Wednesday, May 4, 2016

Successful Treatment of B-Cell ALL Using Adoptive Cell Transfer

The standard treatment of cancer patients has consisted of an approach involving some combination of surgery, chemotherapy and radiation.  Admittedly, these methodologies have grown sophisticated over the years especially in the areas of surgery and radiation.  However, chemotherapy is a “shotgun approach” employing powerful drugs that target any dividing cells.  The nature of these drugs cause significant side-effects in the patients that they are administered to.

There are a new family of drugs that have been developed to combat certain types of cancers that are more highly targeted.  Among these are Imatinib Mesylate – Gleevic –(see illustration below).  Gleevic has been used in the treatment of acute lymphoblastic leukemia (ALL) and gastrointestinal stromal tumor.  This drug specifically targets the enzyme tyrosine kinase that has been implicated in certain cancers. 


 Gleevic


Another drug that has shown promise in trastuzmab – Herceptin (see illustration below).  Herceptin has been used to treat patients with HER2+ breast cancer and adenocarcinoma of the stomach, for example.  Herceptin is a monoclonal antibody (mAb) that targets the HER2 protein found on certain types of cancer cells.


Herceptin

The new approach to fighting certain types of cancers involves a methodology referred to as adoptive cell transfer (ACT).  ACT actually employs the patient’s own immune system in fighting the cancer cells.  Impressive results using this methodology has been shown in clinical trials involving patients with advanced B-cell ALL.

The cancerous B-cells in ALL have a protein on their cell surface that is referred to as CD19.  This protein makes an appropriate target for immunotherapy.  The rationale behind ACT is to utilize the patient’s own T-cells to selectively kill ALL B-cells bearing this marker.  The following steps have been successfully employed in this treatment –

  • CD4 and CD8 lymphocytes are harvested from the patient’s blood.  CD4 T-cells are so-called helper cells and the CD8 T-cells are the cytotoxic cells.
  • These cells are genetically modified using an engineered retrovirus.  The genetic information that is introduced results in the production of a protein referred to as a chimeric antigen receptor (CAR) that binds to a specific cancer cell surface protein (see illustration below).  Structurally, CARS are modified mAbs.  In the case of ALL, the target protein is CD19.
  • Once the patient’s CD4 and CD8 lymphocytes have been successfully modified in the laboratory, they are grown out into billions of cells.  These are the cells that are reintroduced into the patient via infusion.  



CAR Signaling


There is one significant side effect from this approach; it is referred to as cytokine release syndrome that is a direct result of the modified T cells actively involved in killing their cancerous targets.  However, this is a manageable condition.


The results to date in clinical trials have been extraordinary.  This methodology shows great promise that eventually may have broader applications in the treatment of cancer.

Thursday, April 7, 2016

New Danger Posed by the Increasing Abuse of the Opioid Drug - Fentanyl

Opioids are a class of compounds that relieve pain by binding to opioid receptors found on neurons that send signals to the brain that the brain interprets as pain; this binding results in a significant reduction in these signals.  In addition, opioids also bind to analogous receptors within the brain that reduces the emotional response to painful stimuli.
 
According to the National Institute of Drug Abuse, “Medications that fall within this class (opioids) include hydrocodone (e.g., Vicodin), oxycodone (e.g., OxyContin, Percocet), morphine (e.g., Kadian, Avinza), codeine, and related drugs. Hydrocodone products are the most commonly prescribed for a variety of painful conditions, including dental and injury-related pain. Morphine is often used before and after surgical procedures to alleviate severe pain. Codeine, on the other hand, is often prescribed for mild pain. In addition to their pain-relieving properties, some of these drugs—codeine and diphenoxylate (Lomotil) for example—can be used to relieve coughs and severe diarrhea.”

These receptors pre-exist in nature for they bind to certain endogenous opioids such as dynorphins, enkephalins and endorphins.  The structures of morphine and an endorphin are shown below


Morphine


Endorphin

Opioids are readily found in nature and, as such, have been used to relieve for thousands of years within human populations.  Biochemically, opioids receptors (see illustration below) that are imbedded in the outer cell membranes of target cells are G-protein coupled, and activate inhibitory G-proteins. Once bound to the appropriate receptor, they trigger a series of cascading chemical events within the cell resulting in the suppression of neuronal signaling.




Opioid Receptor

Although opioids play a very important role in modern medicine for the relief of acute pain, there is an alarming rise of abuse of these substances especially since they are highly addictive.  Of special concern is the increased street-use of a very potent opioid – fentanyl (see structure below)
Fentanyl
Fentanyl is, often used in anesthesia to prevent pain after surgery or other procedures.  According to DEA administrator Miechele M. Leonhard "Drug incidents and overdoses related to fentanyl are occurring at an alarming rate throughout the United States and represent a significant threat to public health and safety."

