Introduction:
There is a limit to the effect of current therapies for chronic hepatitis B virus (HBV) infection on viral gene expression and replication. However, improved technology has allowed induction of RNA interference (RNAi) in mammalian cells by using short interfering RNA duplexes (siRNA). This is a new approach to inhibit HBV infection and replication.
During RNAi, sequence-specific silencing of homologous genes is directed by double-stranded RNA. The HepG2.2.15 cell line, which is a derivative of the human HepG2 hepatoma cell line which has been stably transformed with several copies of the HBV genome, has been used to study the effect of RNAi on HBV replication. This line serves as an in vitro model for HBV replication.
Method:
1. Purify the endotoxin-free plasmid DNA using the EndoFree Plasmid Kit
2. Grow HepG2 and HepG2.2.15 cells in Dulbecco's modified Eagle's medium (DMEM)
3. Transfect cells in the presence of siRNA at 70% confluency
4. Inject plasmid DNA and siRNAs into mice via the tail vein
5. Disect the liver of the mice into pieces and freeze them immediately for preservation
6. Stain the tissue section of the formalin-fixed mice livers with hematoxylin and eosin
Northern Blot Analysis:
7. Digest total RNA extracted from the cells with DNase
8. Separate RNA with agarose gel electrophoresis
9. Probe the blots with 32P-labeled HBV DNA
Southern Blot Analysis:
7. Load samples containing the entire amount of isolated DNA onto an agarose gel
8. Transfer the gel to a GeneScreenPlus membrane
9. Hybridize the gel with a 32P-labeled HBV cDNA probe
10. Extract DNA from serum of the mice and subject them to Polymerase Chain Reaction (PCR)
11. Dot blot the PCR mixture
12. Hybridize the mixture with a 32P-labeled 424bp DNA fragment
13. Quantitate the viral particles in mice sera by comparing the spot intensities with a standard curve
Results and discussion:
It is proven that HBV gene expression and viral replication in both HepG2.2.15 cells and in vivo in a mouse model can be inhibited by the double-stranded RNA duplex siRNA-1 directed against HBV sequences in the s antigen/polymerase region. Besides inhibiting viral RNA, siRNA-1 had a dramatic effect on viral DNA production, rendering HBV DNA undetectable in sera of treated mice within 2 days after injection.
The effect of siRNA-1 on viral DNA production is indirectly caused by the suppression of both the RNA template and the polymerase enzyme, which are essential for viral DNA synthesis.
The advantage of using RNAi in the therapy of HBV is that the effect appears to be specific for the targeted gene and side effects are minimized. siRNAs simultaneously targeted to different regions of the virus may be a way to increase the efficiency of the treatment and prevent the appearance of viral revertants resistant to the treatment.
Conclusion:
In conclusion, siRNA can induce an antiviral effect on HBV replication and gene expression both in vitro and in vivo. This is useful in future studies that will assess the therapeutic potential of siRNAs for treatment of acute and chronic infections by a pathogen.
Scientific journal:
http://www.nature.com/mt/journal/v8/n5/full/mt2003238a.html
Friday, 3 February 2012
Wednesday, 4 January 2012
WHAT IS HEPATITIS B???
Hepatitis B is an infectious illness which leads to liver inflammation. It is caused by a blood-borne hep B virus (HBV) from the dsDNA hepadnaviridae genus.
Information from these sources:
Photos from these sources:
Structure of the virus
A newly acquired infection is known as acute hepatitis B.
Symptoms include:
- Liver inflammation
- Fever
- Fatigue
- Abdominal pain
- Jaundice
- Gastrointestinal symptoms like loss of appetite, nausea, vommiting and light-coloured stool
However, 90-95% of the infected people usually manage to fight off the infection successfully after a short time. These symptoms will disappear after weeks to months. They will also develop immunity against hepatitis B which lasts a lifetime meaning they will not get the infection again.
Someone who is infected with hepatitis B for more than 6 months would be considered a carrier even if he/she does not show any symptoms of it. They will be able to transmit it to others.
Transmission is usually from an infected person to another via infectious blood or fluids contaminated with infected blood such as semen and vaginal fluids.
This can occur through:
- Direct blood-to-blood contact (e.g. during blood transfusions)
- Using unsterile needles
- Unprotected sex during sexual contact
- Perinatal (from an infected mother to her child during delivery)
However, hepatitis B cannot be spread by physical methods such as holding hands, hugging, kissing, coughing, sneezing, breastfeeding or sharing food and utensils.
Unfortunately, some people whose body cannot get rid of the infection after 6 months will then develop chronic hepatitis B which is more severe.
Chronic hepatitis B is an on-going infection which lasts for more than 6 months. When a person develops chronic hepatitis B, he/she would not be cured of it.
