2 MuSK loxP/Ϫ mice were used for DNA injection; a total of 40 fibers were injected; 28 fibers expressed agrin and rapsyn-GFP; no AChR clusters were detected on the injected fibers. (C) Expression of agrin, Cre, rapsyn-GFP, and MuSK as indicated. AChR induced in the injected fiber form complexes with rapsyn-GFP and AChR clusters appear yellow in the overlay. The Cre-mediated inactivation of the MuSK gene was rescued by expression of transgenic MuSK (3 MuSK loxP/Ϫ mice were used for DNA injection; a total of 62 fibers were injected and 33 fibers expressed agrin and rapsyn-GFP. Endogenous MuSK inactivation was rescued in 22 fibers, which contained AChR/rapsyn-GFP complexes.) (D) Graphic summary of experiments described in A-C shows that Cre-mediated inactivation of endogenous MuSK prevents the formation of AChR clusters. The conditional MuSK gene knock out is rescued with high efficiency by transgenic MuSK: AChR clusters are found in 73% of transgeneexpressing fibers.
indicated already in Fig. 5). MuSK and rapsyn-GFP were injected ectopically to induce AChR clusters and the distribution of rapsyn-GFP relative to AChR clusters was analyzed by confocal microscopy. Single sections revealed that rapsyn-GFP was strictly colocalized with AChR stained by r-bgt (Fig. 8, A-C). In contrast, when MuSK-GFP and rapsyn were injected, MuSK-GFP displayed a different distribution and was spatially distinct from AChR clusters (Fig. 8, E-G).
This was confirmed by a colocalization analysis using all sections of the confocal image series (see Materials and methods). Fig. 8, D and H, show two-dimensional graphs from colocalization histograms of the MuSK/rapsyn-GFPand MuSK-GFP/rapsyn-injected fibers, respectively. The intensity of the GFP fluorescence is plotted on the x-axis and r-bgt fluorescence on the y-axis; the intensity contrast of the two channels was scaled similarly. Data points located near the diagonal line (running through the origin, slope of 1) thus indicate colocalization. From Fig. 8 D it can be seen that all data points were located proximal to the diagonal and are therefore in accordance with the view that rapsyn-GFP and AChR form stoichiometric complexes. In Fig. 8 H, the data points displayed no high degree of codistribution, indicating that MuSK-GFP and AChR were not colocalized. This suggests that a physical link between MuSK-GFP and rapsyn is not a prerequisite for the formation of AChR clusters, which are induced by MuSK kinase activity.

Discussion
Using the gene transfer method, in which plasmid DNA is directly injected into individual muscle fibers of adult rats (Jones et al., 1997;Sander et al., 2000), we tested whether Figure 6. MuSK lacking the ectodomain induces AChR clusters. Rat muscle fibers were injected with DNA of rapsyn and MuSK mutants. After 21 d, muscles were excised. MuSK-induced AChR clusters were visualized by r-bgt. (A) Expression of MuSK-GFP and rapsyn, as indicated, induces AChR clusters. Several different AChR cluster patterns are presented and demonstrate that size and shape of MuSK-induced clusters may vary, probably depending on the concentration of transgenic MuSK expressed upon direct gene transfer. (B) Expression of ⌬ectoMuSK-GFP and rapsyn, as indicated, induces AChR clusters similar to that shown in A (4 rats were used for DNA injection; a total of 47 fibers were injected and AChR clusters were detected on 28 fibers). (C) Diagram: ⌬ectoMuSK-GFP induces AChR clusters as observed in A and B. In all transgene-expressing fibers, AChR clusters are formed (3 rats were used for DNA injection; a total of 48 fibers were injected; 28 fibers expressed ⌬ectoMuSK-GFP and AChR clusters were detected on all 28 fibers). (D) Diagram: kinase-inactive ⌬ectoMuSK K608A -GFP fails to induce AChR clusters (2 rats were used for DNA injection; a total of 25 fibers were injected and 14 ⌬ectoMuSK K608A -GFP-expressing fibers were detected; none of the fibers had ectopic AChR clusters). MuSK, as a component of the putative agrin receptor complex, could induce postsynaptic specializations at ectopic sites in a ligand-independent manner. Overexpression of receptor tyrosine kinases has been shown to lead to their ligand-independent activation, presumably by increased formation of dimers (Schlessinger and Ullrich, 1992;Hubbard et al., 1998). A similar mechanism may activate MuSK when it is expressed ectopically in muscle fibers. Our results show that the expression of MuSK, but not MuSK K608A , leads to the appearance of AChR clusters and ⑀ subunit gene transcripts at the site of gene transfer. Furthermore, AChE aggregates were colocalized with the AChR clusters. So far, no kinase-signaling pathways have been detected that respond to agrin/MuSK activation. However, the fact that overexpression of MuSK in the absence of neural agrin leads to the ectopic appearance of ⑀ subunit mRNA indicates that MuSK-mediated signals could influence gene transcription of the AChR subunit genes and possibly other synaptically expressed genes. Whether this gene induction is directly dependent on MuSK kinase activity or on proteins that bind to activated MuSK (Zhou et al., 1999;Herbst and Burden, 2000) remains to be investigated. There is also the possibil-ity that signaling was indirectly mediated by erbB receptors, which appear to become concentrated at the MuSK scaffold (Burden, 1998;Meier et al., 1998). Recent findings showed that erbB3-deficient mice (Riethmacher et al., 1997), as well as erbB2-deficient mice with a heart-specific erbB2 rescue (Morris et al., 1999), display only minor deficits in early postsynaptic development, suggesting that erbB receptor kinase signaling may not be linked to MuSK to activate AChR subunit gene expression.
