The molecular genetics era allowed a better understanding of mutations in single genes and their effects on individuals and families. With fulfilment of Human Genome Project the mass data now available has moved molecular genetics into genomics, with the potential technique such as microarrays to analyse hundreds to thousands of genes and mRNAs simultaneously. Proteomics technology strategies require to determine the function of proteins. In parallel with development of genomics has been the increasing requirement for bioinformatics in storing, handling and analysing large data sets (Trent.R.J, 2005). Molecular genetics are useful in understanding the disease pathogenesis. For example, cystic fibrosis illustrates how our understanding of this disorder has changed through molecular genetics. The DNA-based knowledge has shown that a number of sporadic conditions are part of cystic fibrosis spectrum. This include meconium ileus in the fetes, male infertility related to congenital bilateral absence of vas deferens (CBAVD) amd chronic idiopathic pancreatitis. The mild forms of cystic fibrosis is due to mutations in the cystic fibrosis gene known as CFTR (Fuchs.J, Podda.M, 2005).
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This essay discusses how a hypothetical disease outbreak might be investigated, and how the application of molecular genetics, proteomics, and genomics could be used to investigate the pathogenesis of the disease and suggest possible therapy. The hypothetical disease was reported to have the following symptoms that are patients get primary symptoms such as flu, followed by anaemia, acute bone pain with accompanying lethargy. After some months, patients die from opportunistic infections, due to impaired immune response. Further investigation revealed that patient had elevated white blood cells, is adding to highly metastatic lymphoma of T cell origin.
The epidemiological studies revealed that the hypothetical disease is rarely seen in children before puberty or in middle aged elderly peoples. This disease is highly seen in people with ages of 18-25 and the disease did not affect the Afro-Caribbean peoples. This hypothetical disease is rarely seen in homosexual males, but distributed amongst bi-sexual and heterosexual communities. The hypothetical disease shows primary symptoms; first the patients are affected by flu- like symptoms and followed by anaemia and acute bone pain with fatigue. These symptoms show the disease may be of infection through viruses. The diseases explained below have similar symptoms like hypothetical disease symptoms. For example, sickle cell disease, the symptoms include flu, anaemia, and pain in bones. Patients infected with sickle cell disease develop pneumococcal pneumonia and other infections. Such as viral infections, in particular, can decrease blood cell production, so anaemia becomes more severe. The disease Transient aplastic crisis is caused by B19 virus. This disease shows symptoms like severe anaemia and bone pain. The patients with this disease have high turnover rate of new red blood cells, a disruption of these cells can lead to severe anaemia. Otherwise the hypothetical disease symptoms can be related with this disease such as, pure red-cell aplasia, this disease develops both in inmunocompromised individuals, the patients with congenital and acquired immune diseases, including congenital immunodifiency, AIDS, leukemia. The inability to produce antibodies against B19 leads to a persistent infection, leading to anaemia. However, symptoms may vary in accordance to the progression of a disease or share the same characteristics of many diseases.
Further evidence linking the primary symptoms to a viral disease, for example it may be due to Epstein-Barr virus, Human Immuno deficiency virus or Hepatitis B virus. The half of patients with primary symptoms of the hypothetical disease recovered within 4-6 weeks, and other half patients continued with fever and weight loss. After few months, patients invariably die due to impaired immune response (http://www.uihealthcare.com/). The disease may be due to Hepatitis B virus (HBV), because chronic Hepatitis B virus infection is due to weak immune response to Hepatitis B virus. Regulatory T cells (Treg) can suppress the function of effector T cells and this may lead to impaired immune response. Changes in regulatory T cells and its function, decreases the antiviral response in HBV patients. Higher percentage of T regulatory cells, such as CD25, CD4, CD45RO and cytotoxic T lymphocyte- associated antigen-4 positive cells was found in CD+ cells of chronic HBV patients, this leads to enhanced proliferation after stimulation with HBV core antigen. T regulatory cells have an immune suppressive effect on some HBV helper cells. The T regulatory cells present in HBV contribute an inadequate immune response against the virus, which leads to chronic infection (Stoop.N.J, 2007). The infections also show the secondary symptoms of the hypothetical disease. This EBV virus is a common virus belongs to herpesviruses family, most of the adults are exposed to and infected by it, young age children are less likely to have been infected by EBV. This EBV virus is associated with infectious mononucleosis. This virus attacks the B-lymphocytes for their replication. This virus infects the immune system and leads to impaired immune response. This virus remains in the infected patient's body and it can be reactivated. The mononucleosis infected by virus leads to Burkitts's lymphoma, a monoclonal B cell lymphoma with germinal centred markers. By seroepidemiological studies and detection of the EBV genome in tumour cells. Elevated EBR antibody titres have also been demonstrated in patients with malignant lymphomas, and chronic lymphatic leukaemia (Ten Napel et al, 1978). This virus can cause EBV-lympoproliferative disorder, like cancer than an infection. This EBV virus explains that in addition to impaired immune response, it finally leads to cancer like infection, chemotherapy cannot access the viruses latently persisting in quiescent B cells or the viruses in tumour cells.