"Often laced in heroin, fentanyl and fentanyl analogues produced in illicit, clandestine labs are up to 100 times more powerful than morphine and 30 to 50 times more powerful than heroin," she added.  In addition, the DEA has noted that, “ingestion of even small doses ― as small as 0.25 mg ― can be fatal. Its euphoric effects are indistinguishable from those of morphine or heroin.”

Of additional concern is the fact that fentanyl can be absorbed directly through the skin, or unknowingly inhaled as an airborne powder; this is especially of critical importance for law enforcement personnel.  Another cause for concern is that fentanyl - like other compounds in the opioid class - can be readily synthesized. In an appropriately equipped laboratory.


The abuse of opioid substances, especially fentanyl is a very real, immediate and important public health issue and deserves the attention of not only government agencies but concerned citizens.  Rather than focusing on criminal punishment of offenders, greater emphasis and resources should be brought to bear on drug rehabilitation and education as a way to help prevent such abuse from happening in the first place.

Monday, April 4, 2016

Creation of a Synthetic Organism with the Smallest Complement of Genes

The tools available to molecular biologists especially in regard to gene sequencing and assembly allow investigators to produce nucleotide sequences that incorporate specific genes and gene clusters into DNA created in-situ.  A team of investigators headed by Craig Venter from the J. Craig Venter Institute in La Jolla California in effect created a microorganism containing 473 genes (Syn 3.0).  The purpose of this investigation was to determine the minimal amount of genetic material required to sustain life as an autonomous organism and successfully reproduce. 



In 2010, Venter and his colleagues created an entire chromosome from the bacterium, Mycoplasma mycoides (this organism has only one chromosome) and demonstrated that this synthetic chromosome was completely functional.  They did this by stripping out the naturally occurring DNA from the mycoplasma, M. capricolum and replacing it with the synthetic chromosome.  The modified organism was called Syn 1.0 and with its complement of 901 genes was shown to be completely viable and capable of reproduction.

With this material in hand, the investigative group sought to assemble Syn 3.0 by methodically whittling down the DNA in Syn 1.0 to the smallest number of genes required to sustain life. The result of this painstaking work was Syn 3.0


What makes this current result so remarkable is that this organism is entirely new.  Of the 473 genes, 149 (31.5%) are of unknown function; therefore, additional work will focus on the discovery of the function of these apparently essential genes.  Syn 3.0 may prove to be an invaluable tool in understanding the evolution of life on planet earth.

Tuesday, March 15, 2016

What is the A1C Test?

The disease diabetes mellitus occurs in two different forms – juvenile or adult-onset.  In either case, the source of the illness is lack of or reduced production of the hormone insulin (see image below) whose role is to enhance the uptake of glucose circulating in the blood by tissue cells, especially adipose and skeletal muscle.  Insulin (as seen below) is referred to as a globular protein




Insulin is normally produced by specialized beta cells resident in the Islets of Langerhans within the pancreas.  It has been established that juvenile diabetes is an auto-immune disease in which the immune system of the patient attacks these beta cells.  Adult-onset diabetes, on the other hand, has a strong association with obesity.

This inability to transfer glucose to tissue cells where it is utilized for energy, leads to high concentrations of glucose in the blood (hyperglycemia).  Over a prolonged period of time, this hyperglycemic state results in very serious and ultimately life-threatening complications including blindness, impaired kidney function, cardiovascular issues leading to heart trouble and leaving victims prone to amputation.  These deleterious side effects arise as the excess glucose in the blood reacts with proteins in various tissues throughout the body – this biochemical reaction is referred to as glycosylation.

Once the cause of diabetes was discovered, it was realized that an obvious therapeutic approach is to give the patient insulin from an external source.  Before the advent of DNA recombinant technology, patients were given insulin harvested from cow pancreas (bovine insulin).  Bovine and human insulin are close enough in structure to allow bovine insulin to have an efficacious effect.  Currently, of course, human insulin is readily available.

Over many years of treating diabetic patients with human insulin, it was realized that periodic administration of insulin is not comparable to the body’s finely tuned regulation of insulin production so as to maintain optimal levels of blood glucose.  In response to this deficit, a technology arose to employ the use of an insulin pump in order to maintain a steady stream of insulin along with careful monitoring of blood glucose levels.  In addition, considerable emphasis has been placed on nutrition, exercise and weight control especially in regard to adult-onset diabetes.

Another important tool in the treatment of diabetes is the so-called A1C test.  This test provides information as to the average level of blood glucose over a 3-month time span; it is also referred to as the glycohemoglobin test.  This test measures the amount of glycosylated hemoglobin.  Hemoglobin is the specialized protein in red blood cells that is designed to carry oxygen to the tissues.  This protein reacts with excess glucose in the blood.  Since red blood cells are recycled by the body over a 3-month period, the test provides information about the average level of glucose in the blood over this period of time.  The result of this test is reported as a percentage.  The normal value is about 5.7%.  The higher the percentage, the higher the blood glucose level has been over the past 3 months.


