Saturday, August 4, 2012

Glycan gimmickry

I created this presentation on glycan gimmickry some time during the first quarter of 2010. Helminths have glycans on their surface, and it seems to be that these glycans are able to manipulate the host immune system to shift towards a less aggressive TH2 approach instead of the inflammatory TH1 mode. The shift toward the anti-inflammatory TH2 response is evidenced by the increase in the production of the IL-4 cytokine, which is a primary indicator of the TH2 response, and a decrease in the production of interferon-gamma (IFN-g), which is one of the primary cytokine markers for the TH1 response. Two cases are demonstrated: that of Brugia malayi and Caenorhabditis elegans, and that of Schistosoma mansoni. This presentation is primarily based on the paper by van Die and Cummings in 2009:

van Die I, Cummings R. 2009. Glycan gimmickry by parasitic helminths: A strategy for modulating the host immune response? Glycobiology. 20:2-12.

The following is an unformatted, plain-text transcription of the presentation:

Glycan Gimmickry by Helminths
Outline
Introduction
Case 1: B. malayi and C. elegans
Case 2: S. mansoni
Conclusion
Introduction
Section Outline
Helminths
Dendritic Cells
Th1 and Th2 Responses
CLRs
Helminth Modulation
Questions and Significance
Helminths
Four billion people infected by parasites.
Helminths are soft-bodied, elongated invertebrate animals.
Helminths are large animals relative to microbes.
Helminths are masters in regulating the immune response.
Most helminths influence the immune system to mount on a Th2 cell response.
“Glycan gimmickry” is a proposed strategy used by parasites to manipulate the immune response.
Dendritic Cells
http://en.wikipedia.org/wiki/File:Antigen_presentation.jpg
Dendritic Cells
http://en.wikipedia.org/wiki/File:Lymphocyte_activation_simple.png
Th1 and Th2 Responses
Th1 response – characterized as pro-inflammatory with the production of cytokines IFN-.
Promotes cellular immune response
Th2 response – characterized as anti-inflammatory with the production of cytokines IL4, IL5, IL10, IL13.
Promotes humoral immune response
Th1 and Th2 Responses
Cytokines are extracellular signal proteins or peptides that act as a local mediators in cell–cell communication.
CLRs
DC-SIGN recognizes the following terminal fucose patterns:
LeX Galβ1-4[Fucα1-3]GlcNAc-
LeY Fucα1-2Galβ1-4[Fucα1-3]GlcNAc-
LDNF GalNAcβ1-4[Fucα1-3]GlcNAc-
MGL recognizes α- or β-linked GalNAc residue patterns:
LDN GalNAcβ1-4GlcNAc-
Tn GalNAcα1-O-Thr/Ser) antigens
MR recognizes patterns containing mannose.
CLRs
(van Die and Cummings 2009)
CLRs
(van Die and Cummings 2009)
Helminth Modulation
Helminths attenuate Th1 responses and induce Th2 and regulatory responses to promote their survival within hosts.
Modulation is somehow related to the expression of certain helminth glycans.
“Glycan gimmickry” is proposed as the strategy helminths employ to promote their survival whereby the expression of certain glycan patterns is actively used to target immune cell lectin receptors to skew the host’s immune response toward less inflammatory reactions.
Helminth Modulation
Glycan gimmickry as an alternative strategy to molecular mimicry as first proposed by Damian (1965).
Glycan gimmickry is thought to be beneficial to the host as well who may otherwise not survive the chronic infection brought about by the strong Th1 immune response.
Questions and Significance
Questions:
What are the underlying mechanisms involved in helminth-induced Th2 responses?
Why has the immune system evolved to tolerate this kind of glycan gimmickry?
Significance
Understanding the mechanisms involved in glycan gimmickry can help further our knowledge of how the immune system functions. This increased knowledge can then be used to develop new drugs to treat or prevent diseases.