Chronic hepatitis B causes fatal conditions like:
- Long term inflammation leading to permanent liver damage
- Scarring of the liver known as cirrhosis (hardening of the liver causing the tissue to scar and stop working)
Also, about two-thirds of people with chronic hepatitis B are chronic carriers. They do not know of their infection since their body does not show any symptoms. However, they actually do have the virus and can transmit it to other people.
(Click on photo for larger image)
Hepatitis B is the most common and serious liver infection in the world. It causes 80% of all primary liver cancers. 15-20% of chronic hepatitis B carriers die from liver disease. More than 600,000 chronic carriers die from liver-related disease every year.
Information from these sources:
http://www.emedicinehealth.com/hepatitis_b/article_em.htm
http://www.hepb.org/hepb/about_hepatitis_b.htm
http://www.hepb.org/hepb/about_hepatitis_b.htm
Photos from these sources:
http://www.abcam.com/index.html?pageconfig=resource&rid=13135
http://www.cdc.gov/Features/dsHepatitisAwareness/?source=govdelivery
http://depts.washington.edu/psworks/glossary/http://www.cdc.gov/Features/dsHepatitisAwareness/?source=govdelivery
HOW TO PREVENT/TREAT HEPATITIS B???
HEPATITIS B PREVENTION
Hepatitis B Vaccine
Hepatitis B immune globulin is given along with the hepatitis B vaccine to unvaccinated people who have been exposed to hepatitis B.
Acute hepatitis B infection:
Nucleoside/nucleotide analogues (NAs):
OTHER HEPATITIS B THERAPY
Hepatitis B Vaccine
There is a vaccine against the hepatitis B virus. It is safe and works well to prevent the disease. A total of 3 doses of the vaccine are given over several months. Hepatitis B vaccine is also produced as a combination product which includes other common childhood vaccinations.
The following groups of people should be vaccinated for hepatitis B:
The following groups of people should be vaccinated for hepatitis B:
- All health care and public safety workers who may be exposed to blood.
- People who have hemophilia or other blood clotting disorders and receive transfusions of human clotting factors.
- Travellers to countries where HBV infection is common. This includes most areas of Africa, Southeast Asia, China and Central Asia, and Eastern Europe.
- People who inject illegal drugs.
- People who have multiple sex partners or have ever had a sexually transmitted disease, like HIV
- People who have a sexual partner who is an HBV carrier.
- Household contacts of persons who are carriers of HBV.
- These include close contacts of people with HBV infection, health care workers who are exposed to HBV-contaminated blood, and infants born to mothers infected with HBV.
- Prevents transmission of the disease in 80% to 90% percent of cases.
- Practice safe sex. Correct use of latex condoms can help prevent transmission of HBV.
- If you inject drugs, don't share needles or other equipment.
- Don't share anything that might have blood on it, such as a razor, toothbrush, fingernail clippers, etc.
- Think about the health risks if you are planning to get a tattoo or body piercing. You can become infected if the artist or person piercing you does not sterilize needles and equipment, use disposable gloves, or wash hands properly.
- If you are pregnant or think you might be pregnant, tell your health care practitioner if you have any of the risk factors for HBV infection.
HEPATITIS B TREATMENT
Acute hepatitis B infection:
- Infection is not treated with antiviral medications.
- If the infected person is dehydrated from vomiting or diarrhea, a doctor may prescribe IV fluids to help them feel better. Medications may also be used to control these symptoms.
- People with mild symptoms can be cared for at home.
Chronic hepatitis B infection:
The degree of liver damage is related to the amount of active, replicating virus in the blood and liver. Regularly measuring the amount of HBV DNA in the blood gives your physician a good idea of how fast the virus is multiplying.
- Antiviral agents, while the best therapy known for chronic hepatitis B, do not work in all individuals with the disease.
- Antiviral therapy is not appropriate for everyone with chronic HBV infection. It is reserved for people whose infection is most likely to progress to active hepatitis or cirrhosis.
- The decision to treat is guided by results of liver function tests, HBV DNA tests, and, frequently, liver biopsies after a complete history and physical examination.
HEPATITIS B MEDICATIONS
They reduce the ability of the virus to reproduce in the body and give the liver a chance to heal itself. However, they are not a cure for hepatitis B.
Pegylated interferon:
- Slows the replication of the virus and boosts the body's immune system to fight the infection.
- Works best in people who have relatively low levels of HBV DNA.
- It is usually not given to people whose liver damage has progressed to cirrhosis, because it can make the liver damage worse.
- The side effects are similar to having the flu. For most people, it is so severe that they cannot continue taking the medication.
- Liver function tests and HBV DNA tests are used to check how well the treatment is working.
- Interferon appears to stop the liver damage in up to 40% of people although relapse is possible.
Nucleoside/nucleotide analogues (NAs):
- They are compounds that mimic normal building blocks for DNA. When the virus tries to use the analogues, it is unable to make new viral particles.
