It has been estimated that 30–40 percent of all cancers can be prevented by lifestyle and dietary measures alone. Obesity, nutrient sparse foods such as concentrated sugars and refined flour products that contribute to impaired glucose metabolism (which leads to diabetes), low fiber intake, consumption of red meat, and imbalance of omega 3 and omega 6 fats all contribute to excess cancer risk. Intake of flax seed, especially its lignan fraction, and abundant portions of fruits and vegetables will lower cancer risk. Allium and cruciferous vegetables are especially beneficial, with broccoli sprouts being the densest source of sulforophane. Protective elements in a cancer prevention diet include selenium, folic acid, vitamin B-12, vitamin D, chlorophyll, and antioxidants such as the carotenoids (α-carotene, β-carotene, lycopene, lutein, cryptoxanthin). Ascorbic acid has limited benefits orally, but could be very beneficial intravenously. Supplementary use of oral digestive enzymes and probiotics also has merit as anticancer dietary measures. When a diet is compiled according to the guidelines here it is likely that there would be at least a 60–70 percent decrease in breast, colorectal, and prostate cancers, and even a 40–50 percent decrease in lung cancer, along with similar reductions in cancers at other sites. Such a diet would be conducive to preventing cancer and would favor recovery from cancer as well. The field of investigation of the role of nutrition in the cancer process is very broad. It is becoming clearer as research continues that nutrition plays a major role in cancer. It has been estimated by the American Institute for Cancer Research and the World Cancer Research Fund that 30–40 percent of all cancers can be prevented by appropriate diets, physical activity, and maintenance of appropriate body weight . It is lMost of the research on nutrition and cancer has been reductionist; that is, a particular food or a nutrient has been studied in relation to its impact on tumor formation/regression or some other end point of cancer at a particular site in the body. These studies are very helpful in seeing the details of the mechanisms of disease. However, they do not help give an overall picture of how to prevent cancer on a dietary level. Even less, they tell little of how to eat when a person already has a cancer and would like to eat a diet that is favorable to their recovery.This review will focus on those dietary factors which has been shown to be contribute to increased risk of cancer and then on those additional protective dietary factors which reduce cancer risk. Finally, some whole-diet studies will be mentioned which give a more complete picture of how these individual factors work together to reduce cancer risk.Eating too much food is one of the main risk factors for cancer. This can be shown two ways: (1) by the additional risks of malignancies caused by obesity, and (2) by the protective effect of eating less food.Obesity has reached epidemic proportions in the United States. Sixty-four percent of the adult population is overweight or obese . About 1It was estimated in a recent study, from a prospective cancer prevention cohort, that overweight and obesity accounted for 14 percent of all cancer deaths in men and 20 percent of those in women . SignifiOn the other side, careful menu planning brings about an approach entitled CRON-Calorie Restriction with Optimal Nutrition. The basic idea is to eat a reduced amount of food  while still consuming all of the necessary amounts of vitamins, minerals, and other necessary nutrients. The only restriction is the total amount of energy  that is consumed. While being difficult to practice, this approach has a lot of scientific merit for being able to extend average life spans of many species of animals including rats, mice, fish, and possibly primates (currently being tested). Along with this life span extension is a reduction in chronic diseases that are common to mankind, reviewed in Hursting et al . A recenRefined sugar is a high energy, low nutrient food – junk food. "Unrefined" sugar (honey, evaporated cane juice, etc) is also very concentrated and is likely to contribute to the same problems as refined sugar. Refined wheat flour products are lacking the wheat germ and bran, so they have 78 percent less fiber, an average of 74 percent less of the B vitamins and vitamin E, and 69 percent less of the minerals (USDA Food database, data not shown). Concentrated sugars and refined flour products make up a large portion of the carbohydrate intake in the average American diet. One way to measure the impact of these foods on the body is through the glycemic index.The glycemic index is an indication of the blood sugar response of the body to a standardized amount of carbohydrate in a food. The glycemic load takes into account the amount of food eaten. An international table of the glycemic index and glycemic load of a wide variety of foods has been published .Case-control studies and prospective population studies have tested the hypothesis that there is an association between a diet with a high glycemic load and cancer. The case control studies have found consistent increased risk of a high glycemic load with gastric , upper a1c) is a time-integrated measurement of glucose control, and indirectly, of insulin levels. Increased risk in colorectal cancer was seen in the EPIC-Norfolk study with increasing HbA1c; subjects with known diabetes had a three-fold increased risk of colorectal cancer [1c, BMI > 30 kg/m2, or who used medications to control diabetes [Perhaps the dietary glycemic load is not consistently related to glucose disposal and insulin metabolism due to individual's different responses to the same glycemic load. Glycated hemoglobin  is low in fiber. In prospective health studies low fiber was not found to be a risk for breast cancer . It is pRed meat has been implicated in colon and rectal cancer. A Medline search in February 2003 uncovered 26 reports of human studies investigating the link between diet and colon or colorectal cancer. Of the 26 reports, 21 of them reported a significant positive relationship between red meat and colon or colorectal cancer ,41-64. AOmega 3 fats  have been shown in animal studies to be protect from cancer, while omega 6 fats (linoleic acid, arachidonic acid) have been found to be cancer promoting fats. Now there have been several studies that have tested this hypothesis in relation to breast cancer, summarized in Table Flax seed provides all of the nutrients from this small brown or golden hard-coated seed. It is an excellent source of dietary fiber, omega 3 fat , and lignans. The lignans in flax seed are metabolized in the digestive tract to enterodiol and enterolactone, which have estrogenic activity. In fact, flax seed is a more potent source of phytoestrogens than soy products, as flax seed intake caused a bigger change in the excretion of 2-hydroxyestrone compared to soy protein .Ground flax seeds have been studied for its effect on cancer, including several excellent studies by Lilian Thompson's research group at the University of Toronto. In one study the flax seed, its lignan fraction, or the oil were added to the diet of mice who had previously been administered a chemical carcinogen to induce cancer. All three treatments reduced the established tumor load; the lignan fraction containing secoisolariciresinol diglycoside (SDG) and the flax seed also reduced metastasis . In anotMore recently Thompson's research group studied mice that were injected with human breast cancer cells. After the injection the mice were fed a basal diet (lab mouse chow) for 8 weeks while the tumors grew. Then one group continued the basal diet and another was fed a 10% flax seed diet. The flax seed reduced the tumor growth rate and reduced metastasis by 45% .Flax seed has been shown to enhance mammary gland morphogenesis or differentiation in mice. Nursing dams were fed the 10% flax seed diet . After weaning the offspring mice were fed a regular mouse chow diet. Researchers then examined the female offspring and found an increased number of terminal end buds and terminal ducts in their mammary glands with more epithelial cell proliferation, all demonstrating that mammary gland differentiation was enhanced . When thoil intake or high blood levels of alpha-linolenic acid and prostate cancer risk [Other researchers have tested flax seed and prostate cancer. In an animal model using mice, Lin et al  found thcer risk . It is qOne of the most important messages of modern nutrition research is that a diet rich in fruits and vegetables protects against cancer.  There are many mechanisms by which fruits and vegetables are protective, and an enormous body of research supports the recommendation for people to eat more fruits and vegetables.Block et al  reviewedSteinmetz and Potter reviewed the relationship between fruits, vegetables, and cancer in 206 human epidemiologic studies and 22 animal studies . They foThere are many substances that are protective in fruits and vegetables, so that the entire effect is not very likely to be due to any single nutrient or phytochemical. Steinmetz and Potter list possible protective elements: dithiolthiones, isothiocyanates, indole-32-carbinol, allium compounds, isoflavones, protease inhibitors, saponins, phytosterols, inositol hexaphosphate, vitamin C, D-limonene, lutein, folic acid, beta carotene (and other carotenoids), lycopene, selenium, vitamin E, flavonoids, and dietary fiber .A joint report by the World Cancer Research Fund and the American Institute for Cancer Research found convincing evidence that a high fruit and vegetable diet would reduce cancers of the mouth and pharynx, esophagus, lung, stomach, and colon and rectum; evidence of probable risk reduction was found for cancers of the larynx, pancreas, breast, and bladder .Many of the recent reports from prospective population-based studies of diet and cancer have not found the same protective effects of fruits and vegetables that were reported earlier in the epidemiological and case-control studies [reviewed in ]. One exIt must be noted that upper intakes of fruits and vegetables in these studies are usually within the range of what people on an American omnivorous diet normally eat. In the Nurses Health Study the upper quintiles of fruit and vegetable intake were 4.5 and 6.2 servings/day, respectively . SimilarCruciferous vegetables (broccoli, cauliflower, cabbage, Brussels sprouts) contain sulforophane, which has anti-cancer properties. A case-control study in China found that intake of cruciferous vegetables, measured by urinary secretion of isothiocyanates, was inversely related to the risk of breast cancer; the quartile with the highest intake only had 50% of the risk of the lowest intake group . In the Broccoli sprouts have a very high concentration of sulforophane since this compound originates in the seed and is not made in the plant as it grows ,96. One Selenium is a mineral with anti-cancer properties. Many studies in the last several years have shown that selenium is a potent protective nutrient for some forms of cancer. The Arizona Cancer Center posted a selenium fact sheet listing the major functions of selenium in the body . These f1. Selenium is present in the active site of many enzymes, including thioredoxin reductase, which catalyze oxidation-reduction reactions. These reactions may encourage cancerous cells to under apoptosis.2. Selenium is a component of the antioxidant enzyme glutathione peroxidase.3. Selenium improved the immune systems' ability to respond to infections.4. Selenium causes the formation of natural killer cells.5. P450 enzymes in the liver may be induced by selenium, leading to detoxification of some carcinogenic molecules.6. Selenium inhibits prostaglandins that cause inflammation.7. Selenium enhances male fertility by increased sperm motility.8. Selenium can decrease the rate of tumor growth.A serendipitous randomized, double-blind, controlled trial of a 200 μg/day selenium supplement in the southeastern region of the USA (where soil selenium levels are low) found that the primary endpoints of skin cancer were not improved by the selenium supplement, but that other cancer incidence rates were decreased by selenium ,99. TherOverall, it appears that poor selenium levels, especially for men, are a cancer risk. If a person has low selenium levels and other antioxidant defenses are also low the cancer risk is increased even further. Women do not appear to be as