Case 1: B. malayi and C. elegans
Section Outline
Introduction
Materials
Results and Discussion
Introduction
Salah T, Laetitia LG, Fahimeda A, Baxter M, Allen J. 2004. Both free-living and parasitic nematodes induce a characteristic Th2 response that is dependent on the presence of intact glycans. Infection and Immunology. 72:398–407.
Brugia malayi is a roundworm nematode that is a causative agent of lymphatic filariasis in humans. Lymphatic filariasis, also known as elephantiasis, is a condition characterized by swelling of the lower limbs.
Caenorhabditis elegans is a free-living, transparent nematode about 1 mm in length, which lives in temperate soil environments. It is used extensively as a model organism in research.
Introduction
The purpose of the study was to test the hypothesis that glycans on B. malayi induce the Th2 response.
Moreover, the study sought to investigate whether such induction may be common to all nematodes including non-parasitic species.
http://en.wikipedia.org/wiki/File:Elephantiasis.jpg
Materials
B. malayi antigen (BmA) or mixed-stage C. elegans antigen (CeA) were prepared by homogenizing each worm population in sterile ice-cold PBS on ice by using a glass-glass homogenizer.
Groups of mice were immunized with a single dose of native CeA or native BmA in CFA (experimental groups) or with PBS in CFA (control group).
Freund's adjuvant is a water in oil emulsion. The so-called complete form (CFA) is composed of inactivated and dried mycobacteria, usually Mycobacterium tuberculosis (the pathogenic agent of tuberculosis).
Materials
BALB/c mice
http://en.wikipedia.org/wiki/File:Lightmatter_lab_mice.jpg
Results and Discussion
Worm antigens caused CD4+ T cells to proliferate and secrete higher levels of IL4.
Results and Discussion
Periodate-treated B. malayi antigens (pBma).
Lane 1 is pBma; Lane 2 is mBma (mock).
Periodate treatment modified glycan structures but maintained protein backbone integrity
Results and Discussion
pBmA and pCeA did not increase IL4 levels as mBmA and mCeA did.
Results and Discussion
IL-4 classically induces sequential switching to immunoglobulin G1 (IgG1) and IgE, whereas IFN- is associated with switching to IgG2a.
Immunization with mBmA increased IgG1 levels.
Case 2: S. mansoni
Section Outline
Introduction
Materials
Results and Discussion
Introduction
van Liempt E, van Vliet SJ, Engering A, Garcia Vallejo JJ, Bank CM, Sanchez-Hernandez M, van Kooyk Y, van Die I. 2007. Schistosoma mansoni soluble egg antigens are internalized by human dendritic cells through multiple C-type lectins and suppress TLR-induced dendritic cell activation. Mol Immunol. 44:2605–2615.
The parasitic helminth Schistosoma mansoni is the causative agent of the chronic disease schistosomiasis, which affects 300 million people worldwide, particularly in tropical countries. The disease is characterized by granulomatous reactions around viable eggs entrapped in host tissues.
Introduction
The early stage of infection with S. mansoni leads to a Th1 response. As the infection progresses and eggs are released by the adult worms, the Th1 response declines and switches towards a Th2 response, driven by the egg antigens.
http://en.wikipedia.org/wiki/File:Schistosomiasis_itch.jpeg
Introduction
The ability of monocyte-derived iDCs pulsed with SEA or copulsed with SEA and TLR-ligands to mature and induce polarized T cell responses was examined.
The interaction of SEA with DC-expressed CLRs was also studied.
Results showed that DC-SIGN, MGL as well as the mannose receptor play a role in binding and subsequent internalization of SEA.
Co-localization of SEA with MHC II in the lysosomal compartments suggests that antigen processing and presentation can occur.
Materials
Lysophosphatidylserine (LPS) – induces Th1/Th2 response via TLR2/4-dependent mechanism.
Polyinosine–deoxycytidylic acid (Poly-I:C) – induces Th1 response via TLR3-dependent mechanism
Results
SEA does not promote DC maturation.
Results
SEA prevents DC maturation caused by LPS or Poly-I:C in a dose-dependent manner.