- They reduce the amount of virus in the body.
- Side effects are less common than with pegylated interferon. NAs have been associated with changes in body fat distribution, reduced blood cell counts, and increased levels of lactic acid in the blood.
- HBV may become resistant to NAs over time.
- NAs do not cure the infection. Relapse is possible even in patients who have had a good response to treatment.
No herbs, supplements, or other alternative therapy is known to work as well as antiviral medication in slowing HBV replication and promoting liver healing in hepatitis B. At this time, no specific herb or herbal preparation is recommended.
THERE IS NO SURGICAL THERAPY FOR HEPATITIS B.
- If liver damage is so severe that the liver starts to fail, liver transplant may be recommended.
- Liver transplant is a major process and surgery with an extended recovery period but also depends on the availability of a matching donor liver.
http://www.emedicinehealth.com/hepatitis_b/article_em.htm#Hepatitis B Overview
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Michelle's Story: Breaking the Cycle of Hepatitis B Infections from Mother-to-Child
Michelle is one of 1.25 million Americans who live with chronic hepatitis B. Michelle was born in 1969 to an American father and Vietnamese mother, who had met and married during the Vietnam War.
She grew up healthy and happy in Kentucky, unaware that her mother had unknowingly passed on the hepatitis B virus to her at birth. When Michelle was born, there was no hepatitis B vaccine available to prevent this infection. Had she been immunized within 12 hours of birth, she would be free of infection today.
“I found out about my infection through a routine blood test during my first pregnancy in 2000,” Michelle said. Kentucky is one of the few states in the country that require pregnant women to be screened for hepatitis B. “The nurse called me at home to tell me my hepatitis B test had come back positive. I immediately thought it was a lab error.”
“Eight years earlier, I had donated blood and was told that I had hepatitis B. I was re-tested and they told me I had never been exposed to hepatitis B and was free of infection,” she recalled.
But after the hepatitis B test came back positive during her pregnancy, Michelle went to see a specialist for more tests. This time, he confirmed she had chronic hepatitis B. Most teens and adults infected with the hepatitis B virus experience only a brief or acute infection. However, when newborns like Michelle are infected, they face a 90 percent risk of developing a chronic or life-long hepatitis B infection.
But after the hepatitis B test came back positive during her pregnancy, Michelle went to see a specialist for more tests. This time, he confirmed she had chronic hepatitis B. Most teens and adults infected with the hepatitis B virus experience only a brief or acute infection. However, when newborns like Michelle are infected, they face a 90 percent risk of developing a chronic or life-long hepatitis B infection.
“Needless to say, after my diagnosis I experienced the emotional succession of denial, depression and then acceptance of my hepatitis B infection,” she said.
Her immediate concern was to make sure her newborn daughter would not be infected with hepatitis B.
“During my delivery, I made sure the hospital staff was aware of my hepatitis B, and I constantly reminded them to make sure my baby received the hepatitis B vaccine within 12 hours of her birth, which prevents mother-to-child infection 90 percent of the time.
Fortunately, the hospital staff was on top of things and my daughter was vaccinated properly and today is free of hepatitis B.”
Michelle’s husband also tested negative for hepatitis B after her diagnosis and was quickly vaccinated.
After much pressuring, Michelle had her parents tested for hepatitis B. “When my mom asked her doctor to test her for hepatitis B, her doctor asked ‘Why?’ He saw no reason to test her, even though Asian-Americans are at extremely high risk of hepatitis B.”
Her mother’s hepatitis B test came back positive. Later that year, Michelle found out that her maternal grandmother, who lives in the United States, also tested positive for hepatitis B. “After this revelation, our family history came pouring out,” Michelle explained. “I learned that another of my mom's sisters also has hepatitis B, as do other family members.
Vietnam, like other countries in Asia, has very high rates of chronic hepatitis B infection, which is why one in eight Vietnamese-Americans has chronic hepatitis B. Hepatitis B is the second-leading cause of cancer death in Vietnamese-American men because it is often not diagnosed or treated until serious liver disease has occurred.”
Today, Michelle has a second child, who was also promptly vaccinated at birth and remains free of hepatitis B.
“Knowing that both my children were properly immunized against hepatitis B at birth gave me great confidence that they would be free of hepatitis B,” said Michelle. “Sadly though, this means that only my children’s branch of my family tree will be free of hepatitis B. I wish I could say the same for the rest.”
Source:
Rudolfo's Story: One Man's Personal Quest for a Cure
Rodolfo is a pioneer. In his journey to find a cure for his chronic hepatitis B infection, he has chosen the path less traveled at almost every step of the way.
Today, he practices meditation and yoga to strengthen his body on a daily basis. He takes an oral antiviral drug called tenofovir (Viread), which has not yet been approved for hepatitis B treatment by the U.S. Food and Drug Administration (FDA). Recently, at age 42 years, Rodolfo participated in a highly experimental stem cell treatment available only in Europe.