sensitive to selenium, as breast cancer has not been found to be influenced by selenium status in several studies -104, altAll green plants also contain chlorophyll, the light-collecting molecule. Chlorophyll and its derivatives are very effective at binding polycyclic aromatic hydrocarbons (carcinogens largely from incomplete combustion of fuels), heterocyclic amines (generated when grilling foods), aflatoxin (a toxin from molds in foods which causes liver cancer), and other hydrophobic molecules. The chlorophyll-carcinogen complex is much harder for the body to absorb, so most of it is swept out with the feces. The chemoprotective effect of chlorophyll and its derivatives has been tested in laboratory cell cultures and animals ,108. TheVitamin B-12 has not been proven to be an anti-cancer agent, but there is some evidence indicating that it could be beneficial. The form of administered vitamin B-12 may be important.Some experimental cancer studies have been carried out with various forms of vitamin B-12. Methylcobalamin inhibited tumor growth of SC-3 injected into mice , and cauLaboratory mechanistic evidence for the effects of vitamin B12 were seen in a laboratory study with vitamin B-12 deficient rats. Choi et al  found thFolic acid is the dark green leafy vegetable vitamin. It has an integral role in DNA methylation and DNA synthesis. Folic acid works in conjunction with vitamin B-6 and vitamin B-12 in the single carbon methyl cycle. If insufficient folic acid is not available uracil is substituted for thymidine in DNA, which leads to DNA strand breakage. About 10% of the US population (and higher percentages among the poor) has low enough intakes of folic acid to make this a common problem . As showFolate may be more important for rapidly dividing tissue, like the colonic mucosa. Therefore, the cancer risk associated with low folate intake is probably higher for colon cancer than for breast cancer. Most of the breast cancer studies only found a protective effect of folate among women who consumed alcohol D has been shown to inhibit growth of colonic epithelial cells , primary2.The mortality rates for colon, breast, and ovary cancer in the USA show a marked north-south gradient . In ecolCarotenoids have been studied vigorously to see if these colorful compounds can decrease cancer risk. In ecological studies and early case-control studies it appeared that β-carotene was a cancer-protective agent. Randomized controlled trials of β-carotene found that the isolated nutrient was either neutral  or actuaHowever, there is a large body of literature that indicates that dietary carotenoids are cancer preventative  were associated with a 35% lower risk of prostate cancer. Furthermore, lycopene was even more protective for advanced stages of prostate cancer, with a 53% decrease in risk . A more In addition to the two reports above a nested case control study from the Health Professional Follow-up Study with 450 cases and controls found an inverse relation between plasma lycopene and prostate cancer risk (OR 0.48) among older subjects (>65 years of age) without a family history of prostate cancer . Among yIn a nested case-control study from the Physicians' Health Study cohort, a placebo-controlled study of aspirin and β-carotene, there was a 60% reduction in advanced prostate cancer risk (P-trend = 0.006) for those subjects in the placebo group with the highest plasma lycopene levels, compared to the lowest quintile. The β-carotene also had a protective effect, especially for those men with low lycopene levels .In addition to these observational studies, two clinical trials have been conducted to supplement lycopene for a short period before radical prostatectomy. In one study 30 mg/day of lycopene were given to 15 men in the intervention group while the 11 men were in the control group were instructed to follow the National Cancer Institute's recommendations to consume at least 5 servings of fruits and vegetables daily. Results showed that the lycopene slowed the growth of prostate cancer. Prostate tissue lycopene concentration was 47% higher in the intervention group. Subjects that took the lycopene for 3 weeks had smaller tumors, less involvement of the surgical margins, and less diffuse involvement of the prostate by pre-cancerous high-grade prostatic intraepithelial neoplasia . In anotVitamin C, or ascorbic acid, has been studied in relation to health and is the most common supplement taken in the USA. Low blood levels of ascorbic acid are detrimental to health  and vitWhile vitamin C is quite possibly an effective substance, the amounts required for these therapeutic effects are obviously beyond dietary intakes. However, intravenous ascorbate may be a very beneficial adjuvant therapy for cancer with no negative side effects when administered properly.There are many more substances that will have some benefit for cancer therapy. Most of these substances are found in foods, but their effective doses for therapy are much higher than the normal concentration in the food. For example, grape seed extract contains proanthocyanidin, which shows anticarcinogenic properties  and produce some B vitamins in the small intestine where they can be utilized. Beneficial bacteria help with food digestion by providing extra enzymes, such as lactase, in the small intestine. Beneficial bacteria help strengthen the immune system right in the gut where much of the interaction between the outside world and the body goes on. Beneficial bacteria can help prevent food allergies. They can help prevent cancer at various stages of development. These good bacteria can improve mineral absorption, maximizing food utilization.Lactobacillus species and Eubacterium aerofaciens, both producers of lactic acid, were associated with the populations with the lower risk of colon cancer, while Bacteroides and Bifidobacterium species were associated with higher risk of colon cancer [However, the balance of beneficial and potentially pathogenic bacteria in the gut is dependent on the diet. Vegetable fiber encourages the growth of beneficial bacteria. A group of Adventist vegetarians was found to have a higher amount of beneficial bacteria and lower amount of potentially pathogenic bacteria compared to non-vegetarians on a conventional American diet . Differen cancer There is a solid theoretical basis for why probiotics should help prevent cancer, especially colon cancer, and even reverse cancer. Probiotics produce short chain fatty acids in the colon, which acidify the environment. Lower colon pH is associated with lower incidence of colon cancer. Probiotic bacteria reduce the level of procarcinogenic enzymes such as beta-glucuronidase, nitroreductase, and azoreductase .L. casei was used in two trials of patients with superficial bladder cancer. In the first trial, the probiotic group had a 50% disease free time of 350 days, compared to 195 days for the control group [ol group . The secol group .Except for the two studies noted above, most of the research of probiotics and cancer has been done in animals. Studies have looked at markers of tumor growth or at animals with chemically induced tumors.B. longum, but only 29 and 21% by B. longum and inulin alone, respectively [Lactobacilli rhamnosus (LGG) and Bifidobacterium lactis (Bb12) had a significantly lower number of tumors compared to the control group [Studies in rats have shown that probiotics can inhibit the formation of aberrant crypt foci, thought to be a pre-cancerous lesion in the colon. Some of the best results were obtained with a probiotic strain consumed with inulin, a type of fructooligosaccharide. Total aberrant crypt foci, chemically induced, were reduced 74% by the treatment of rats with inulin and ectively . There wol group . A probiol group .Lactobacillus salivarium ssp. Salivarius UCC118, reduced fecal coliform levels, the number of potentially pathogenic Clostridium perfringens, and reduced intestinal inflammation. In this small study two mice died of fulminant colitis and 5 mice developed adenocarcinoma in the control group of 10 mice, while there was no colitis and only 1 mouse with adenocarcinoma in the probiotic test group [In lab mice bred to be susceptible to colitis and colon cancer, a probiotic supplement, st group .The research on probiotics and disease is still an emerging field. There is a high degree of variation of health benefits between different strains of bacteria. As new methods for selecting and screening probiotics become available, the field will be able to advance more rapidly.Many people diagnosed with cancer have digestion or intestinal tract disorders as well. Impaired digestion will greatly hinder a nutritional approach to treating cancer. If the nutrients cannot be released from the food and taken up by the body, then the excellent food provided by the Hallelujah Diet will go to waste. Digestive enzyme supplements are used to ensure proper and adequate digestion of food. Even raw foods, which contain many digestive enzymes to assist in their digestion, will be more thoroughly digested with less of the body's own resources with the use of digestive enzymes. So, the enzymes taken with meals do not have a direct effect upon a tumor, but assist the body in getting all of the nutrition out of the food for healing and restoring the body to normal function. Recently, an in vitro system was used to test the use of supplemental digestive enzymes. The digestive enzymes improved the digestibility and bioaccessibility of proteins and carbohydrates in the lumen of the small intestine, not only under impaired digestive conditions, but also in healthy human digestion .There is evidence that indicates the presence of an enteropancreatic circulation of digestive enzymes . DigestiEnzymes, especially proteases, if they reach systemic circulation, can have direct anti-tumor activity. Wald et al  reportedIn a similar experiment, an enzyme mixture of papain, trypsin, and chymotrypsin, as used in the preparation Wobe-Mugos E, was rectally given to mice that were inoculated with melanoma cells. Survival time was prolonged in the test group (38 days in the enzyme group compared to 24 days in the control mice) and 3 of the 10 enzyme-supplemented mice were cured. Again, a strong anti-metastatic effect of the proteolytic enzymes was seen .Further evidence of the efficacy of oral enzyme supplementation is available from clinical trials in Europe. Two different studies have demonstrated that two different oral proteolytic enzyme supplements were able to reduce high levels of transforming growth factor-β, which may be a factor in some cancers ,173. In Enzyme supplements have also been shown to reduce side effects of cancer therapy. Enzyme supplementation resulted in fewer side effects for women undergoing radiation therapy for carcinomas of the uterine cervix , for patEven though these few studies don't give a lot of evidence of the effectiveness of oral enzyme supplementation, it is clear that there are some circumstances that will be helped by enzyme supplementation, with very little danger of negative side effects. At the least, enzymes will improve digestion and lessen the digestive burden on the body, leaving more reserves for disease eradication. However, as the research indicates, the effect may be much greater than that, with the potential for direct anti-tumor activity.A diet-based cancer therapy, the Gerson Therapy, was used to treat melanoma cancer. The five-year survival rates from their therapy compared very favorably to conventional therapy reported in the medical literature, especially for more advanced stages of melanoma  see Tab.An Italian cohort of 8,984 women was followed for an average of 9.5 years, with 207 incident cases of breast cancer during that time. Their diets were analyzed by patterns – salad vegetables (raw vegetables and olive oil), western (potatoes, red meat, eggs and butter), canteen (pasta and tomato sauce), and prudent (cooked vegetables, pulses, and fish). Only the salad vegetable diet pattern was associated with a significantly lower risk of breast cancer, about 35% lower. For women of normal weight (BMI <25) the salad vegetable pattern was even more protective, about a 61% decreased risk of breast cancer . The oveIn US-based studies the "prudent" diet has been shown to be protective for colon cancer, while the "western" diet has been shown to be detrimental. The "western" dietary pattern, with its higher intakes of red meat and processed meats, sweets and desserts, French fries, and refined grains, was