Results
Cytokine levels are also reduced when SEA is present.
Results
When SEA is added, TH1 response is skewed to TH2.
Results
SEA is internalized and is channeled to the endosomal compartments, then to the lysosomes, and finally to the MHC II molecules.
Conclusion
Conclusion
Helminths are soft-bodied, elongated invertebrate animals that infect about 4 billion people worldwide.
“Glycan gimmickry” is a proposed strategy used by parasites to manipulate the immune response.
Dendritic cells (DCs) are antigen-presenting cells (APCs) that trigger polarization of T-cells.
The Th1 response is pro-inflammatory while the Th2 is anti-inflammatory.
Cytokines are extracellular signals used mediate the type of response elicited.
CLRs are used by DCs to recognize helminth glycans. Some prominent CLRs include DC-SIGN, MGL and MR.
Conclusion
Inflammatory modulation is somehow related to the expression of certain helminth glycans.
Most helminths influence the immune system to mount on a Th2 cell response.
Understanding the mechanisms through which helminths influence the immune system may help us develop new drugs to treat diseases.
Case 1: B. malayi and C. elegans
Glycans in B. malayi and C. elegans induce Th2 response as manifested by the increase in IL4 levels.
Removal of glycans on the worm surfaces removed their capacity to induce the Th2 response.
Conclusion
Case 2: S. mansoni
SEA does not promote DC maturation.
SEA inhibits DC maturation induced by LPS or Poly-I:C.
SEA skews Th1 responses toward more Th2-like responses as evidenced by the shift in cytokine levels from INF- to IL4.
SEA is internalized by DCs and is channeled to the endosomal compartments, then to the lysosomes, and finally to the MHC molecules.
References
Salah T, Laetitia LG, Fahimeda A, Baxter M, Allen J. 2004. Both free-living and parasitic nematodes induce a characteristic Th2 response that is dependent on the presence of intact glycans. Infection and Immunology. 72:398–407.
van Die I, Cummings R. 2009. Glycan gimmickry by parasitic helminths: A strategy for modulating the host immune response? Glycobiology. 20:2-12.
van Liempt E, van Vliet SJ, Engering A, Garcia Vallejo JJ, Bank CM, Sanchez-Hernandez M, van Kooyk Y, van Die I. 2007. Schistosoma mansoni soluble egg antigens are internalized by human dendritic cells through multiple C-type lectins and suppress TLR-induced dendritic cell activation. Mol Immunol. 44:2605–2615.
End


Germline gene therapy

Welcome to nonbonding, where I try to publish articles that are somewhat scientific in nature. As a start, I wrote this argumentative essay in 2010 about germline gene therapy. The essay takes a pro-stand and advocates the use of germline gene therapy. Enjoy reading.

The following is an unformatted, plain-text transcription:

Germline Gene Therapy
Genetic inheritance can both be good and bad. On the good side, a child can inherit his parents’ positive traits such as beauty, intelligence, or physical strength, which would favor his chances of survival and promote a better sense of well-being. On the bad side, a child can inherit his parents’ physical illnesses, which may shorten his life or cause him to experience a demoted quality of life. Any loving parent would want his child to inherit his commendable genetic characteristics, but no loving parent would want to see his child suffer from the same disease he is currently suffering from, most especially if the illness is a seriously debilitating one such as AIDS, systemic lupus erythematosus, or cystic fibrosis. Though a majority of parents would like to ensure that their children are born healthy without genetic defects, most of history has shown that the human race is powerless over the control of the genetic attributes passed on from one generation to another. So far, there has been no method through which humans could regulate what genes  children were allowed to inherit or not.