The MuSK kinase signal is thought not to be sufficient to elicit MuSK action . Additional domains located in the extracellular region of MuSK appear to be required for the clustering of AChR. The ectodomain mediates agrin-dependent activation via the putative receptor MASC and may interact physically with a hypothetical linker, RATL, to allow MuSK to recruit rapsyn-AChR complexes to a primary scaffold in the postsynaptic membrane . However, in myotubes cultured from MuSK Ϫ / Ϫ mutant mice it was observed that AChR clusters are also induced in a ligand-independent manner by MuSK, even without being colocalized with AChR clusters (Zhou et al., 1999). Using cultured MuSK Ϫ / Ϫ myotubes, the authors demonstrated that formation of agrin-induced as well as spontaneous AChR clusters requires MuSK.
Gene transfer experiments in single muscle fibers demonstrate now that AChR clusters can be induced by spontaneous MuSK kinase activity in vivo. Since the AChR-inducing activites of MuSK and ⌬ ectoMuSK are similar, the clustering process appears not to require additional signals mediated by the ectodomain of MuSK. Jones et al. (1999) induced AChR clusters in muscle fibers by expressing transgenic MuSK chimeras. They found endogenous MuSK associated with the ectopic AChR clusters and concluded that endogenous MuSK, recruited by the transgenic kinase activity, induced AChR clustering via its ectodomain. If the ectodomain was actually required for the formation of AChR clusters one would assume that expression of transgenic full length MuSK, which would not need to recruit endogenous MuSK, was more potent in the induction of AChR clusters than the truncated ⌬ ectoMuSK mutant. However, this was not the case, indicating that kinase activity is sufficient to induce AChR clusters.
Nevertheless, endogenous MuSK has to be inactivated to exclude any contribution to the observed changes induced by transgenic MuSK and MuSK mutants. Conditional MuSK gene knock out has been achieved in MuSK loxP/ Ϫ mice by expressing transgenic Cre. Using the agrin-mediated formation of ectopic AChR clusters as bioassay we showed that endogenous MuSK is inactivated, which prevents the formation of ectopic AChR clusters. The fact that the local MuSK gene knock out is rescued by transgenic MuSK supports the view that agrin requires MuSK to induce AChR clusters. The inactivation of endogenous MuSK, combined with the expression of MuSK mutants lacking the entire ectodomain, demonstrates in addition that the ectodomain of MuSK can be deleted or replaced by GFP without obvious effects on the ability to induce AChR clusters. The apparently ligandindependent activation of MuSK is thus sufficient to initiate signals that lead to the formation of AChR clusters and postsynaptic-like specializations. As there are only very few preexisting AChR in extrasynaptic regions of innervated muscle fibers, it is assumed that transgenic MuSK leads to the production of new AChR which are then organized into clusters.
The AChR clusters induced upon expression of MuSK or of MuSK and rapsyn together do not differ significantly in size and shape, indicating that the endogenous pool of rapsyn is sufficient for the formation of ectopic AChR clusters. In heterologous expression systems transgenic rapsyn self-aggregates and promotes clustering of AChR when both proteins are coexpressed (Froehner et al., 1990;Phillips et al., 1991). However, expression of rapsyn in ectopic muscle fiber regions does not result in the appearance of AChR clusters, demonstrating that AChR are not simply recruited and clustered from preexisting AChR by transgenic rapsyn, as may be the case in cultured myotubes.