associated with a 46% increase relative risk of colon cancer in the Nurses' Health Study . SlatterIn an analysis of the colon cancer data from the Health Professionals Follow-up Study, Platz et al  found thA plant-based dietary pattern in being currently tested in the Women's healthy Eating and Living (WHEL) Study. About 3,000 women who were treated for an early stage of breast cancer have been randomized into two groups. The dietary goals for the test group of the study are 5 servings of vegetables, 16 oz of vegetable juice, 3 servings of fruit, 30 g of fiber, and <20% of energy from fat. No guidelines were given for animal product intake, and initial results seem to confirm, since there were no changes in body weight, total cholesterol, or LDL cholesterol , which wWhat is the result when all of these things are put together? What if all of these factors reviewed here were taken into account and put into practice? This anticancer diet would have:• adequate, but not excessive calories,• 10 or more servings of vegetables a day, including cruciferous and allium vegetables; vegetable juice could meet part of this goal,• 4 or more servings of fruits a day,• high in fiber,• no refined sugar,• no refined flour,• low in total fat, but containing necessary essential fatty acids,• no red meat,• a balanced ratio of omega 3 and omega 6 fats and would include DHA,• flax seed as a source of phytoestrogens,• supplemented with ~200 μg/day selenium,• supplemented with 1,000 μg/day methylcobalamin (B-12),• very rich in folic acid (from dark green vegetables),• adequate sunshine to get vitamin D, or use 1,000 IU/day supplement,• very rich in antioxidants and phytochemicals from fruits and vegetables, including α-carotene, β-carotene, β-cryptoxanthin, vitamin C (from foods), vitamin E (from foods),• very rich in chlorophyll,• supplemented with beneficial probiotics,• supplemented with oral enzymesAs reviewed above, reductions of 60 percent in breast cancer rates have already been seen in human diet studies, and a 71 percent reduction in colon cancer for men without the known modifiable risk factors. These reductions are without taking into account many of the other factors considered in this review, such as markedly increased fruit and vegetable intake, balanced omega 3 and 6 fats, vitamin D, reduced sugar intake, probiotics, and enzymes – factors which all are likely to have an impact on cancer. Certainly cancer prevention would be possible, and cancer reversal in some cases is quite likely.Michael Donaldson is a research scientist at the Hallelujah Acres Foundation, a foundation for investigations pertaining to the Hallelujah Diet. Funding for this review was provided by the Hallelujah Acres Foundation.


If the United States' Human Cloning Prohibition Act of 2003 (H.R. 534) becomes law, American researchers practicing any form of cloning could face up to ten years in prison and a minimum $1 million fine. The bill criminalizes a research procedure, called somatic cell nuclear transfer, that involves removing the DNA from a fertilized egg and replacing it with the DNA of a body (soma) cell. While the procedure could theoretically be used to clone a human being, used therapeutically its great promise lies in yielding a renewable source of stem cells to repair and regenerate tissue damaged by disease or injury. Embryonic stem cells appear most suited to this task, but some researchers are finding that adult stem cells could perform similar duties in certain tissues. And adult stem cells, it appears, are responsive to genetic manipulation. H.R. 534 does not threaten researchers working with adult stem cells.The precise origin of adult stem cells is unclear, though some propose that they are “set aside” during embryonic development and sequestered in mature tissue. These cells, which can make identical copies of themselves or give rise to specialized cells, serve primarily to replace damaged or injured cells. Skeletal muscle has a remarkable capacity to regenerate following exercise or injury and harbors two different types of adult stem cells to accomplish the job: satellite cells and adult stem cells that can be isolated as side population (SP) cells. Like embryonic stem cells, the adult cells commit to a certain fate once particular genes are activated.Pax7, plays a crucial role in directing the differentiation of these adult stem cells into skeletal muscle cells.It was thought that only satellite cells could mediate skeletal muscle regeneration until recently, when scientists found that adult stem cells not only participate in muscle tissue regeneration but also spawn satellite cells. A certain population of these stem cells, which are recognized by the cell surface proteins CD45 and Sca1 (stem cell antigen-1), is involved in normal muscle tissue repair, but is only triggered into the muscle cell development pathway by injury. The question then arises: what molecular factors turn these adult stem cells into muscle cells? Now Michael Rudnicki and colleagues have shown that one gene, Pax7 is required to turn adult stem cells into myogenic cells during regeneration. Here, the researchers worked with mouse models and in vitro experiments to investigate which cell populations Pax7 targets and how the gene initiates muscle cell formation in injured tissue. They show that CD45:Sca1 cells taken from regenerating muscle in mice lacking the Pax7 gene could not become muscle cells. And they show that by putting Pax7 back into CD45:Sca1 cells taken from uninjured muscle, they can generate a population of proliferating myoblasts that readily differentiate into muscle cells. When CD45:Sca1 cells engineered to express Pax7 proteins were injected into the muscles of mice lacking dystrophin (the protein defective in muscular dystrophy), the cells differentiated, forming dystrophin-expressing muscle cells in the defective muscle. This shows that engineered “donor cells” can differentiate in living tissue and help repair dystrophic muscle. When the researchers injected Pax7 (using a gene therapy virus) into the damaged muscle of mice lacking Pax7, they observed the production of muscle-forming cells that not only gave rise to differentiated muscle cells, but also aided in tissue repair.In previous studies, Rudnicki's group demonstrated that Pax7 as a key regulator of muscle cell differentiation in specific populations of adult stem cells during muscle tissue regeneration. If therapeutic strategies that activate Pax7 in adult stem cells can turn them into muscle cells, effectively replenishing injured or diseased muscle tissue, there's hope of reversing the debilitating effects of progressive muscle-wasting diseases. Though the clinical efficacy of such an approach will require intensive investigation, the results on these adult stem cells are encouraging—especially in this political climate.The researchers argue that these results “unequivocally establish”


Health professionals, says Herxheimer, must share their understanding of the benefits and harms of any treatment with patients and their families Everything that doctors and other health workers do involves communication about the benefits and harms to be expected from interventions—whether they are therapeutic, diagnostic, or prophylactic. As health-care professionals, we need to share our understanding and perceptions of benefits and harms with patients and their families as fully as we can. We also have to share them with other professionals. When we do so we have to remember that how we personally value particular benefits and harms may well differ from how another person values them.A clinician who recommends an intervention does so in the belief that its benefits outweigh the harms that it can cause. In most consultations there is little time in which to explain in detail what these benefits and harms are, or to find out what the patient thinks about them. Moreover, most clinicians are not trained or practised at describing and explaining benefits and harms clearly to patients, and much of the time they also lack important information about these aspects.The problems begin with the word “risk”. Very often people use it when they mean “harm”, and this causes ambiguities and confusion. The widely used expression “benefit/risk ratio” is meaningless—no such ratio exists. Before a decision is made to use an intervention, its benefits and harms must be weighed, ideally by the clinician and the patient together. Other advantages and disadvantages, such as convenience and cost, may also be relevant. This analysis requires use of the same dimensions for considering both benefits and harms. These dimensions have not been generally recognised or taught, though they seem obvious enough.In this context any benefit or harm has four dimensions (see sidebar). The clinician is expected to know or find out about the nature and probability of each benefit and harm, and how to maximise benefits and minimise harms. A great many clinicians do not meet this expectation, and often that is not their fault. But only patients can say how they regard the hoped for benefits and the possible harms, though they may need help to think clearly about them. Clinicians should identify how much the benefits matter to their patient—for example, are the benefits of taking a medicine or having an operation “worth the trouble”?—and whether a specific harm is particularly threatening or would be intolerable to that particular patient. People's fears, wishes, and priorities differ greatly and unpredictably.The deepening of the voice that occurs with long-term use of tamoxifen for breast cancer, and that is usually irreversible, is an example of a side effect that prescribers, manufacturers, and drug regulators have considered trivial and have largely ignored. While this side effect does not bother most women, for professional or keen amateur singers it is a disaster—it can rob them of what they enjoy most.A patient who is offered a treatment with serious implications needs time and encouragement to think, and to talk to other people, before making a decision. Three major issues are important in helping patients with decision-making: obtaining reliable information about benefits and harms, effectively communicating probabilities to the patient, and determining what to do to reverse or mitigate harmful effects when they occur.Innumeracy is very widespread. Many people cannot handle percentages, and most are unclear about the meaning of “relative risk”, “absolute risk”, “odds ratio”, and “number needed to treat”, including many clinicians who want to tell their patients about the likelihood of benefits or harms . Gigerennature, described by its quality, its intensity, and its time course (onset, duration, and reversibility).Its probability that it will occur.The importance to the person experiencing it.Its maximised, or the harm prevented or minimised.How the benefit can be www.cochrane.org). Nevertheless, there are huge gaps—we still lack reliable evidence about many important interventions.The effectiveness of an intervention  is most readily estimated from controlled clinical trials. With the rise of evidence-based medicine, there are now many more critical analyses, systematic reviews, and meta-analyses of the best evidence, as in the rapidly growing Cochrane Database of Systematic Reviews  can understand what to watch for and what to do if a problem arises. If not, or if examination or lab tests are necessary, then monitoring and follow-up by a nurse or doctor will need to be arranged.Here is a checklist of points for clinicians—and of course also drug regulators—to consider. (1) When an adverse effect occurs, should the dose be reduced, or the drug changed? (2) If reduced, by how much? (3) Is reducing the dose possible and practicable with the available preparations? (4) How and over what time should the effect of the change be observed and assessed? (5) Should the patient, as well as the clinician, keep the records of adverse effect(s) and their intensity and timing? Such notes can help both the patient and current and future doctors. Medication experiences can remain relevant for life. (6) Should an adverse effect be reported to a local or national adverse drug reaction register? (If in doubt, the answer is yes).Effective communication about harms and risks is an essential component of care, and it requires learning, preparation, and rehearsal. The onus lies with professionals to persuade and to teach patients to play their part in coming to an informed decision about treatments.


Tuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM.We developed a cell enzyme-linked immunosorbant assay (Cell ELISA) to quantitatively measure production of antibodies against the 30-kD protein in B cells from CSF of TBM and PTPM patients.The cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM.When induced with the 30-kD protein antigen, B cells derived from CSF of TBM patients respond to IgG production within 24 h while those derived from PTPM patients do not respond. Tuberculous meningitis (TBM) is an infection of the central nervous system (CNS) that is prevalent in both under-developed and developing countries. An increased incidence of TBM has occurred in recent years due to the growing number of people infected with human immunodeficiency virus (HIV). Diagnosis of TBM remains problematic despite many new, advanced diagnostic methods ,2. PreviClinical as well as CSF features are helpful for diagnosing TBM, but they cannot be used to differentiate TBM from other infectious and non-infectious disorders ,7. In paIn an earlier study, we reported the presence of a diagnostic 30-kD protein antigen in CSF of confirmed and suspected TBM patients . ImmunolCellular immune function is characterized by the existence of various types of lymphoid cells. As lymphocytes participate in the production of humoral immunity, they may respond to the 30-kD protein antigen in TBM and PTPM patients. We have developed a cell ELISA to study the response of B cells derived from CSF of TBM and PTPM cases following challenge with the 30-kD protein antigen. The purpose of the present study was to evaluate the antibody response to the 30-kD protein antigen in CSF of TBM and PTPM patients by cell ELISA and to determine whether this method may be used in differential diagnosis of TBM from PTPM.The Central India Institute of Medical Sciences (CIIMS), Nagpur, is a tertiary referral center. CSF was collected from patients who were suspected of having TBM or other infections before they received any treatment. For patients undergoing cranial surgery, analysis of CSF was performed if they were suspected of having meningitis. These patients were already on broad-spectrum antibiotics, such as third-generation cephalosporins and aminoglycosides.To establish a diagnosis of meningitis, 2–5 ml CSF was withdrawn from patients using a lumbar puncture. CSF was then subjected to routine biochemical analysis and pathological analysis including Gram staining, India ink staining, and AFB staining and culturing. One milliliter of CSF was used for the cell ELISA study, and 1 ml was used for detection of the 30-kD protein band by SDS-PAGE analysis in 12 randomly selected TBM and PTPM patients. Diagnosis of TBM and PTPM was based on the criteria described below.Presence of Mycobacterium tuberculosis in CSF by staining and/or culture, ORClinical meningitis with the following observations:A. Sub-acute or chronic fever with features of meningeal irritation such as headache, neck stiffness, and vomiting with or without other features of CNS involvementB. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.5), and/or pleocytosis with lymphocytic predominanceC. Presence of the 30-kD protein band in CSF on SDS-PAGE analysisD. Good clinical response to antituberculous drugsNone of the 12 TBM patients had positive AFB staining.Presence of pathogenic bacteria in CSF by staining and/or culture, ORClinical meningitis with the following observations:A. Fever and/or signs of meningeal irritation , stroke, or head injury and who have received antibiotics), OR High fever and/or signs of meningeal irritation with or without CNS manifestations (patients who received broad-spectrum antibiotics)B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.2), and/or pleocytosis with a predominance of polymorphonuclear cells; CSF may resemble that of chronic meningitis patientsC. Absence of the 30-kD protein band in CSF on SDS-PAGE analysisD. Good clinical response to broad-spectrum antibioticsPeripheral blood samples from six healthy volunteers were also analyzed and included as negative controls.CSF samples obtained from confirmed and suspected TBM cases were subjected to SDS-PAGE. SDS-PAGE was performed with a vertical slab gel electrophoresis system (Broviga, India) using the standard Laemmali method (10). A 4% stacking gel and 10% running gel were used. Electrophoresis was carried out at 250 volts/50 mAmps. Gels were developed by staining with Coomassie brilliant blue GR-250 and the protein profiles were then studied. Band size (i.e., molecular weight) was estimated using molecular weight markers  in a parallel lane.Following separation of proteins from CSF of confirmed TBM patients (AFB-positive) by SDS-PAGE, the 30-kD protein band was sliced out of the gel and pre-equilibrated in elution buffer  and then electro-eluted in a whole gel eluter system (Biotech, India) for 90 min at 30 volts (11). The sample was then harvested from the unit and dialyzed against PBS and the protein content was measured using a Bio Lab KIT. Protein purity was checked using native PAGE and was then used to evaluate the antibody response of B cells derived from CSF of TBM and PTPM patients.One milliliter of CSF collected from TBM and PTPM patients was centrifuged at 400 rpm for approximately 20 min. The supernatant was then discarded and the cell pellet was washed two times with PBS and then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum.Heparinized blood samples were obtained from six healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by standard Ficoll-Hypaque gradient centrifugation. PBMCs were dissolved in PBS and centrifuged at 400 rpm for approximately 15–20 min, and the PBMCs were then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum.2 in a carbon dioxide incubator. The following day, the plates were washed with PBS and horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (1:10,000) was then added to the plates. After a 2-hr incubation at 37°C, the plates were washed again with PBS and 100 μl tetramethylbenzidine (TMB)/H2O2 were added. The TMB/H2O2 served as a substrate for HRP. After a 15-min incubation, 100 μl stop solution (2.5 N sulphuric acid) were added and the plates were then read with an ELISA reader at 450 nm (12).Flat-bottomed, 96-well ELISA plates were coated with 10 μg 30-kD antigen/ml diluted in PBS (pH 7.2). Following overnight incubation, the plates were washed with PBS and then coated with 5% BSA-PBS for 4 h. The plates were then washed five times with PBS. Two-hundred μl of the cell preparation derived from CSF of patients with TBM or PTPM were then added to the wells and coated. Each sample was prepared in duplicate. Plates were maintained overnight at 37°C in 5% CODetailed clinical data for TBM and PTPM patients are presented in Table The ELISA absorbance values of IgG to the 30-kD protein antigen in CSF from TBM and PTPM patients are presented in Figure During the past decade, several conventional immunoassays including ELISA, dot immunobinding assays, immunoblot assays, and various molecular methods such as the polymerase chain reaction (PCR) have been reported as adjuncts in the diagnosis of TBM ,13-15. HM. tuberculosis and may be considered to be a diagnostic marker for TBM. In this study, we used this 30-kD protein antigen to evaluate the IgG antibody response of B cells derived from CSF of TBM and PTPM patients and from peripheral blood samples from six healthy volunteers. A cell ELISA was developed for the quantitative measurement of antibody production against the 30-kD protein antigen by these cells. Higher titers of IgG antibody production were observed in TBM patients compared to PTPM patients. The cells obtained from CSF of TBM patients gave an early response, presumably because they were already sensitized against the TBM antigen. However, when challenged with the 30-kD protein antigen, the cells obtained from PTPM patients and healthy volunteers gave a delayed response since they are not sensitized against this antigen. Therefore, an early response on this time scale is indicative of TBM.We previously used SDS-PAGE to demonstrate the presence of a 30-kD protein antigen in the CSF of TBM patients that is specific to M. tuberculosis (H37Rv strain) received from Colorado State University, Fort Collins USA (data not shown). The only limitation of this method is the time period (24–30 h) involved. However, the sensitivity of the test overcomes this drawback since it the only reported method that can discriminate TBM from PTPM.We have thus shown that cell ELISA is a sensitive technique for the differential diagnosis of TBM from PTPM. This method involves the demonstration of active antibody production by cells, particularly those derived from the affected site . PreviouThe sensitivity and specificity of IgG antibody in differential diagnosis of TBM from PTPM using the 30-kD protein antigen by cell ELISA was found to be 92% (11/12). We have also demonstrated that antibody production against the 30-kD protein antigen is higher in cells derived from CSF of TBM patients compared to PTPM patients.Various methods have been developed in our laboratory that yield a high specificity and sensitivity for diagnosis of TBM, but a small number of false positive results have been observed in pyogenic meningitis cases, particularly PTPM cases ,18. The The cell ELISA method for diagnosing TBM is based on the assumption that local synthesis of humoral antibodies against MTB antigen occurs. Various researchers have shown that CSF-derived cells have a significantly higher proliferation response to purified protein derivative (PPD) in patients with TBM, which is suggestive of an intrathecal immune response ,19.Our data can be summarized by the following observations: first, cell ELISA is a useful method for differentiating TBM from PTPM using the 30-kD protein antigen; second, the method of challenging B cells from CSF of suspected TBM patients with the 30-kD protein antigen can be helpful in confirming a TBM diagnosis; and third, the cell ELISA allows several samples to be analyzed simultaneously. Hence, the cell ELISA should be a very useful tool for the differential diagnosis of TBM from PTPM.M. tuberculosis. We have demonstrated that by using cell ELISA, we can differentiate TBM patients from PTPM patients, which should be helpful for diagnosing TBM. Additionally our results suggest that lymphocytes from CSF of TBM patients when challenged with 30 kD protein give a quick response by producing IgG antibodies when compared with that of PTPM and healthy volunteers. This may be because lymphocytes from TBM patients have already been exposed to 30 kD MTB antigens.The presence of a 30-kD protein antigen in CSF of TBM patients indicates that this protein carries the candidate marker antigen which is specific to The authors declare that they have no competing interests.RSK carried out the study design, data collection, statistical analysis, data interpretation, literature search, and manuscript preparation; NPA, RPK, and RMS assisted in data analysis collection; NHC assisted in data collection, statistical analysis, and data interpretation; HJP participated in the preparation of the manuscript, data interpretation, and study design; GMT provided assistance in preparation of the manuscript, data interpretation, study design, and funds collection; and HFD supervised the study design, statistical analysis, data interpretation, manuscript preparation, and literature search.The pre-publication history for this paper can be accessed here:


Multiple sequence alignment algorithms are very important tools in molecular biology today. Accurate alignment of proteins is central to several areas such as homology modelling, docking studies, understanding evolutionary trends and study of structure-function relationships. In recent times, improvement of existing progressing programs and implementation of new iterative algorithms have made a significant change in this field.We report an alignment algorithm that combines progressive dynamic algorithm, local substructure alignment and iterative refinement to achieve an improved, user-interactive tool. Large-scale benchmarking studies show that this FMALIGN server produces alignments that, aside from preservation of functional and structural conservation, have accuracy comparable to other popular multiple alignment programs.The FMALIGN server allows the user to fix conserved regions in equivalent position in the alignment thereby reducing the chance of global misalignment to a great extent. FMALIGN is available at  The advent of large genome projects has led to an explosion of sequence data in public databases. Analysis of protein families, understanding their evolutionary trends and detection of remote homologues are now the primary objectives. Genome annotation and analysis tools like fold prediction, homology modelling, protein-ligand docking and clustering algorithms rely heavily on accurate multiple alignments to provide a genome-wide perspective.The most popular approach for multiple sequence alignment has been the progressive alignment method . A multiIn this paper, we present an alignment algorithm that combines the properties of both progressive alignment methods and iterative refinement algorithms. This algorithm offers the user the selective advantage of guiding the course of alignment by simultaneous inputs of multiple conserved motif regions that in turn guarantees retention of structure/function in the final alignment. FMALIGN is an alignment server that provides the user to obtain a control over the alignment by providing important conserved regions as input to the alignment program to achieve a more structurally relevant and functionally useful alignment of protein sequences. It employs the sequential branching method to identify the closest pair of sequences and subsequently includes the next closest sequences to generate a guided tree using UPGMA , which iThe algorithm of FMALIGN (Fixed Motif ALIGNment) program combines three main criteria: (a) Progressive global alignment method, (b) substructure  conserved region fixation and (c) iterative refinement of alignment with identification of more conserved regions. The procedure involves three steps: (i) identification and fixing of the specified sequential conserved regions. This alignment method requires specific regions of the sequences to be aligned as anchor and these anchors are generally meant for sequentially conserved parts which do not undergo many changes. (ii) derivation of the progressive multiple sequence alignment guided by the tree. During this step, excluding the fixed anchored regions, the rest of the sequences are divided into several sub-segments that are aligned employing a dynamic alignment algorithm in a sequential order from N to C-terminus. The phylogenetic guide tree has been derived using Unweighted Pair Group Method with Arithmetic Mean (UPGMA)  which isFMALIGN server can accept amino acid patterns for multiple motifs provided by the user. It also provides option to the user to search motifs within their sequences using FINDMOTIF routine or to obtain them for the alignment by providing a link to SMoS , a strucTo assess the performance of FMALIGN in comparison to other programs, Sum-of-Pair-score (SPS) and Column-Score (CS) alignment scoring scheme  are applFMALIGN derived alignments retain high degree of conservation in secondary structures. Six members from the globin family were selected comprising a wide range of sequence identity between them (7% to 61%). Conserved regions for six aligned globin sequences were identified by the FINDMOTIF routine starting from CLUSTALX  alignmenIn order to evaluate the FMALIGN server, the results have been compared with 10 other alignment programs and objective criteria were employed to assess the quality of an alignment. We selected the BAliBASE benchmark alignment database  to compaReference 1 alignments consist of a small number of equidistant sequences of similar length, i.e. the percent residue identity (% ID) between any two sequences is within a specified range and no large extensions or insertions have been introduced.Reference 2 contains alignments of a family of closely related sequences with >25% ID, plus up to three 'orphan' sequences (distant members of the family with <20% ID, sharing a common fold). It is designed to evaluate program accuracy according to two criteria: (i) the stability of the family alignment when orphans are introduced into the sequence set and (ii)the quality of the alignment of the orphan sequences.Reference 3 checks the ability of the programs to correctly align equidistant divergent families into a single alignment. The reference alignments consist of up to four families, with <25% ID between any two sequences from different families.Reference 4 and 5 contain alignments of upto 20 sequences including N/C-terminal extensions (upto 400 residues) and insertions (upto 100 residues), respectively.This dataset is designed to study the effect of sequence length and percentage identity on the performance of the alignment program and provides a basis for the remaining tests. The overall performance of FMALIGN server for this dataset is comparable to the two best performing alignment programs, like PRRP  and CLUSIt is possible to assess the performance of the methods to align divergent 'orphan' sequences (10–20% ID with the family and between orphans) with a family of highly related (>25% ID) sequences using this data set. It is also interesting to observe the disruption of the family alignment due to the introduction of orphans. Figure This allows the assessment of the programs to correctly align approximately equidistant divergent families (<20% ID) composed of highly related sequences (>25% ID) into a single multiple alignment. Figure This dataset includes large N/C-terminal extensions to investigate whether the programs are capable of aligning the core blocks flanking the extensions. No large internal insertions are introduced at this stage. Mostly local alignment strategies out-perform the global methods. PILEUP  is the only program based on a global alignment method which does reasonably well compared to other global methods. Performance of FMALIGN is comparable to the best three methods  are calculated using a standard program kindly provided by the authors of BAliBASE. The inter-motif regions are aligned in FMALIGN by normal progressive alignment using standard substitution matrices like BLOSUM62. The gap penalties used in this version of FMALIGN are all maintained according to standard multiple alignment parameters. Benchmarking at the superfamily level has also been done utilizing both structural and sequential conserved regions. The dataset is wide enough to include proteins from different structural classes and of different length. The average sequence identity for this dataset was less than 30% since the proteins are related at the superfamily level. The performance of FMALIGN has been tested against one of the best performing multiple sequence alignment (T-Coffee) as well as structure based alignment tool (COMPARER). Studies on large and different datasets revealed an overall better performance of FMALIGN server for all categories in BAliBASE benchmarking database. It works especially well at lower sequence identity range, such as superfamily level, where no two proteins are more than 25% identical to each other in comparison to other popular methods like CLUSTALX and T-Coffee. It is well-known that automatic multiple sequence alignments at poor sequence identity are often subject to careful manual validations and improvements to avoid offsets of critical functionally important residues. FMALIGN can sensitively address this issue to avoid manual intervention subsequent to final alignment. FMALIGN-derived alignments also show a high conservation of secondary structural elements and provide better alignments for comparative modelling. The implementation of this alignment algorithm can be used to include new members into an existing protein superfamily with the help of motif regions that provide a reliable approach to connect protein sequences with their structural homologues within a particular superfamily. FMALIGN is available via the following URL S.C. had carried out the benchmarking, was involved in the development of the server and has written the first draft of the manuscript. N.B. had written the initial part of the code and P.A. has written the latter part of the code and developed a web server. R.S. had initiated the idea, was involved in discussions and in the critical reading of the manuscript.Fixed Motif ALIGNmentFMALIGN SPS Sum-of-Pairs Score