Not until recently, advances in science and technology have shown glimpses that it may one day be possible for humans to guide the biological design of its future descendants.  For instance, in 1998, scientists at Chromos Molecular Systems, a Canadian biotechnology company, were able to introduce an artificial chromosome   into a mouse which then gave birth to mice which inherited the inserted chromosome (Stolberg, 2000). In another endeavor, researchers transferred chromosomes from a female monkey with defective mitochondria  into the egg cell of another female with healthy mitochondria. The egg cell was then fertilized with the sperm of a male monkey, so that the resulting offspring had healthy mitochondria even though the bulk of its genetic inheritance was derived from the female with defective mitochondria (Conor, 2009). These among many other experiments give hope that the human species may in the future control the genetic destiny of its race, and not merely be a passive spectator of its evolution.
The prospect of engineering the genetic code of unborn persons has given rise to a new form of medical treatment called germline  gene therapy, a type of gene therapy whereby defective genes are modified so that the diseases associated with the genes are eliminated. Germline gene therapy is to be distinguished from somatic gene therapy in that whereas somatic gene therapy does not pass edited genes to offspring, germline gene therapy does and further transfers the corrected genes to the offspring’s progeny and subsequent descendants (“Gene therapy,” 2007). Consequently, germline gene therapy carries with it the powerful potential of eliminating diseases from the genetic pool of the human race (Frankel & Chapman, 2000).
Despite the captivating benefits that germline gene therapy offers, not everyone has been receptive of the proposed treatment. Various arguments have been raised to prevent the development and implementation of the genetic technology. One argument claims that performing germline gene therapy would enable us to act as if we were God. To the proponents of this reasoning, the genetic fate of future children should at best be left to God or nature to decide (Weiss, 1998). Still another argument contests that legalizing the technology would intensify societal discrimination, as genetically enhanced citizens would tend to belittle those who have no hereditary corrections (Stolberg, 2000). Finally, there are those who contend that replacing “disease” genes with “healthy” genes may result to unforeseen side-effects that would affect future descendants indefinitely (Johnson, 2002). Though these arguments are admittedly valid and are a serious cause for concern, it is the stance of this paper that germline gene therapy should still be implemented but with great caution to improve the health of future generations.
First, those who oppose germline gene therapy argue that its implementation would be immoral as we would be taking the place of God or nature in deciding the genetic fate of our children; they ask, who are we that we should determine whether a child should be born with a disease or not (Weiss, 1998)? In addition, there are those who argue that the genes hold a special value because they define our biological existence, distinguish us from each other as individuals, and are at the very core of our humanness. Therefore, tampering on these inheritable determinants would destroy the underlying fabric of our humanity (Frankel & Chapman, 2000).
Playing God may appear immoral indeed, especially when technology is applied for selfish gains. For example, if in the future it becomes possible to manipulate genes to produce superior athleticism, parents may wish to modify their unborn children’s genes in the hope that when their children mature, they would become outstanding professional athletes who could earn huge amounts of money. In this hypothetical case, the use of germline gene therapy to create exceptionally talented offspring is clearly unethical.
In contrast, when advancements in medicine are utilized to prevent or eliminate sickness, most of us would view such application as humanitarian rather than unethical. For instance, the small pox disease was a deadly viral infection that claimed the lives of millions throughout history. In the 20th century alone, it caused the deaths of about 500 million people (Koplow, 2003). When vaccines were developed so that smallpox was declared completely eradicated by the World Health Organization in December 1979 (Cock, 2001), the world did not complain that we were playing God since we were ensuring that future generations would never acquire the disease again. Rather, we were relieved that this contagious illness would never once more cause suffering for us and our children.
Playing God then to engineer our offspring to fulfill selfish ambitions is unmistakably unethical, but manipulating nature, whether through vaccines or genes, to alleviate affliction brought about by sickness should not be judged as immoral, but rather as noble and charitable. It is therefore in this area of disease elimination that germline gene therapy should be pursued and developed.
Others may argue that even though playing God may occasionally be humanitarian, the implementation of germline gene therapy is still a decision that requires the consent of future generations as it is ultimately them who will be affected. Since it is impossible to obtain the opinions of unborn people on whether they would favor the application of germline treatment to their familial lineage or not, it would be best if the genetic code of our descendents is left unmodified (Kahn, n.d.).