AChR clusters are also induced by agrin in MuSK Ϫ / Ϫ cell cultures transfected with MuSK mutants lacking the putative RATL binding domain, indicating that MuSK-rapsyn/ RATL interactions are dispensable for at least some aspects of MuSK function (Zhou et al., 1999). The finding that the GFP-labeled kinase and AChR clusters can be spatially distinct in muscle fibers lends support to the view that the rapsyn/AChR clustering process does not require a structural linker protein between MuSK and AChR. MuSK signals act locally restricted, since transgenic MuSK-GFP is not transported over a wide distance but is expressed within a spatially limited range at the DNA injection site, where it determines formation of AChR clusters.
These results lead to the hypothesis that MuSK signals mediate an increased expression of AChR subunits which are assembled to form complexes with endogenous rapsyn. Postsynaptic specializations develop only at sites where MuSK is concentrated, under natural conditions at contact sites of nerve and muscle. The clustering of AChR that we observe upon transgenic expression of MuSK may thus reflect a physiological process occurring before synapse formation. It was reported that in topoisomerase-deficient mice, motor axons are not developed. Yet, there are AChR concentrated in the central region of the diaphragm muscle (Yang et al., 2000). This suggests that the accumulation of AChR occurs in the absence of neuronal factors. However, AChR are not accumulated in muscle fibers lacking MuSK, although motor axons are present in these muscles (DeChiara et al., 1996), demonstrating that MuSK is required for the accumulation of AChR. While this paper was under review, two papers were published where AChR expression was investigated in embryonic mutant mice lacking agrin, MuSK, rapsyn, and/or motor nerves (Lin et al., 2001;Yang et al., 2001). The results show that MuSK and rapsyn are required for early postsynaptic differentiation which occurs in absence of agrin or motor axons. In this scenario (Fig. 9) MuSK accumulates in the central, developmentally oldest region of muscle fibers. Reaching a critical threshold, spontaneous kinase activity is strong enough to induce accumulations of AChR, similar to those we observe upon transgenic expression of MuSK. In a second step, agrin (and possibly other factors) released by incoming motor axons might favor the formation of denser accumulations by locally activating MuSK. The local action of MuSK and the local secretion of (2) Agrin released by incoming motor axons supports the formation of denser accumulations by locally activating MuSK, possibly by mechanisms suggested in previous models (Zhou et al., 1999). The local action of MuSK and the local secretion of agrin reinforce each other to generate the extreme density of postsynaptic proteins encountered at the neuromuscular junction. See Discussion for further information.


s2fieldsofstudy: Biology Medicine

text:
Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice.

Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185neu antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8+ lymphocytes. Specific anti–HER-2/neu antibodies were produced and a nonpolarized activation of CD4+ and CD8+ cells secreting IL-4 and interferon (IFN)-γ were evident. A central role for IFN-γ in the preventive effect was proven by the lack of efficacy of vaccination in IFN-γ gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-γ is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-γ knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu–expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

Introduction
Immunological prevention of tumors is a concept based on the ability of immunity to arrest tumor progression in healthy individuals with a high risk of cancer. Successful re-sults have been reported in mice treated with chemical carcinogen (1) and in transgenic mice that develop mammary (2) or prostate (3) carcinomas by using IL-12 (1,2), cellular vaccines (4), DNA vaccines (5,6), recombinant proteins (7), and adoptive immunotherapy (3).
While these studies provide significant proofs of concept, the protection afforded was markedly influenced by the model adopted. Balb/c female mice transgenic for the transforming rat HER-2/neu oncogene (Balb-neuT mice) display an early development of multifocal mammary carcinomas. In these mice, final tumor incidence was not affected but only significantly delayed by IL-12 systemic treatment, while a 50% reduction in tumor incidence was observed in FVB mice transgenic for rat HER-2/neu protooncogene (FVB-neuN), which develop fewer tumors with a longer latency period (2). Both cytokines and antigen-specific vaccines were effective, albeit through markedly different reaction mechanisms (1)(2)(3)(4)(5)(6)(7). It should be noted that in HER-2/neu transgenic mice the existence of immunological tolerance to HER-2/neu, similar to what is observed in patients with neu-expressing breast cancer, has been documented (6,8).