Personal Digital Assistants (PDAS) are rapidly becoming popular tools in the assistance of managing hospitalized patients, but little is known about how often expert recommendations are available for the treatment of infectious diseases in hospitalized patients.To determine how often PDAs could provide expert recommendations for the management of infectious diseases in patients admitted to a general medicine teaching service.Prospective observational cohort studyInternal medicine resident teaching service at an urban hospital in Dayton, Ohio212 patients (out of 883 patients screened) were identified with possible infectious etiologies as the cause for admission to the hospital.Patients were screened prospectively from July 2002 until October 2002 for infectious conditions as the cause of their admissions. 5 PDA programs were assessed in October 2002 to see if treatment recommendations were available for managing these patients. The programs were then reassessed in January 2004 to evaluate how the latest editions of the software would perform under the same context as the previous year.PDAs provided treatment recommendations in at least one of the programs for 100% of the patients admitted over the 4 month period in the 2004 evaluation. Each of the programs reviewed improved from 2002 to 2004, with five of the six programs offering treatment recommendations for over 90% of patients in the study.Current PDA software provides expert recommendations for a great majority of general internal medicine patients presenting to the hospital with infectious conditions. PDAs  are becoming widely used in medicine. A survey done by the America College of Physician predicted that 67% of physicians would be using PDAs by the end of 2002 . These dOne medical specialty which has focused on PDA's application in medicine is infectious diseases . There aMiami Valley Hospital is an 827 bed secondary and tertiary referral center, in Dayton, Ohio. It is an urban hospital that averages over three-thousand patient admissions per month. The internal medicine resident teaching service consists of two teams, each team with 2 senior residents and 2 interns who are supervised by an attending physician. There is a senior resident and intern on call in the hospital at all times. Patients were eligible to be admitted to resident's service if they were established within the Medical-Surgical Health Center of Miami Valley Hospital, if they were uninsured or if they had insurance but were without a local physician (a.k.a. private unattached patients). The general medicine teaching service averages approximately eight admissions per twenty-four hour period.Mandell, Douglass and Bennett's Principles and Practice of Infectious Disease (PPID) (8) (Table The patients were screened prospectively over a four month period (July 2002 through October 2002). The admitting senior resident prospectively recorded the chief complaint and initial differential diagnosis for each patient. One of us (SDB), as the chief resident during these months, gathered the data during morning report. Patients were considered appropriate for the study if they had a leading diagnosis or active alternatives that suggested an infectious disease at the time of admission. Cases selected were then assigned categories based on the "major clinical syndromes" from 8) Table . Syndrom (SG) [ (JHABx) [) (5MID) [) (5MCC) [) (PMID) [) (QID) [A Sony Clie T615C  was the device used to access the software, but all programs evaluated were also available in the Pocket PC format . Five PDA programs were initially searched to determine if expert recommendations were available. Software was chosen based on its availability to both the Palm OS and the Pocket PC and at the time of the initial software evaluation were the primary infectious disease programs available (since 2002 numerous other titles have been released to address infectious diseases). The PDA programs included: Sanford's Guide to Antimicrobial Therapy  (SG) , John's ) (5MID) , 5-Minut) (5MCC) , and Poc) (PMID) . The ini[) (QID)  was alsoA minimum of three attempts were used to locate conditions within each program. Searching was done using either the disease name or clinical problem that had led to the patient's admission. Synonyms were used when appropriate to increase the possibility of locating a condition within each program (for example: "urinary tract infection" was the first term searched and if no results available, then "pyelonephritis" was searched next and if still no results then "kidney infection" was entered into the database). Programs that were organized according to organ system (JHABx, QID only) were searched within the appropriate organ system, while programs that listed diagnoses alphabetically were searched accordingly. Expert recommendations were considered to be present and thus counted as a positive if the software had treatment recommendations present, whether related to antibiotic decision or "supportive care."In order to determine the change in software capabilities over a 14 month period, the same patient data set was used to assess software programs in January of 2004. A comparison of the programs was then performed comparing the availability of expert recommendations.The data was collected by a single physician (SDB) and then validated independently by a second physician (TEH). The data was analyzed using descriptive statistics with simple frequencies and the confidence intervals were calculated according to standard formulas.Over the four month period, from July through October of 2002, there were 883 patients admitted to the resident service, of whom 212 had syndromes that were suspected on admission to be infectious in etiology (202 of which could be assigned to the pre-defined categories)  familiar with both the PDA and the aforementioned software looking for management recommendations. This usually included antibiotic recommendations and occasionally recommendations regarding further evaluation. The possibility that we did not find recommendations which were indeed available seems unlikely because of the previously mentioned points as well as a systematic approach using multiple synonyms. Furthermore, the data was independently reviewed by a second physician and no inconsistencies were identified.A third potential limitation is that of reproducibility. Since searching was done by an experienced user, searching errors would be more likely with less experienced user(s). Many of the programs require persistent use to learn the nuances and style of the programs and how to best access information. Therefore, in order for others to duplicate our results, users will need to develop some profiency with the software and be willing to perform searches using multiple synonyms.Lastly, our study may have limits to its applicability. Our patients were admitted to an inpatient general internal medicine teaching service at an urban hospital in the United States. These data probably apply to similar inpatient services elsewhere within North America, but may not apply to either other specialty services (Pediatrics, Surgery, etc) or hospitals in other parts of the world.While the limitations mentioned above apply to this study, they can also be expanded to include limitations in the software. No physician can access a software program for the first time and be expected to take full advantage of the recommendations available. It takes time and effort to maximize the clinical utility that is available on the PDA, which unfortunately, many clinicians do not accomplish. This paper was focused on treatment recommendations and did not evaluate the ability of the individual programs to assist with diagnosis. The diagnostic data and information other than treatment recommendations varies from program to program. This could be a topic for future research or may be available in other formats such as software reviews in PDA magazines.This study demonstrates that these infectious disease programs have improved over time table . The reaDespite these potential limitations, our data suggests that PDAs provide expert recommendations for the majority of infectious clinical conditions encountered in practice by non-infectious disease general physicians. Since a majority of patients admitted to the hospital receive care from clinicians who are not specialists in infectious diseases, PDA's have the potential to have an impact on the quality of initial care by guiding not only the choice of antibiotics but also diagnostic testing and when to involve infectious disease specialists. Further research is needed to examine the efficiency of use; evidence supporting the recommendations, and the clinical impact of the use of these resources.


Norwalk virus and related noroviruses cause a short but unpleasant illness known variously as stomach flu, viral gastroenteritis, and winter-vomiting disease. They are the major causative agent of epidemic gastroenteritis worldwide. Norwalk virus was first described in 1968 when teachers and pupils at a school in Norwalk, Ohio, succumbed to acute gastroenteritis. The causative agent was identified in 1972, and since then, many other Norwalk-like viruses have been recognized. Noroviruses are usually picked up from contaminated food or water but can also be spread by person-to-person contact. There are no cures for norovirus infections and no vaccines. Worse still, noroviruses survive freezing, heating to 60 °C, and the amounts of chlorine added to public water supplies. Little surprise, then, that around 23 million Americans get a norovirus infection annually.To reduce this disease burden, better prevention and control strategies for noroviruses are urgently needed. However, the development of such strategies has been hampered by the inability of scientists to find a way to grow noroviruses in cultured cells. Like all viruses, noroviruses replicate inside host cells, and they are choosy about which cells they will grow in. The discovery by Herbert Virgin and colleagues that a mouse norovirus can be grown inside dendritic cells and macrophages, two types of immune system cells, opens the door to a better understanding of norovirus biology and better disease control strategies.In 2003, Virgin's team discovered MNV-1, a mouse norovirus, and reported that the innate arm of the immune system (as opposed to the adaptive arm) was important in combating MNV-1 infection. The adaptive immune system involves cells that respond to a disease-causing bacterium or virus by making “adaptations” to their genomes that result in specific anti-pathogen responses. The innate immune system, on the other hand, contains cells that recognize general features of pathogens and rapidly attack them when they enter our bodies. It is our first line of defense against bacteria and viruses, and Virgin and coworkers found that mice with defective innate immune systems were particularly sensitive to MNV-1.Now, by examining tissues taken from mice infected with MNV-1 infection, the researchers show that in live animals the virus infects macrophages (cells that engulf and kill pathogens) and dendritic cells (cells that display pathogen proteins to the adaptive immune system). This observation provided the clue needed to grow a norovirus in cultured cells for the first time: when the researchers took uninfected dendritic cells or macrophages out of animals with defective innate immune systems and grew them in the laboratory, they found that MNV-1 could replicate within these cells. The researchers then used physical and biochemical techniques to verify that what they were growing in culture was indeed MNV-1 and also determined the cellular factors that control MNV-1 growth in culture, thereby confirming that the innate immune system is important for combating norovirus infection. Analysis of the sequence of a virus attenuated by growth in vitro identified an important part of the viral capsid that plays a role in MNV-1 virulence, potentially opening up an avenue to vaccination with attenuated viruses.The researchers speculate that dendritic cells in the gut may provide the route by which noroviruses gain access to cells deep within the lining of our guts and thus cause disease. The next step will be to see whether human noroviruses can also grow in macrophages and dendritic cells. If they do, researchers should at last be able to elucidate the lifecycle of human noroviruses and identify the cellular factors needed for their replication. Hopefully, this new knowledge will swiftly suggest ways to prevent and control the human diseases caused by noroviruses.