Indeed, executing germline modifications for future generations without their consent may sound improper and offensive, but do we not do the same process when we implement economic, social and political policies that affect unborn people regardless of what their opinions will be in the future? An example that illustrates this practice is the Great Green Wall of China which seeks to cease the unrestrained expansion of the Gobi Desert through the planting of trees that would fence the circumference of the desert. The project, which is estimated to last for 70 years and is projected to be completed beyond the year 2050 (China’s, 2001), will definitely affect future citizens of the country as agricultural land area may be preserved if the desert growth is controlled, or reduced if the trees fail to hold back the desert. Either way, lawmakers decided to push through with the project regardless of the consent of unborn generations, because they thought that this was the best strategy to contain the desert and that the lives of future inhabitants would be improved if the project was implemented.
Similarly, we do not need the consent of future generations to apply germline gene therapy if we know that such application would lead to the improvement and even the salvation of their lives. Future citizens would probably even be enraged if they knew that we had the capacity to save their lives from a debilitating illness, but we chose not to because we did not have the ability to determine their consent on whether to utilize germline technology or not.
A second major contention raised by those who oppose germline gene therapy projects that a ‘Gattaca’-like world would emerge if the technology is legalized, in reference to Andrew Niccol’s 1997 film, Gattaca, wherein society was divided into those who were genetically enhanced and those who were not. The genetically engineered, termed as ‘valids’ in the film, enjoyed special privileges such as ease in finding employment, while those with no inheritable alterations – the ‘invalids’ – were virtually unemployable as employers perceived them as prone to illnesses. Society then was highly discriminatory with the ‘valids’ conveniently realizing their aspirations in life, and the ‘invalids’ relegated to whatever jobs society deemed as insignificant (Maslin, 1997).
Indeed, there is a strong possibility that if germline gene therapy is implemented, discrimination will arise as those engineered to be less susceptible to disease may be more highly regarded by society. Moreover, those treated with germline therapy may tend to demean those without inheritable improvements. As it is the rich who will most likely be the ones who will be able to afford the genetic technology, the gap between the rich and the poor will be further aggravated (Weiss, 1998).
Despite the undeniable threat of heightened discrimination that germline gene therapy may cause, an analysis of the existing dynamics in society reveals that several practices at present already cause and demonstrate prejudice. Employment, for instance, in the Philippines is highly biased and unfair as employers tend to hire graduates from three prestigious universities, namely, University of the Philippines, Ateneo de Manila University, and De La Salle University.  Job seekers then, who do not belong to the three reputable schools, often find it difficult to gain employment despite their competence. Moreover, as it is the rich who are frequently the subset of the population able to afford the exorbitant tuition fees dictated by these well-esteemed educational institutions, the disparity between rich and poor is exacerbated (Ortega, Drilon, and Mier, 2008).
With or without germline gene therapy then, injustice thrives in civilization as a result of varying degrees in the quality of service received from institutions, customs or practices. In the education example above, employers evolved to become biased as their historical assessments disclosed that graduates from the three schools regularly performed better in job interviews and competency examinations. To facilitate the hiring process then, they developed the habit of overlooking the résumés of applicants not belonging to the top three universities (Ortega et al., 2008).
If improvements in education, medicine, or other societal functions eventually lead to discrimination, does this mean that we should cease all advancements as they ultimately widen the gap between the rich and the poor? Certainly not. From a medical perspective, should we stop the search for the cure for AIDS, arthritis, or other complicated diseases, simply because in due course, it will only be the rich who will be able to afford the vaccines or the drugs that will be invented? And from the educational viewpoint, should we halt as well the pursuit of incubating excellent learning institutions because in the end, it will only make the rich richer and the poor poorer? To these questions, the logical answer seems to be a definite no.