This paper reports the efficacy of combined allogeneic tumor cell vaccination and IL-12 treatment in the prevention of mammary carcinomas in virgin Balb-neuT mice. Within the first 5 mo of age the tissue-specific expression of HER-2/neu causes a progression in all their ten mammary glands from atypical hyperplasia to an invasive carcinoma that closely resembles human neoplasia (9). The combination of cell vaccines and cytokine treatment we have chosen inhibited tumor onset in a model of rapid progression to malignant mammary carcinoma.

Materials and Methods
Mice. Balb-neuT female mice (H-2 d ) overexpressing the transforming activated rat HER-2/neu oncogene under control of the mouse mammary tumor virus promoter (2) were bred under specific pathogen-free conditions by Charles River Laboratories. IFN-␥ gene knockout Balb/c mice from Jackson Immu-noResearch Laboratories were crossed with Balb-neuT, and IFN-␥ knockout Balb-neuT mice (IFN-␥ Ϫ / Ϫ Balb-neuT mice) selected by PCR analysis. The total number of backcrosses of Jackson IFN-␥ knockout mice to Balb/c mice was 10. When spleen cells from IFN-␥ knockout Balb-neuT mice were used as stimulator in mixed lymphocyte cultures, no significant activation of proliferative response of Balb-neuT responder T lymphocytes was ever found. Only individually tagged virgin females were used in experiments described here and were treated according to the European Community guidelines. Mammary glands were inspected weekly, and tumor masses measured with calipers in two perpendicular diameters. Progressively growing masses of Ͼ 3 mm in mean diameter were regarded as tumors. Growth was monitored until all 10 mammary glands displayed a tumor or until a tumor exceeded a mean diameter of 1.5 cm, at which time mice were killed for humane reasons.
Vaccination and IL-12 Treatment. Starting at the sixth week of age Balb-neuT mice received successive 3-wk courses of four twice-weekly intraperitoneal vaccinations with 2 ϫ 10 6 allogeneic Neu/H-2 q mammary carcinoma cells in 0.4 ml of PBS, followed by five daily intraperitoneal administrations of recombinant mouse IL-12 (provided by S. Wolf, Genetics Institute, Andover, MA) in the third week. In the first course the five injections were of 50 ng of IL-12 in 0.2 ml of PBS supplemented with 0.01% mouse serum albumin (MSA; Sigma-Aldrich), the subsequent injections were of 100 ng. After 1 wk of rest, the course was repeated until mice were killed or were 1 y old. Control Balb-neuT mice received either mock vaccination or MSA administration. Their tumor progression mirrored that of untreated mice.

Results
Prevention of Mammary Carcinogenesis. Vaccination with allogeneic mammary carcinoma cells (Neu/H-2 q ) expressing high surface levels of both p185 neu and H-2 q class I molecules ( Fig. 1) followed by intraperitoneal administration of IL-12 was used to halt mammary carcinogenesis in Balb-neuT mice. IL-12 was chosen because of its ability to significantly delay tumor onset (1,2). Beginning when the mice were 6 wk old, they received Neu/H-2 q cells twiceweekly in the first and second week, followed by five daily administrations of IL-12 in the third week. After 1 wk of rest this course was repeated until mice were killed or reached the age of 1 y.
This combined treatment effectively inhibited the development of mammary carcinomas (Fig. 2). All control mice developed their first mammary tumor within 22 wk, when all mice treated with Neu/H-2 q cells plus IL-12 were completely tumor free. Within 30 wk all control mice developed tumors in all 10 mammary glands. At 52 wk (end of the study) 88% of mice treated with Neu/H-2 q cells plus IL-12 were still completely tumor free; their lifetime was more than doubled.
Weight of the Treatment Components. IL-12 alone produced a significant delay in tumor latency and a significant reduction in tumor multiplicity, but did not affect tumor incidence ( Fig. 2), in agreement with our previous findings (2). Vaccination with Neu/H-2 q cells alone delayed tumor onset, but did not result in tumor-free mice ( Fig.  2), thus indicating that IL-12 markedly contributes to the efficacy of the combined treatment. Vaccination with the Neu neg /H-2 q cells alone was ineffective and its combina-  tion with IL-12 resulted in a protection similar to that afforded by IL-12 alone (Fig. 2). Therefore, p185 neu recognition appears to play a central role in halting HER-2/neu carcinogenesis.