The A1C test is an important tool in determining the efficacy of treatment for diabetic patients.  

Tuesday, March 1, 2016

The Role of the Large Protein Titin in Dilated Cardiomyopathy

There is a serious heart condition referred to as dilated cardiomyopathy (DCM) that is a major cause of heart failure and often results in premature death; this disease is found in one in two-hundred and fifty adults (0.4%).  DCM can originate either as a result of an underlying vascular problem or can have a genetic origin.  This report will focus on the progress that has been made in regards to the genetic implications of this condition.

Through the work of Dr. John T. Hinson at the Division of Cardiovascular Medicine at the Brigham and Women’s Hospital, Boston MA and his colleagues from many diverse institutions, it has been show that mutations of a large protein that constitutes one-half of the sarcomere (a structural unit of a myofibril in striated muscle) are the most common cause for DCM.  This protein is referred to as titin (TTN) (see image below) and the mutations involved result in a truncated version of TTN.  These genetic variants of TTN are referred to as TTN-truncating variants (TTNtvs).




Since the functional significance of TTN within the muscle sarcomere was unclear, the investigators involved in this research, applied the tools of molecular biology to better define the role played by TTN within heart muscle.  To accomplish this, they grew out cardiac micro-tissue cultures comprised of cardiomyocytes derived from pluripotent stem cells (iPS) that were harvested from the patients studied.  When these patient-derived cells were compared to those derived from normal individuals (the controls), it was discovered that, “certain missense mutations like TTNtvs diminish contractile performance and are pathogenic.”

Furthermore, these patient-derived cardiomyocytes also demonstrated sarcomere insufficiency, reduced responses to mechanical and biochemical stress as well as impairment in critical cell signaling pathways.  All of these results, when taken together, point to TTNtvs as playing a causative role in genetically-induced DCM.  This is a significant finding with broad implications.

Thursday, February 11, 2016

Molecular Disease

There are a host of diseases that are a direct result of a mutation in a single gene. Examples of this kind of disease are many including sickle cell anemia, severe combined immunodeficiency disease (SCID) and many others. The world famous chemist, Linus Pauling (1901-1994) coined the term Molecular Disease to refer to this type of illness. He was awarded the Nobel Prize in Chemistry for his work in 1954.

 We shall use sickle cell anemia as a case in point to illustrate how a singular change in the molecular structure of a gene can have profound consequences for the organism. A patient with sickle cell anemia presents the following symptoms often beginning at 4 months old:


  • Painful episodes that can last hours or days Attacks of abdominal pain 
  • Bone pain 
  • Breathlessness 
  • Delayed growth and puberty 
  • Fatigue 
  • Fever 
  • Jaundice 
  • Paleness 
  • Rapid heart rate 
  • Ulcers on the lower legs (in adolescents and adults). 


Other symptoms include:

  • Chest pain 
  • Excessive thirst 
  • Frequent urination 
  • Painful and prolonged erection (priapism - occurs in 10 - 40% of men with the disease) 
  • Poor eyesight/blindness 
  • Strokes 
  • Skin ulcers. 


This disease was thought to have a genetic etiology based upon the epidemiological data which showed its prevalence among individuals of African descent (one in twelve African Americans are heterozygous for this trait). Furthermore, these data also pointed to a recessive trait i.e. both alleles have to possess the altered gene for the symptoms to appear.

The disease presents with a singular characteristic – misshapen red blood cells (See Figure 1). This change in morphology from the normal disc-shaped cell to crescent-shaped is a direct result of the altered tertiary structure of the hemoglobin molecule (referred to as Hemoglobin S). Normal hemoglobin has a globular tertiary structure (See Figure 2).



Figure 1 





Figure 2   

Hemoglobin is the protein found in circulating red blood cells (RBC) that is responsible for transporting oxygen through the blood stream and ultimately to all the tissues of the body. This change in the structure of hemoglobin is a direct result of a replacement of one of its constituent amino acids. In this case the sixth amino acid in the protein polypeptide chain has been changed from glutamic acid to valine. This singular modification causes the deoxygenated form of the protein to clump together (See Figure 3). This replacement results in a molecule that is no longer readily soluble in the cell cytoplasm of RBC. As a result of this one change, the overall morphology of RBC is changed from the normal disc shape to crescent shape and they can no longer flow readily through the bloodstream as shown in Figure 1.




 Figure 3   

Since the sequence of all proteins is determined by the specific sequence of nucleotides in the genes responsible for their production, sickle cell anemia is a direct result of a single point mutation in the gene carrying the information for the production of hemoglobin found on human chromosome number 16 – hence the term, molecular disease.