A more reasonable approach to the problem of prejudice would be to establish laws, campaigns, or institutions that discourage discrimination or help the unprivileged gain equal opportunities with the more fortunate citizens. For example, in the hypothetical case that germline gene therapy is implemented in the future and employers start practicing bias in their recruitment processes, then the government may pass a law prohibiting the use of genetic information as the sole basis for employment selection. Indeed, it would be unfair to turn down a good-fitting job applicant for the lone reason that he carries an uncorrected gene that makes him vulnerable to heart failure. For those who are unfortunate to receive germline gene therapy, government could subsidize the costs for the distribution of this treatment so that a greater fraction of the population is benefited. In addition, non-government organizations could also be established to raise funds for the charitable administration of gene therapy to the poor.
Once again, we ask ourselves how future inhabitants would react if they are terribly affected by some dreaded disease which could have easily been treated using germline technology, but we at the present chose not to perform the treatment because we thought that it would intensify discrimination. They would most likely be infuriated as they would rationalize that the prejudice caused by genetic engineering could have been simply solved by enacting regulations or establishing charitable institutions that promote equality and justice. But then, we at the present were too lazy to debate and iron-out the details of these laws, and chose instead to rashly dismiss the idea of genetic treatment.
Finally, one of the strongest opposition against germline gene therapy focuses on the safety of the proposed treatment. The argumentation is two-fold. First, elimination of disease genes may remove certain benefits that these deleterious genes ironically bestow on their victims. For example, as heterozygous  possession of the gene for sickle-cell anemia  has been shown to increase resistance to malaria, populations in geographical regions where malaria is prevalent have been observed to carry a higher frequency of the sickle-cell gene (Stryer, 1995). Therefore correction of the sickle-cell gene would result to subsequent increases in malaria infections. Second, replacing defective genes with corrected ones may inadvertently result to unexpected side-effects that affect future generations indefinitely (Johnson, 2002).
Undeniably, these concerns pose a very serious threat to the health of future inhabitants, so that it is very tempting to completely close ourselves to the idea of germline gene therapy. However, before shutting ourselves from the thought, we should think twice about the benefits we could gain from the technology. If the benefits outweigh the disadvantages, then it would be unreasonable not to legalize the therapy.
To understand why the advantages may be greater than the negative consequences, one has to realize that germline gene therapy is simply the same evolutionary process that we humans have been using since the germination of our existence (Wadman, 1998). In the phenomenon of evolution, all genes are subject to mutations resulting from errors incurred from DNA replication and recombination. These mutations in turn lead to diversity in our species. However, due to the course of natural selection, some evolved species are too weak to survive in their environments so that they fail to propagate their progeny successfully through time. The theory of evolution then is a generator of randomized inheritable characteristics where feebly developed creatures are eventually plucked out of existence so that only those who are well adapted to the environment survive in the long run (Watson, Hopkins, Roberts, Steitz, and Weiner, 1987).
In the same way, germline gene therapy works by the introduction of mutations into the genetic transcripts of species. However, unlike evolution which relies on an entirely stochastic methodology leading to an abundance of deleterious genes and consequently weak organisms, germline gene therapy uses a directed approach to establish corrected genes that strengthen the evolutionary family lines. In essence then, the gene technology is natural evolution itself with the exception that specific mutations intended for good are used instead of randomly-picked alterations that are apathetic to what is beneficial or not to the survival of the species.
Therefore we should ask ourselves the question on which approach has a higher probability of promoting the continuation of the human lineage. If in the future, a disease arises that is so devastating that it relentlessly wipes out populations one after the other, would leaving our species to the hands of randomized mutations be enough to save us? Would not a more focused strategy toward mutational production be a more efficient solution for our survival?
The scenario is similar to the chances of a man meeting a certain woman at a tea house on a specific date and time. If the man does not first call the woman to invite her, then his chances of meeting the woman at the precise location and time are close to null. In an analogous manner, if we leave our chances of survival from a future merciless pandemic to pure natural evolution, it would probably take a billion years before our mutations finally converge to a winning formula potent enough to resist the epidemic. But the killer disease probably would not need a billion years to consume the whole species of Homo sapiens. It would probably need much less.