As the block of cell proliferation by MitC may reduce the immunogenicity of a vaccine (15), while vaccination with replicating cells poses practical issues, the need for live and replicating cells was also evaluated. The combination of MitC-treated Neu/H-2 q cells with IL-12 resulted in a block of carcinogenesis and all mice were tumor free at 1 yr (Fig. 3). Vaccination with MitC-treated cells allowed us to evaluate also the efficacy of syngeneic Neu/H-2 d cells, which otherwise would be tumorigenic; no protection was obtained with such cells alone, whereas the delay in tumor occurrence induced by Neu/H-2 d cells plus IL-12 was not longer than that elicited by IL-12 alone (see Fig. 2 for comparison). These data point to a requirement for both allogeneic MHC molecules and p185 neu recognition for an effective protection.
Pathological Evolution of the Mammary Gland. By the third week of age foci of atypical hyperplasia were evident in the numerous side buds (9, 16) of control mice and subsequently extended to all 10 mammary glands. Foci of carcinoma in situ first apparent around the fifteenth week ( Fig.  4 A) evolved to invasive lobular carcinomas by the twentieth. 10 wk later invasive lobular carcinomas were present in all the glands. Treatment with IL-12 alone slowed down this progression. The atypical foci grew much more slowly and the onset of in situ and invasive carcinomas was delayed by 5-6 wk. The lesions were invaded by reactive cells (Fig. 4 B), whereas a similar infiltrate was barely visible in the controls. At the fifteenth week of age the side buds and the terminal ductal end buds (TEBs) of mice treated with Neu/H-2 q cells plus IL-12 were carpeted with a single layer of epithelial cells and surrounded by reactive cells. The few small hyperplastic foci were the scene of intense inflammation (Fig. 4 C). After the fifth course (at 27 wk), the mammary tissue was mainly composed of small ducts with few TEBs. Remaining side buds and TEBs were slightly hyperplastic and surrounded by reactive cells. These were sometimes found inside the lobular structures among the epithelial cells ( Fig. 4 D-F).
In mice treated with Neu/H-2 q cells plus IL-12, attenuated inflammation was present at 35, 40, and 45 wk of age after the eighth or tenth course. In very few mice only invasive lobular carcinomas developing from hyperplastic foci were microscopically detectable. Their lobules were less compact than in the controls, while the neoplastic epithelial cells were poorly coherent and frequently disaggregated, and gave rise to fissures. The stroma inside and around the tumor was more abundant and occupied by a distinct inflammatory infiltrate. Reactive cells were numerous in the stroma of both the hyperplastic and the tumor lesions, and occasionally intermingled with the neoplastic cells after crossing or damaging the basal membrane.
Immunohistochemical examination after the second course (fifteenth week of age) revealed a reduction of hyperplastic foci and TEBs containing epithelial cells expressing p185 neu . At 27 wk, the mammary tissue of mice treated with Neu/H-2 q cells plus IL-12 was only constituted by small ducts lined by a single layer of epithelial cells without or with a slight p185 neu expression confined to the cytoplasm ( Fig. 5 A, C, and E) associated with a poor positivity to PCNA (Fig. 5 B, D, and F). By contrast, a marked cytoplasmic and membrane p185 neu expression, similar to hyperplastic and neoplastic lesions from untreated and IL-12treated mice, was displayed by the few carcinomas detected in mice treated with IL-12 and cell vaccine.
A conspicuous macrophage, neutrophil, and CD8 ϩ lymphocyte infiltrate was present in the hyperplastic foci and in the tumor stroma of mice treated with Neu/H-2 q cells plus IL-12 (Table I). The number of dendritic and NK cells also increased, though it was not higher than in mice treated with IL-12 only. Recruitment of reactive cells was accompanied by overexpression of endothelial adhesion molecules in the small vessels.