Plasmodium falciparum sporozoites, these antibodies are predominantly directed to the major repetitive epitope (Asn-Pro-Asn-Ala)n, (NPNA)n. In individuals immunized by the bites of irradiated Anopheles mosquitoes carrying P. falciparum sporozoites in their salivary glands, the anti-repeat response dominates and is thought by many to play a role in protective immunity.The circumsporozoite surface protein is the primary target of human antibodies against P. falciparum infected Anopheles stephensi was recapitulated in a phage display library. Following affinity based selection against (NPNA)3 antibody fragments that recognized the PfCSP repeat epitope were rescued.The antibody repertoire from a protected individual immunized by the bites of irradiated H3 and VκI families for heavy and light chain respectively with moderate affinity for the ligand.Analysis of selected antibody fragments implied the response was restricted to a single antibody fragment consisting of VH/VL pairing (PfNPNA-1). The affinity for the ligand was in the μM range. If anti-repeat antibodies are involved in the protective immunity elicited by exposure to radiation attenuated P. falciparum sporozoites, then high circulating levels of antibodies against the repeat region may be more important than intrinsic high affinity for protection. The ability to attain and sustain high levels of anti-(NPNA)n will be one of the key determinants of efficacy for a vaccine that relies upon anti-PfCSP repeat antibodies as the primary mechanism of protective immunity against P. falciparum.The dissection of the protective antibody response against the repeat epitope revealed that the response was apparently restricted to a single V Plasmodium sporozoites, the infective stage of the malaria parasite, confers protective immunity [Plasmodium falciparum is contained within the repeat tetramer peptide (Asn-Pro-Asn-Ala)n, (NPNA)n [P. falciparum sporozoites, protected individuals had significant elevations of anti-repeat antibodies (>19 μg/ml) [Malaria threatens public health in regions of the world where more than a third of the human population lives ,2. It haimmunity ,4. The rimmunity  in a rod (NPNA)n . In some9 μg/ml) .P. falciparum sporozoite induced protection, a phage display library of antibody gene fragments isolated from the peripheral blood lymphocytes of such a protected donor (WR5) [3 epitope were selected. Recognition was restricted to a single antibody designated PfNPNA-1, encoded by VH3 and VκI families. This restricted humoral response has implications for rational vaccine design and the potential use of this human monoclonal antibody to prevent P. falciparum infection.With the advent of recombinant combinatorial antibody technology ,9 and phor (WR5)  was asseP. falciparum infected Anopheles mosquito's and subsequently shown to be protected against a non-irradiated parasite challenge, donated lymphocytes by leukophoresis five days after a booster challenge (appropriate informed consent was obtained) for details see Egan et al., [A human volunteer (WR5), who was previously exposed to the bites of γ-irradiated  et al., . The irr et al., . The equH) and the kappa chain (κ) were accessed by RT-PCR and combined by overlap extension PCR, resulting in shuffling of the VH and the VL domains. The VH PCR amplification was carried out with the cDNA template generated using the 3'HuVH primer. The VH domains were amplified using 5'HuVHA and 3'HuVH-Link 3' designed to anneal with the sequence corresponding to the first β-strand of the CH1 domain and overlap with the 5'HuVk primer. The κ chains were amplified using 5'HuVk and the 3'Hukappa primers. The VH and the κ chain PCR products were combined by overlap extension PCR using a VH flanking primer 5'HuVHB (to introduce a NheI site) and the 3'HuKappa primer.The genes encoding variable heavy (V5'-TATTAGCGGCCGCCCAACCAGCCATGGCCGAEFIJLOPETGACBCAGTCTCC-3' (where B=G+C+T, S=G+C, E = 50%A+33%C+17%T, F = 83%A = 17%G, I = 83%T+17%C, J = 50%T+33%C+17%G, L = 67%G+17%T+17%C, O = 67%T+17%A+17%C, and P = 83%G+17%C)5'-TCCTGAAGCTTGACGACCTTCGATCTCTCCCCTGTTGAAGCTCTT-3'5'-SAGGTGCAGCTGSTGSAGTCTGG-3'5'-GGCTGGTTGGGCGGCCGCTAATATGGAGGAGGGTGCCAGGGGGAAGAC-3'5'-GTTTCGCTAGCGTAGCTCAGGCTSAGGTGCAGCTGSTGSAGTCTGG-3' The procedural steps are illustrated in Figure H/κ products were digested with restriction enzymes NheI and HindIII, and ligated into pORFES [E. coli transformed with the ligation mixture was plated with and without carbenicillin selection, to determine the number of functional inserts. The VH/κ coding sequences are directionally inserted for expression between an OmpA leader peptide (to direct the polypeptide into the periplasm), and the β-lactamase. Functional full-length VH/κ β-lactamase fusion polypeptide is secreted into the periplasm. Bacteria harbouring plasmids conferring antibiotic resistance may be positively selected. The VH/κ coding insert may be readily transferred as a XbaI-HindIII fragment into the JC-M13-88 phage vector to display the insert polypeptide as a gpVIII fusion. The selected "functional" library of VH/κ inserts were excised from pORFES using XbaI and HindIII, ligated into pre-digested JC-M13-88 [E. coli (XL1-Blue: Stratagene). Phage was produced overnight at 37°C in the presence of 1 mM IPTG, unless otherwise stated. A schematic outline of the vectors is shown in Figure The PCR amplified Vo pORFES . An aliqC-M13-88 , and tra3C (Chiron Mimotopes Peptide Systems, San Diego, CA.) was conjugated to BSA using Imject Activated Immunogen kit (Pierce, Rockford, IL) according to the manufacturers guidelines. ELISA plates (Dynatech Immunlon I, Alexandria, VA) were coated with BSA or (NPNA)3C-BSA and used in phage panning experiments essentially as described elsewhere [10 plaque forming units (pfu) of the phage library in dilution buffer (PBS pH 7.2, Tween-20 0.05%, BSA 0.1%, NaN3 0.02%) was added (1 × 1010 plaque forming units (pfu) per well). After 4 h the wells were washed and the bound phage were eluted by applying either 0.1 M glycine-HCl, pH2.2 or a solution of the free peptide (NPNA)3 (~8 μM) dissolved in dilution buffer, for 15 min at ambient temperature. An aliquot of the phage elute was titered, and the remainder was used to propagate phage for further rounds of panning. The three-domain single chain antibody retains the kappa constant domain thus permits plaques filter lifts to be probed with anti-human kappa chain antibodies for immunodetection. VH and VL coding sequences were determined by sequencing of replicative form (rf) phage DNA prepared from κ-positive plaques, using the oligonucleotides primers:The peptide (NPNA)lsewhere . To bloc3'Seq VH-JC130 (5'-CGGCCATGGCTGGTTGGGCGGCC-3') and3'Seq VL-JC128 (5'TTCAACTGCTCATCAGATGGCGG-3').XbaI-HindIII sites introduced upstream of a sequence encoding a six histidine tail. Insertion of VH/κ coding sequence selected by phage display as XbaI-HindIII fragment would result in the expressed polypeptide being secreted into the periplasmic space with a hexa-histidine tag. The plasmid pAbHIS was constructed by PCR modification of pUC18 using the primers PUCSpe-JC127(5'-TCATCATACTAGTAACGACACCCGCCAACACCC-3') and M13-JC118 (5'-AAGCTTATGATGTCTAGAGCTGTTTCCTGTGTGAA-3'). A pair of annealed oligonucleotides designed to encode a 6×His tag were ligated into the HindIII digested plasmid to complete pAbHIS. The selected PfNPNA-1 VH/κ gene was excised from the rf JC-M13-88 DNA by digestion with XbaI and HindIII and ligated into similarly digested pAbHIS. An additional 6×His-coding pair of oligonucleotides was ligated into the PfNPNA-1 VH/κ linker sequence as NotI-NcoI insert. The expression of PfNPNA-1 VH/κ in E. coli D29A1 cells at 25°C, and the isolation of bacterial periplasmic material was performed as described [2 (20 mM) were added, the bacterial suspension was incubated on ice for a further 20 min before final centrifugation step. The periplasmic extract was passed over Ni-NTA resin (Qiagen), washed and the PfNPNA-1 VH/κ was eluted with 300 mM imidazole. SDS PAGE and western blotting were used to asses purity and integrity of the expressed VH/κ polypeptide during the purification procedure (data not shown). Purified PfNPNA-1 VH/κ was quantified spectrophotometrically assuming an OD at 280 nm of 1 = 0.72 mg/ml protein.The expression vector pAbHIS, was constructed by modification of pUC18. The β-galactosidase coding region was removed and escribed  with mod3C-BSA (10 μg/ml). Dilutions of the peptide (NPNA)3 were made in dimethyl formamide (DMF) before mixing with the PfNPNA-1 VH/κ diluted in PBST. Aliquots of 0.1 ml were added to duplicate wells, incubated for 2 h at 37°C. In all wells the final concentration of DMF was 1% (v/v). After washing 4 times with PBST, anti-human kappa chain alkaline phosphatase conjugate diluted 1:1000 in PBST was added and incubated as before. The wells were washed 4 × with PBST and rinsed 1× with PBS and substrate p-nitrophenyl phosphate was added, the absorbance was determined at 405 nmELISA Plates (Dynatech Immunlon I) were coated with (NPNA)H/κ to R32tet32, recombinant hepatitis core containing (NANP)4 peptide sequence and (NPNA)3C-BSA conjugate coated microtiter plate well was determined by ELISA essentially as described above. The serum(s) and the recombinant PfNPNA-1 VH/κ were diluted 1/16 and 1/10 respectively.The binding of immune serum (WR5), non-immune serum and PfNPNA-1 V12 pfu/ml in dilution buffer were applied (0.1 ml/well) to duplicate wells coated with (NPNA)3-C-BSA or BSA (10 μg/ml). After incubation at ambient temperature for 4 h, plates