Rodolfo’s non-traditional pursuit of a cure is fueled by a fierce drive to recover the health and energy he had before suffering from acute hepatitis B five years ago, when he was living in New York City. The liver infection initially devastated him physically and emotionally. “I was trying to launch a theater career and loving the high energy of New York City,” he recalled, “and then hepatitis B hit me like a ton of bricks.”
Suddenly, he was exhausted and aching all the time. A simple blood test showed that he had acute hepatitis B. Follow-up tests showed that the virus was not going away, and he was then diagnosed as having chronic hepatitis B more than six months later.
Rodolfo’s doctor wanted to perform a liver biopsy to see whether he had any liver damage and whether he would be a good candidate for treatment. “I avoided getting a liver biopsy for almost a year because I was in denial, I didn’t want to face the fact that my life would be permanently changed by this,” Rodolfo said.
Finally he relented and underwent a needle liver biopsy, which is a procedure that involves the removal of a small sample of liver tissue for examination. The biopsy revealed cirrhosis – serious scarring of the liver.
Rodolfo returned to his hometown of Miami and to the embrace of his close knit Cuban-American family as he attempted to reassemble his life. During this time, however, he refused to accept that the fatigue, pain and liver damage caused by his chronic hepatitis B infection was something he couldn’t beat.
“My symptoms are the reason why I am willing to be so experimental in pursuing treatment,” he said quietly. “And the fact that I may be advancing research that could lead to a successful hepatitis B treatment is simply so satisfying that it enriches my life,” he added.
Initially, his doctor recommended treatment with the oral antiviral drug called lamivudine (Epivir-HBV). But Rodolfo viewed it “as a palliative, a drug I would have to be on it for the rest of my life.” He wanted a treatment that had the potential to produce a cure, regenerate his liver and return the energy he had before being struck down with hepatitis B.
Although Rodolfo decided to try lamivudine, after one year of treatment his viral load rebounded due to drug resistance – that is, the virus stopped responding to the drug and started reproducing actively again. From this disappointing result, he started doing extensive research on his own and learned about tenofovir, an antiviral drug that has been FDA-approved to treat HIV, but also appears to be quite effective against hepatitis B.
While tenofovir has not yet been approved for hepatitis B, Rodolfo found a liver specialist who was willing to prescribe it “off-label” since the drug is in phase III clinical trials for hepatitis B. For the past three years his viral load has dipped to undetectable levels and his liver damage has subsided.
But Rodolfo was still in quest of a complete cure and maintained an active “hope file,” where he compiled news of experimental treatments that might some day vanquish the virus and regenerate his embattled liver.
“I have a fighter spirit in me,” he admitted with a shy smile. “I don’t just settle for what is available.” He didn’t want to assume that tenofovir would always be able to keep the hepatitis B virus in check.
He read about a doctor in England who was conducting experiments that used stem cells to regenerate the liver. According to the research literature, human embryonic stem cells have the potential to develop into many different cell types in the body. Serving as a sort of cellular repair system, they can continuously subdivide and their “offspring” cells have the potential to become another type of cell, such as liver cells that could potentially repair a liver that has been scarred or damaged by chronic hepatitis B.
Stem cell research has been limited by politics in the United States, despite early successes in Europe and Asia, because stem cells can be obtained from aborted embryos, as well as cloned or cultivated from a patient’s own white cells. “The minute I read about an experimental trial using stems cells to regenerate the liver, I decided I had to get into it,” Rodolfo said, “this could be a possible cure for chronic hepatitis B.”
He emailed the London researchers and in early 2005 was one of five people accepted into the trial. “They used my white blood cells to cultivate about one million stem cells, which they infused into my portal vein. It is hoped that the stem cells will multiply in my liver, take on the characteristics of healthy liver cells, and proceed to repair and regenerate it,” he explained.
In London, the doctors drew blood from one of his arms, removed the white blood cells, and then pumped the blood back into him through his other arm. “You feel quite weak during the process,” he said. The major risk of the treatment was a side effect from a drug used to boost the body’s ability to create stem cells from white blood cells. It could cause a rupture in the spleen if too many stem cells were produced.
Since undergoing the highly experimental stem cell treatment, Rodolfo has experienced no ill effects from the treatment. It will be several months before researchers can tell if the transplanted stem cells were successful in generating new, healthy liver tissue.
In the meantime, Rodolfo has returned home to Miami and is recovering from the procedure. He admits it is sometimes hard to be a “medical guinea pig”. “Family members told me I was out of my mind to try this, and my dad was very anxious, but it is very important to me to find an effective treatment for myself and for others who live every day with the debilitating effects of chronic hepatitis B,” he said. “There has to be a way to beat this infection. And I’m determined to find it.”
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