Significantly fewer blood vessels were found in mammary glands of mice treated with Neu/H-2 q cells plus IL-12 and of those receiving IL-12 only than in the controls. By contrast, proinflammatory cytokines and chemokines were more evident in mice treated with Neu/H-2 q cells plus IL-12 than in mice receiving IL-12 only and were almost absent in mammary glands from untreated controls. Cell-mediated Reactivity. At the fifteenth week of age, after the second course, groups of 3-5 control and treated mice were killed to evaluate spleen cell reactivity. Both total spleen cell yield and the absolute number of CD4 ϩ cells were significantly higher in mice treated with Neu/H-2 q cells plus IL-12. However, CTL assays against HER-2/ neu-positive or -negative targets showed only %01ف of specific lysis of HER-2/neu-positive targets by spleen cells from Balb-neuT mice vaccinated with Neu/H-2 q cells plus IL-12. These spleen cells did not display any protective activity when they were admixed with a tumorigenic dose of Neu/H-2 d syngeneic mammary tumor cells in a Winntype neutralization assay in Balb-neuT mice (data not shown). On the other hand, an increased spontaneous proliferation was displayed in vitro by the CD4 ϩ lymphocyte subpopulation. It was further increased in an antigen-specific manner by the addition of mitomycin-blocked syngeneic Neu/H-2 d cells. Moreover, total spleen cells and CD8 ϩ lymphocytes of mice repeatedly treated with Neu/ H-2 q cells plus IL-12 spontaneously released more IFN-␥ than leukocytes from control or IL-12-treated mice (Fig.  6). Large amounts of IFN-␥ were released by total and CD8 ϩ spleen cells after restimulation by Neu/H-2 q cells. IL-4 was secreted spontaneously or after restimulation by both total spleen cells and CD4 ϩ lymphocytes (but not by CD8 ϩ lymphocytes) from mice treated with Neu/H-2 q cells plus IL-12. This cytokine release pattern was triggered by the recognition of both H-2 q allogeneic MHC glycoproteins and p185 neu since IFN-␥ and IL-4 were produced not only in response to p185 neu plus alloantigens (Neu/ H-2 q ) but also by the presentation of p185 neu on syngeneic cells (Neu/H-2 d ) as well as by alloantigens alone (Neu neg / H-2 q ) ( Table II).
Role of IFN-␥. In IFN-␥ knockout Balb-neuT mice the combined administration of Neu/H-2 q cells plus IL-12 was totally ineffective (Fig. 7). This fading of the protection points to the crucial role of IFN-␥ in the prevention observed. Along with its numerous immunomodulatory activities, IFN-␥ may also directly modulate tumor cell phenotype and the in vivo behavior of tumor cells. Our results suggest that induced IFN-␥ can affect the behavior of Neu/H-2 d cells by inhibiting their angiogenic phenotype and the HER-2/neu-mediated signaling pathways leading to the neoplastic behavior (11). In vitro, IFN-␥ inhibited the proliferation of Neu/H-2 d cells, downmodulated their membrane expression of p185 neu (which was even reduced by 80% by the combination of IFN-␥ and TNF-␣) and the production of the proangiogenic protease MMP-9, while it are surrounded by an evident infiltrate composed of reactive cells that sometimes are found within epithelial cells after crossing or damaging the basal membrane. Immunohistochemistry reveals that granulocytes (E) and CD8 ϩ lymphocytes (F) are present not only in the stroma but also inside the lobular structure after crossing the damaged basal membrane (arrowheads). A-C, original magnification: ϫ200; D, original magnification: ϫ400; E and F, original magnification: ϫ630. strongly upmodulated the production of antiangiogenic cytokines such as IP-10 and MIG (Fig. 8).
Antibody Response. High-titer antibodies binding Neu/ H-2 q cells were detected in sera from mice vaccinated with allogeneic Neu/H-2 q cells ( Fig. 9 A). Staining of Neu Neg / H-2 q cells was much lower than Neu/H-2 q cells (mean fluorescence intensity 61 Ϯ 9 vs. 332 Ϯ 11), thus suggesting that sera of H-2 d mice vaccinated with Neu/H-2 q plus IL-12 contained p185 neu -reactive antibodies along with alloreactive anti-H-2 q antibodies. Neither untreated control mice nor mice treated with IL-12 alone produced antibody against Neu/H-2 q cells ( Fig. 9 A). To circumvent the issue of alloreactivity we performed a Western blot analysis ( Fig.  9 B), which showed that antibodies of mice vaccinated with Neu/H-2 q plus IL-12 specifically recognized p185 neu (Fig. 9 B). Only sera from mice receiving cell vaccines resulted cytotoxic against syngeneic Neu/H-2 d tumor cells, whereas no lysis was observed against syngeneic p185 neunegative H-2 d lymphocytes (Fig. 9 C). The lysis was stronger when Neu/H-2 q cell vaccine was combined with IL-12 administration.
A much lower antibody response was displayed by IFN-␥ knockout Balb-neuT mice treated with Neu/H-2 q cells plus IL-12. Cytofluorometric analysis of serum binding to Neu/H-2 q cells showed a mean fluorescence activity of 124 Ϯ 44 for IFN-␥ knockout Balb-neuT mice versus 396 Ϯ 39 for Balb-neuT mice (P Ͻ 0.01).