Conclusion
Germline gene therapy is a proposed method for introducing inheritable enhancements whereby genes of germline cells are modified so that resulting progeny and subsequent descendants carry the altered gene fragments indefinitely. Though the proposed treatment shows promise on the possibility of eliminating diseases from the human gene pool, not everyone is enthusiastic with the treatment citing numerous robust arguments against the development and legalization of the technology. First, opponents of the genetic therapy contest that we would be playing the role of God as we would be determining the diseases our future children are allowed to die from or not. Second, those against the technology assert that societal discrimination would intensify as those who are genetically enhanced would have a sizeable advantage over those with no modifications. Lastly, a major contention against germline gene therapy centers on the harmful and unanticipated medical consequences that the treatment may bring about.
Though all three contention points are valid and not to be dismissed without due consideration, germline gene therapy still ought to be realized but with utmost prudence for the improvement and even the salvation of human lives. First, though playing God certainly does not sound comfortable, history has shown that we had taken this role time and again in executing the policies and projects that we thought would tremendously benefit future inhabitants. Second, while it cannot be denied that a scenario of heightened societal discrimination is very feasible, a more logical approach to this complication would be to establish laws that prohibit prejudice and to encourage institutions to promote justice and equality for all. Finally, to comprehend why the therapy still ought to be done despite its inherently hazardous nature, one has to realize that germline gene therapy uses specifically directed mutations so that the chances of survival of the human race from a potentially extinction-causing disease are magnified without limit.
References
China’s Great Green Wall. (2001, March 3). BBC News. Retrieved March 4, 2010, from http://news.bbc.co.uk
Conor, S. (2009, August 27). Gene therapy for the unborn. The Independent. Retrieved February 25, 2010, from http://www.independent.co.uk
Cock, K. (2001). The eradication of smallpox: Edward Jenner and the first and only eradication of a human infectious disease. Nature Medicine 7, 15-16.
Frankel M., & Chapman A. (2000, September). Human inheritable genetic modifications: Assessing scientific, ethical, religious, and policy issues. Retrieved March 2, 2010, from http://www.aaas.org/spp/sfrl/projects/germline/report.pdf
Gene therapy and children. (2007, October). Retrieved February 25, 2010, from http://health.msn.com/kids-health/articlepage.aspx?cp-documentid=100151202
Johnson, M. (2002). Germ-line gene therapy: Hubris, playing God, or a future panacea? Bios, 73(1), 16-18.
Kahn, J. (n.d.). Genetic fixes and future generations. CNN.  Retrieved March 2, 2010, from http://www.cnn.com
Koplow, D. (2003). Smallpox: The fight to eradicate a global scourge. California: University of California Press.
Maslin, J. (1997, October 24). The next bigotry: privilege by genetic perfection. The New York Times. Retrieved March 4, 2010, from http://www.nytimes.com
Ortega, R., Drilon, C., & Mier, A. (2008, October 12). Job applicants: The school matters. Philippine Daily Inquirer. Retrieved March 4, 2010, from http://www.inquirer.net
Platt O. S., Brambilla, D. J., Rosse, W. F., Milner, P. F., Castro, O., Steinberg, M. H., et al. (1994). Mortality in sickle cell disease -- life expectancy and risk factors for early death. The New England Journal of Medicine, 330(23), 1639-1644.
Stolberg, S. G. (2000, January 1). Visions: Biology: A genetic future both tantalizing and disturbing; a small leap to designer babies. The New York Times. Retrieved February 25, 2010, from http://www.nytimes.com
Stryer, L. (1995). Biochemistry (4th ed.). New York: W. H. Freeman and Co.
Wadman, M. (1998). Germline gene therapy ‘must be spared excessive regulation.’ Nature, 392, 317.
Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz, J. A., & Weiner, A. M. (1987). Molecular biology of the gene (4th ed.). California: The Benjamin/Cummings Publishing Company, Inc.
Weiss, R. (1998, March 22). Engineering the unborn. The Washington Post. Retrieved February 25, 2010, from http://www.washingtonpost.com