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Interferons are secretary proteins produced by leucocytes, fibroblasts and lymphocytes. The name comes from the initial discovery that interferon's 'interfere'' with virus multiplication.
Human interferon's are divided into three major groups based on characteristics such as antigenicity, biological and chemical based traits. These groups are called alpha, beta and gamma interferon's. (Arbabi et al, 2003)
It has a broad range of biological activities including specific antiviral activities, potentiation of the antiviral activities of interferon alpha and interferon beta, and preventing the growth of neoplastic cells and macrophages. It also boosts the production of the major histocompatibility complex (MHC) on macrophages and other cells. (Arbabi et al, 2003)
Gamma interferon is an important cytokine in the host defence against infection by viral and microbial pathogens. It induces a variety of physiologically significant responses that contribute to immunity.
Cellular proteins regulated by Interferon Gamma are involved in the host response to infection and according to Shtrichman and Samuel (2001), a study of mice deficient in proteins of the Interferon Gamma response pathway establishes its importance.
Cells that produce it include natural killer cells and CD8 cytotoxic lymphocytes and T lymphocytes.
The interferon Gamma gene is located on the long arm of chromosome 12 in the human.
The gene possesses four exons and three introns, the mature Interferon Gamma mRNA is 1.2 Kb and encodes a protein of 17 Kda and functions as an N-glycosylated homodimer. (Shtrichman et al, 2001) The final protein product is thus a glycoprotein consisting of 146 amino acids containing 30% carbohydrates. (Arbabi et al, 2003)
The Interferon Gamma cDNA sequence has been identified and in a study conducted by Arbabi et al, 2003, it has been cloned into a prokaryotic expression vector and it was then proved that the recombinant product has the same immunological and chemical properties as authentic Interferon Gamma. The aim in the study was to clone the human interferon gamma encoding cDNA in a suitable prokaryotic expression vector in order to produce the protein in E.coli.
The cloning procedure proceeds as follows.
Human blood lymphocytes are isolated and a viability assay is performed using a standard haemocytometer chamber. About 105 - 106 cells are added to each well of a 24 well cell culture plate containing culture medium and 10 % fetal calf serum.
Total RNA is extracted from the stimulated T.Lymphocytes, and then mRNA extraction is performed using an mRNA extraction kit. As interferon gamma is involved with the immune response, this is the reason T. Lymphocytes are used, as this is the best source of the mRNA.
cDNA synthesis is performed next, again using a cDNA synthesis kit according to the correct protocol. Briefly, purified PolyA mRNA is used in a reverse transcription reaction mixture containing Oligo-dT primer, reverse transcriptase buffer, dnTP, RNase inhibitor and reverse transcription enzyme, this is incubated at 37o c for one hour.
Two gene specific primers (FA1 and FA2) are designed using the human gamma interferon DNA sequence available in the Genbank database.
PCR Amplification of interferon gamma encoding gene:
SacI and NotI restriction enzymes sites are introduced in the FA1 and FA2 primers, respectively.
Standard PCR protocol using the synthesized cDNA , gene specific primers (FA1 and FA2), PCR buffer, dnTP and Taq polymerase, is carried out for 35 cycles altogether including 94 0 c for 1 minute, 57 0 c for 1 minute and 720 c for 1 minute for each cycle and the products are then analysed through the use of agarose gel electrophoresis.
The PCR products are digested in a reaction buffer containing 10 x buffer and the 2 restriction enzymes and this is incubated at 37 o c for 5 hours.
The vector, a pUC derivative is also digested with the same enzymes under similar conditions as described in the previous point.
The digested products are then purified using a DNA purification kit and a vector-insert ratio of 3:1 is used for the ligation reaction. This reaction contains 10 x ligase buffer and T4 DNA ligase enzyme and incubated at 18 o c overnight.
The ligated material is used for transformation of the E.coli cells according to standard protocol.
Cloning Screening by PCR:
The ampicillin resistant colonies are used as a DNA template in a standard PCR protocol and cycled 35 times. (as an ampicillin resistant gene is present for selection purposes on the vector)
The amplified PCR products are digested with a DraI restriction enzyme.