 Since this disease has such a serious impact on the mortality for those who are afflicted, it might be asked why natural selection has not deleted this deleterious gene over the course of evolution. There is an explanation for this seeming contradiction. Those individuals that are heterozygous for this trait – where only one of the alleles has the mutation – have a subset of RBC that is misshapen. The remaining cells are normal, and these individuals are symptom free. Those who are homozygous for the trait – where both alleles have the mutation – have the full-blown disease. It appears that those who are heterozygous for the trait are protected from the malaria parasite whose host is RBC. Therefore, natural selection would favor those who are heterozygous for sickle cell anemia (carriers). 

In the past, this type of illness has been impervious to the possibility of a cure, since its origin resides in the very makeup of an individual’s heredity as expressed through the genes. However, since, red blood cells originate in the bone marrow, sickle cell anemia can be cured by bone marrow transplantation, but this approach has its own set of significant risks.

Recent advances in molecular biology and gene therapy have demonstrated that this daunting limitation can be effectively breached using gene therapy. SCID is a particularly devastating and ultimately fatal disease in which the affected child has no defense against infections. Through the ground breaking work of Dr. Alessandro Aiuti, ten patients suffering from SCID are still alive. The mutated gene in this condition is the ADA gene responsible for the production of the enzyme adenosine deaminase - ADA. The laboratory of Dr. Aiuti from the San Raffaele Institute for Gene Therapy in Milan successfully used the following procedure: bone marrow cells from the patients involved were incubated with a specially engineered virus containing the normal ADA gene. These engineered cells were reintroduced into the patients. Positive results were seen almost immediately following treatment. A similar approach has been used in the treatment of a disease characterized by a congenital degeneration of the retina. In this study four of six patients had a notable improvement of vision.

The latest advance has been made with Adrenoleukodystophy (ALD), a disease linked to the X chromosome. This is a severe neurodegenerative disease that leads to destruction of myelin, the outer membrane of nerve cells, in the brain and severe nervous system dysfunction. This disease is caused by a mutation in the ABCD1 gene. The first successful clinical test using gene therapy for ALD has recently been reported by Dr. Nathalie Cartier and her colleagues from the University of Paris-Descartes in Paris, France. The approach used was to take hematopoietic stem cells (HCS) from two young male patients and incubate their cells with a virus that was engineered to carry the normal ABCD1 gene. These modified cells were then reintroduced to the patients. Eventually, blood cells with the normal gene were found distributed throughout each patient’s body. Within 14 to 16 months post treatment, cerebral demyelination was arrested and neurological and cognitive functions remained stable. The patients’ own cells were used in this procedure; this avoids any need for a donor and obviates any concern of possible rejection. This is an extraordinary result and has profound implications for the future of gene therapy in medicine and may eventually find application in the treatment of sickle cell anemia.

Tuesday, February 9, 2016

Viruses Shown to Transfer Immunity between Infected Cells

Interferons (IFN) are known to play a critical role in the innate immune response to viral infection.  The molecular mechanism responsible for eliciting this response has been well studied.  It has been shown that IFN production is initiated by signaling pathways activated by molecular sensors in the presence of viral particles including cytosolic DNA sensors.  One of these DNA sensors is Cyclic GMP-AMP synthase (cGAS).  This enzyme, when activated, catalyzes the synthesis of a second messenger referred to as cyclic GMP-AMP (cGAMP).  This messenger subsequently activates transcription factors that “turn on” the genes responsible for the production of IFN (STING).

This cascading sequence of steps occurs within the infected cell.  This activation of IFN may also be spread to nearby cells connected via a gap junction.  However, Dr. A. Bridgeman and his colleagues from the medical Research Council Human Immunology Unit at the Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, suspected that this immunity could also be transferred by the viral vector to whatever additional cells it was infecting.  They proposed that the infecting virus might actually incorporate and transfer the cGAMP second messenger.  This suggestion has some precedence in that the human immunodeficiency virus 1 (HIV-1) has been shown to incorporate host-derived substances.  Given this known behavior, the investigators hypothesized that cGAMP could be picked up by infecting virions, incorporated and subsequently passed on to additional host cells and in that way actually inadvertently spread the immune response.

In order to test this proposal, they used modified and attenuated virions to infect a human embryonic kidney cell line that expressed STING and that were able to induce the production of IFN in response to cGAMP.  When these same viruses were subsequently exposed to a target cell line, they were shown to have induced the expression of IFN suggesting that their working hypothesis was correct.  The investigators painstakingly ruled out other possible variables that might explain this phenomenon.  Even virus-like particles stripped of their RNA genome still induced IFN production in target cells.   Finally, they went on to demonstrate unambiguously that cGAMP was, in fact, packaged into the virus studied.


This is an interesting finding that may have clinical value.  In light of this evidence, the authors suggest that “using viral vectors with cGAMP therefore holds promise for vaccine development.”