were washed with PBST. The bound phage was detected with sheep anti-M13 antibodies (5'-prime 3'-prime), followed by rabbit anti-sheep alkaline phosphatase antibodies in PBST added sequentially for 1 h at 37°C. Plates were washed and developed as described above.Phage at 1 × 10H/κ was compared with a well-characterized murine monoclonal anti-Pf repeat antibody 2A10 [Plasmodium falciparum NF54 strain sporozoites were either fixed in ice cold acetone for 10 min or used unfixed. Slides were first blocked with 4%BSA in PBS for 1 h. Antibodies diluted in PBST were applied for 2 h, then slides were washed 4× with PBS and fluoroscein-conjugated anti-human kappa chain or anti-mouse immunoglobulin (Sigma) was applied, diluted 1:25 in PBST. After 2 h slides were washed as above and mounted in SlowFade anti-fade reagent (Molecular Probes, Eugene, OR) and viewed by fluorescence microscopy.The PfNPNA-1 Vody 2A10 ,18 in IF3 sequence of the P. falciparum CSP was provide as whole ascitic fluid (a kind gift from Dr P. Sinnis New York University). Concentration of the whole IgG was estimated using a standard antibody capture ELISA. Immune IgG (denoted (Vol-IgG) was purified from serum of the immune volunteer (WR5), donated at the time of lymphophoresis using Protein A Sepharose (Pharmacia) and quantified assuming OD at 280 nm of 1.0 represents 0.8 mg/ml IgG. Within the Vol-IgG, the proportion of (NPNA)3 specific IgG with κ or λ light chains were determined by ELISA (data not shown).The murine mAb 2A10 ,18   containeo pORFES  and an aC-M13-88  with tenH/κ-phage library were subjected to four rounds of panning on (NPNA)3C-BSA coated wells. Both the acid and peptide elution strategies yielded significantly greater numbers of phage after four cycles of panning on (NPNA)3C-BSA when compared to panning on BSA alone  were positive in the phage ELISA for binding to (NPNA)3C-BSA and were encoded by an identical sequence, henceforth denoted PfNPNA-1. Prior to panning ten kappa positive clones were randomly selected for sequencing 3C-BSA were all identical to PfNPNA-1. Despite extensive sampling of phage that were positive in the phage ELISA for binding to (NPNA)3C-BSA (n = 25), only the PfNPNA-1 sequence was observed.Samples of the Vne Table . Analysi NP 04, 1, 13 wereH/κ polypeptide was carried out on Ni-NTA agarose beads, yielding 0.5 mg of the 38 kDa VH/κ polypeptide/L bacterial culture.The PfNPNA-1 sequence was transferred to the expression vector pAbHIS  with P. falciparum sporozoites . The murine antibody and the recombinant PfNPNA-1 VH/κ molecule both labelled the parasites.Anti-sporozoite activity of the PfNPNA-1 Vs Figure . The hum50 value used to approximate the affinity of binding. Binding affinity of the monovalent PfNPNA-1 for (NPNA)3 compared favourably with values previously reported for a panel of conventional murine monoclonal antibodies directed against the repeat epitope [Competitive ELISA was carried out and the IC epitope , which a15]2, R32tet32 [4 epitope contained within the hepatitis B virus nucleocapsid (C75CS2) [3C-BSA conjugate is probably due to the multivalent array of the capture ligand (i.e. multiple peptides coupled per BSA molecule), favouring more efficient retention of the antibody.Analysis of the fine specificity of the antibody PfNPNA-1 revealed weak binding to the repeat based [NVDP(NANP)R32tet32 , whilst C75CS2)  was stroCS2  was 6) sequences were subsequently transferred to the phage display vector. Panning with a functionally enhanced library resulted in very efficient enrichment and recovery. Previously it had been demonstrated that manipulating the conditions of phage production results in modulation of the density of antibody display on phage [The recombinant antibody library construction differed from conventional antibody phage display library assembly -13, a pron phage . The phaon phage  prior toP. falciparum sporozoite antibody response by combinatorial antibody library phage display would permit individual selected antibodies to be evaluated for protective potential and the information generated could be used in vaccine design. In particular, attention was focused on antibodies against the structural motif (NPNA)n. Despite using two different strategies for the elution of repeat region peptide specific antibodies (acid and peptide specific) it would appear that the anti-structural repeat response by this protected individual is restricted to a single VH/VL combination observed in the panel of selected phage (n = 25). Sequencing of randomly picked phage prior to panning revealed that a diverse range of VH and VL families were represented in the library as shown in Table H/VL was not represented in the sampling and was only detected after enrichment.Induction of protective immunity against sporozoite challenge by exposure to radiation attenuated malaria sporozoite has been demonstrated in humans ,7,22. PrP. falciparum sporozoites indicates that they recognize the repeat epitope(s) with equivalent affinities [H and VL genes suggesting antigen driven affinity maturation. Based on these observations, PfNPNA-1 may be a good candidate to develop and evaluate as a protective antibody.Comparison of the monovalent PfNPNA-1 molecule with the conventional bivalent murine mAb, such as the in vitro inhibitory 2A10 against finities . The seqP. falciparum sporozoites under natural conditions in the field, immunization with irradiated P. falciparum sporozoites induces in general higher levels of antibodies against the PfCSP repeats, and does induce sterile protective immunity [P. falciparum sporozoites. The generally accepted explanation for the lack of protection in the one volunteer is that the volunteer did not receive an adequate immunizing dose of irradiated sporozoites . Howeve bites )had signve bites ) H and VL domains for further in vitro and in vivo evaluation. The use of a human monoclonal antibody as a preventive measure against P. falciparum malaria, would be independent of factors which hinder active vaccination, such as adjuvant effects, the requirement to be effectively presented in a diverse range of human leukocyte class I and II molecules, and immunlogical antagonism [P. falciparum isolate that does not contain the (NPNA)n repeats on the PfCSP [P. falciparum. If this restricted antibody response to the repeat epitope plays a role in preventing P. falciparum infection, PfNPNA-1 may be a useful prophylactic agent. Moreover, if PfNPNA-1 is shown to be protective in passive immunization in humans or monkeys as previously demonstrated for anti-P. vivax CSP murine mAb, NVS3 [Efforts are being directed towards producing a fully human IgG based on the PfNPNA-1 Vtagonism ,44. In phe PfCSP , and theAb, NVS3 , it woulH/VL pairing, designated PfNPNA-1 encoded by VH3 and Vκ I families (with evidence of somatic mutations). The affinity for the ligand was in the μM range, which in the context of a whole antibody may be more than sufficient for retention on a polyvalent surface such as the P. falciparum CSP. It is speculated that the induction and the maintenance of high circulating levels of antibodies against the structural PfCSP repeat may be more important than intrinsic high affinity for the ligand for protection against P. falciparum infection. The absence of high affinity anti-repeat antibodies is in concordance with the expected response against a multivalent antigen (i.e. sporozoite surface). Under physiological conditions a whole IgG antibody and a multimeric ligand result in bivalent binding. Such complexes can have avidities estimated to be approaching the product of two independent monomeric interactions. In this case, the 1 × 10-6M monovalent affinity of PfNPNA-1 may approach a theoretical higher avidity (1 × 10-12 M) in the context of a whole antibody. This implies that further affinity maturation either in vivo or in vitro may not necessarily increase physiological effectiveness of the whole IgG antibody. Public health officials have acknowledged the urgency for development of an effective anti-P. falciparum malaria vaccine. One of the key criteria of such a putative vaccine may be the induction and maintenance of high levels of anti-(NPNA)n antibodies. The fully human PfNPNA-1 IgG could be used as a positive control in evaluating sera from immunized donors, or possibly be developed as a prophylactic agent that could be used alone or in combination with various vaccination strategies. One immediate hurdle for the development of such an antibody as a prophylactic would be the anticipated high cost of commercial manufacture in mammalian cells. However, advances in alternative antibody production technology may one day provide some more cost effective solutions [Over the past 25 years the antibody response against the PfCSP repeat epitope has been pursued as a target for active vaccination, with encouraging results . Our attolutions ,49.P. falciparum infected Anopheles mosquitoes, it should be possible to further dissect the antibody response against "other" sporozoite antigens [With the availability of an antibody phage display library constructed from a protected individual immunized via bites of irradiated antigens .JAC was the postdoctoral researcher on this project. WOR and SLH co-investigators. ASK was the PI and recipient of the Department of Army award. All authors read and approved the final manuscriptThe views and opinions expressed herein are those of the author and do not purport to reflect those of the U.S. Navy or the Department of Defense, Sanaria Inc or Avanir Pharmaceuticals Inc.