The positive colonies from the PCR screening experiment are grown and plasma preparation is performed.
The prepared plasmids are digested with the same enzymes used for cloning and analysed using gel electrophoresis.
The recombinant clones are sequenced and compared to the known sequence of the gene using the Genbank database.
All the above steps serve to prove that the correct gene was cloned before expression of the protein product.
The final steps include the sub-cloning of the gene into an expression vector with the gene segment being under the control of the T7 promoter in the vector and the expressed protein can be accumulated in the cytoplasm.
There is also a His Tag at the end of the cloned gene fragment for purification and detection purposes.
To compare human and recombinant gamma interferon, primary rabbit antibody anti-sera against interferon gamma and secondary antibody conjugate goat anti-rabbit HRP can be used in Elisa and Western Blotting analysis to determine if both forms of interferon gamma can be recognised with the antibodies.
In a study conducted by Arbabi et al, 2003, it was found that both forms were recognised by the antibody indicating a positive outcome in the area of recombinant drugs using interferon gamma. This shows that the recombinant form of the protein is very close to the natural form, thus making it very successful for therapeutic purposes.
This study of osteopetrosis with the treatment of Interferon Gamma 1B was carried out to determine the long term treatment of this drug. This study had been previously carried out on 8 patients with osteopetrosis over a 6 month period which proved to have a positive response in the condition; this was then carried out on a larger number of patients to determine the efficacy of the drug over a 6 an 18 months period.
20 patients were initially evaluated, the patients were evaluated on:
Presence of dense bones (radiographs )
Excess of trabecular bone.
Presence of cartilaginous remnants of bone.
13 of the 14 patients had a history of infections which required the treatment of intravenous antibiotic therapy.
6 patients had been receiving calcitriol therapy.
2 adults and one child had undergone bone marrow transplant which had a temporary positive response.
5 patients receiving prednisone were discontinued.
By the end of the evaluation process 14 patients were eligible for the treatment; this was approved by the FDA and the institutional review board of South Caraliona.
The 14 patients were treated with 1.5 ug (per kilogram of weight) of Interferon Gamma 1B, this was self administered subcutaneous injection which was taken 3 times a week, prior to the administration of the Interferon Gamma 1B and every 4 to 6 hours for 24 hours patients took 15 mg of acetaminophen to limit hyperpyrexia (causes an extremely elevated temperature that may lead to acute infectious diseases).
The effects of Interferon Gamma 1B were assessed by :
Audiologic and ophthalmologic examinations.
Bone turnover- measuring calcium, phosphorus, and alkaline phosphatase.
24-hour urinary excretion of calcium, hydroxyproline, and N-telopeptide, a specific marker of the degradation of bone collagen.
Bone density- measured by dual energy x ray absortiometery.
Blood counts- determine changes in haematopoiesis.
*During the 18 month therapy- 3 patients were removed due to lack of improvement.
The 11 patients who had received the 18 month treatment had an increase in bone resorption, bone marrow space and the diameter of optic-nerve foramina.
9 of the 11 patient had an increase in there platelet count.
The cartilaginous islands decreased in size or disappeared and the bone spicules were more uniformly stained, suggesting more normal mineralization.
Computed tomography revealed a significant increase in the cross-sectional area of the optic foramina and auditory canals during therapy.
One patient had an increase in hearing.
Increase in the volume of bone marrow in each patient.
All patients had at least one episode of an elevated temperature -above 38C.
2 patients had severe hyperpyrexia.
One had severe diarrhoea. (Both of these 2 side effects disappeared after reduction in treatment by 50%.
2 patients had hypocalcemic tetany- this was resolved with a 25 % increase in calcium intake.
The study confirms the efficacy of Interferon Gamma 1B in patients with osteopetrosis. Treatment of this drug over a period of 18 months showed stability or improved of the condition of all patients treated for 18 months, none of the 14 patients treated over a 6 month period died.
The study evaluated that treatment with Interferon Gamma 1B showed biochemical evidence of increased bone resorption; this treatment also decreased the number of infections.
Treatment of osteopetrosis with Interferon Gamma 1B is a suitable treatment for those patients who are not candidates for a bone marrow transplant.