Analysis of Ig subclasses in sera from Balb-neuT transgenic mice treated with the cell vaccine or with the vaccine plus IL-12 is shown in Fig. 10. The combined treatment with Neu/H-2 q cells plus IL-12 significantly (P Ͻ 0.05) increased the level of IgG1, IgG2a, and IgG2b. By contrast, in IFN-␥ knockout BALB-neuT mice receiving the vaccine plus IL-12 the treatment failed to increase the level of all Ig subclasses except IgG1.

Discussion
Combination of an allogeneic cell vaccine with systemic IL-12 prevented the onset of mammary carcinoma in tumor-prone Balb-neuT mice. Their lifespan was more than doubled, while the quality of their life was not impaired. The protection afforded was much more effective than those previously obtained in the same mice with the exogenous cytokine alone (2) or DNA vaccination (12). It was even stronger than that elicited by cytokines (2), DNA, or protein vaccines (5)(6)(7) in other lines of HER-2/neu transgenic mice which develop fewer tumors with a longer latency. The treatment combined different immunological stimuli, namely p185 neu , allogeneic class I MHC glycoproteins, and IL-12. Their separate evaluation showed that all were required. Balb-neuT mice receiving allogeneic Neu/H-2 q cells or IL-12 alone displayed a slower carcinogenesis, but all eventually succumbed to carcinoma. Only mice receiving the combination were protected from carcinoma for up to 1 y.
To analyze the contribution of cell-bound determinants we replaced Neu/H-2 q cells in the vaccine either with Neu neg /H-2 q cells, a p185 neu loss variant, or with Neu/ H-2 d cells, expressing p185 neu on a syngeneic background. The various cell lines derive from independent transgenic carcinomas (11,12), therefore the possibility that other factors may contribute to their different vaccine effect cannot be ruled out. The markedly higher protection afforded by IL-12 in combination with Neu/H-2 q cells, as opposed to the poor resistance of mice vaccinated with IL-12 and Neu neg /H-2 q cells, underlines the crucial importance of p185 neu recognition. Similarly, the weight of allogeneic MHC signal was evident when the effective protection from carcinogenesis of mice receiving IL-12 and allogeneic Neu/H-2 q cells was compared with the scarce efficacy of treatments with IL-12 and syngeneic Neu/H-2 d cells. Allorecognition may provide a favorable cytokine microenvironment, but also induce a rapid destruction of  the vaccine and facilitate the cross-presentation of p185 neu by host APCs (17).
Tumor prevention appeared to rest on the activation of multiple immune mechanisms. The dissection of their in vivo role was made difficult by the complexity of the reaction elicited and the duration of treatments, however dis-    tinct immune responses were conspicuously present or absent in association with protection from tumor onset. IL-12 alone significantly delays tumor progression through its effects on cell-mediated immunity and angiogenesis (18,19). Tumors arising in Balb-neuT mice receiving IL-12 alone were infiltrated by dendritic cells, NK cells, and CD8 ϩ lymphocytes. The local release of IFN-␥ induced the expression of tertiary antiangiogenic mediators such as IP-10 and MIG, and the microvessel density was decreased. Similar findings have been obtained in other experimental models where IL-12 was employed for cancer prevention (1,2).
A stronger inflammatory response was evident in mice receiving IL-12 and Neu/H-2 q allogeneic cells. Local production of MIP-2 and the expression of endothelial adhesion molecules correlated with the recruitment of granulocytes which may damage tumor vessels and mediate antibody-dependent cytotoxicity (20).
The combined treatment, but not IL-12 alone, elicited a marked specific humoral immune response. Anti-p185 neu antibodies in the sera of treated mice may impair carcinogenesis by inducing a functional block of p185 neu receptor function, downregulating its expression on the cell membrane (21), and impeding its ability to form the homo or heterodimers that spontaneously transduce proliferative signals to the cells (21,22). The reduced number of p185 neupositive cells, the intracytoplasmic confinement of p185 neu , and the diminished nuclear positivity to anti-PCNA monoclonal antibody in the mammary glands of mice treated with Neu/H-2 q cells plus IL-12 after the second course point to a direct inhibitory activity by anti-p185 neu antibody. The mammary glands of treated mice displayed fewer and fewer side buds after each course. This is a critical issue in tumor prevention since progression leading to the formation of invasive lobular carcinomas originates from side buds proliferating cells (PCNA-positive) that highly express p185 neu (9,16).
Activation of CD4 ϩ and CD8 ϩ cells by the combined treatment was associated with cytokine release within the mammary gland and in the spleen. Moreover, infiltrating lymphocytes were present in the stroma surrounding neoplastic cells and penetrated the basal membrane to interact with p185 neu -positive neoplastic epithelial cells. This type of tumor infiltration by lymphocytes was not observed in Balb-neuT receiving IL-12 alone (2,23) or Neu/H-2 q cells alone. In human carcinomas and melanoma, the presence Figure 9. Antibody production by Balb-neuT mice. (A) Cytofluorometric analysis of serum binding to Neu/H-2 q cells. Each bar represents the mean Ϯ SEM of sera (diluted 1:65) from three mice at the fifteenth week of age bled after the second course of the indicated treatment. (B) Neu/H-2 q and Neu neg /H-2 q cell extracts were immunoprecipitated by sera from three mice treated with Neu/H-2 q cells plus IL-12, PBS as negative control, and anti-rat p185 neu monoclonal antibody 7.16.4 as positive control (Neu mAb). Western blot analysis was performed using the antineu polyclonal antibody C18. (C) Complement dependent cytotoxicity against syngeneic Neu/H-2 d cells and Balb-neuT lymphocytes, which do not express p185 neu (H-2 d ). The percentage of viable lymphocytes was determined after a 30-min incubation with sera of mice bled after the second course of treatment (diluted 1:10) followed by a 30-min incubation with rabbit low-tox complement (diluted 1:10). Results are expressed as 100 Ϫ % viable lymphocytes. of a reactive infiltrate intermingled with tumor cells, as opposed to the presence of leukocytes at the periphery of tumor nests, often correlates with a more favorable prognosis (24,25).
In vitro no evidence of a major involvement of specific CTL activity was found. Even though we cannot rule out the possibility that CTL activity could contribute to the preventive effect, data reported here show that in vitro cytotoxicity and Winn-type tumor neutralization assay were not predictive of treatment efficacy. The absence of a measurable CTL activity in lymphoid organs does not exclude direct killing of HER-2/neu positive mammary cells at the tissue level, but on the whole, it is unclear if vaccination elicited T cells against HER-2/neu.
A major contribution of T cells, whether alloreactive or recognizing HER-2/neu, was the copious production of IFN-␥. The high IFN-␥ secretion by CD8 ϩ T cells from mice receiving Neu/H-2 q allogeneic cells plus IL-12 and the loss of efficacy of this combined treatment in IFN-␥ knockout Balb-neuT mice point to a central role of IFN-␥ in the inhibition of carcinogenesis. Along with its numerous activities, IFN-␥ directly modulates tumor cell phenotype and immunogenicity (26,27). It is also the crucial downstream mediator elicited by IL-12 (20) that induces the expression of tertiary antiangiogenic mediators such as IP-10 and MIG, and decreases the density of tumor microvessels (19). Our in vitro data (28) suggest that IL-12induced IFN-␥ modulates the behavior of Neu/H-2 d cells by inhibiting their angiogenic phenotype and the HER-2/ neu-mediated signaling leading to the neoplastic behavior (11). IFN-␥ is also required for the complete activation of effector T cells (29) and is crucial for the induction of C-fixing IgG2a and IgG2b subclasses (30,31).
In principle IFN-␥ might also favor tumor growth through the downmodulation of tumor antigens (32), however, this effect is probably irrelevant in HER-2/neu carcinogenesis, because antigen p185 neu expression is required for tumor growth (11), and the major consequence of its downmodulation would be the loss of tumorigenicity, rather than the enhancement of tumor growth resulting from reduced tumor recognition by T cells. Other contradictory effects of IFN-␥ in vivo, such as the enhancement of metastatic spread (33), are exerted only on late stages of tumor progression, thus do not play a role in the early steps of mammary carcinogenesis which were prevented by the combined treatment used here.
In summary the lack of efficacy of the combined treatment in IFN-␥-deficient HER-2/neu transgenic mice demonstrated that IFN-␥ is an essential component of the immune response leading to mammary carcinoma prevention. The breeding of Balb-neuT mice selectively deficient for other immune functions will allow to better appraise the weight of immune responses that were either strongly induced by the combined treatment (e.g., humoral immunity), or of uncertain value in determining the protective effect (e.g., CTL activity).
In conclusion each component of the combined treatment played a role, and a complete protection was ob-tained when all components were included to induce a wide spectrum of immune responses. Their concerted action stopped a devastating cancer-prone condition affecting all the ten mammary glands, and more than doubled the lifespan of Balb-neuT mice. This impressive result suggests that a similar approach might prevent tumors in persons with a high risk of cancer.