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Further clinical and laboratory investigations reveal that, in addition to anaemia, patients have an elevated white cell count and a highly metastatic lymphoma produced from T cell origin, and spreads throughout the vital organs. This cancer is quite refractory to conventional chemotherapy and radiotherapy. The investigations for the hypothetical disease shows metastatic lymphoma, the similar symptoms are also seen in transient lymphocytosis. Human T-cell leukemia-lymphoma virus (HTLV-1) may produce transient lymphocytosis. Most patients recover, however, some develop T cell leukemia in adults. In lympocytosis, the level of lymphocytes in the blood increases. Lymphocytosis may result from infectious mononucleosis, or cancers such as leukemia or Non-Hodgkin's lymphoma, increase number of leukocytes or white blood cells produce lymphocytosis. It is caused by increases in the number of precursor cells that grows into mature WBCs. This leads to infections like cancer, bleeding from digestive tract, or destruction of red blood cells (http://www.bloodindex.org). Non-Hodgkin's lymphoma (NHL) is a cancer of lymph system. Lymphoma occurs when cells in the lymph grow uncontrollably, which forms tumour. HIV/AIDS-related NHL is the cancer associated with HIV/AIDS. There are many sub types in NHL; one of them is intermediate and high-grade lymphoma. It is estimated that 4 to 10% of people with HIV/AIDS develop NHL. From the above mentioned details, the patients with elevated white cell count and highly metastatic lymphoma may be, due to Human T-cell leukemia-lymphoma virus (Ko et al, 2008)
The main mechanism of the hypothetical disease may be infection of viruses in to our body cells, they accumulate in our immune system, and so the lympocytosis takes place, the level of white blood cell count increases in the blood and leads to metastatic lymphoma. This disease cannot be cured by chemotherapy and radiotherapy. So the methods used to cure viral infection disease, can be implemented to diagnose and to treat the hypothetical disease.
Viral infection can be diagnosed using molecular diagnostic techniques. Viruses sush as human immunodeficiency virus (HIV), which fail to proliferate in standard cell cultures. Now it may be detected and identified by assay of their nucleic acids. Molecular techniques may be the ideal approach for direct demonstration of the presence of nonculturable viruses. This techniques are also proving an approach for detecting a wider range of viruses. Various nucleic acid detection a powerful tool for use in detecting viruses (Lennete.H.E & Smith.F.T, 1999).
The molecular techniques amplify the target nucleic acid (the viral DNA). PCR is an target amplification technique. PCR allows many copy of a nucleic acid sequence. This may be very helpful in analyzing the samples that contain few virions or are typically small in size. The technique does not rely on viability of the virus or even on the presence of intact genome (Lennete.H.E & Smith.F.T, 1999).The viral respiratory infections is generally identified by isolation of virus in cell culture and immunofluorescent assays. RT PCR used in detection of respiratory RNA viruses. A rapid real-time multiplex PCR was developed for the detection of viruses like influenza viruses (influenza A, influenza B), human respiratory syncytial virus (RSV), parainfluenza virus in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens ( Templeton et al., 2004). The diagnosis of human T-cell lymphotrophic virus type1 (HLTV1) or type 2 infection is generally made using serological assays that shows the presence of specific antibodies such as, HLTV-1 and HLTV-2. Particular PCR analyses and discharge the presence of HTLV-1 and HTLV-2 infection in patients. This HLTV-1 and HLTV-2 PCR was performed with CD4 counts, using nested primers against pol region that permits typing of these viruses. This assay has shown a high specificity, and the sensitivity is approximately 10 copies of HTLV-1 and HTLV-2 per PCR (Bassani et al., 2005). The EBV can be easily identified using PCR, characterization and identification of EBV strains has also been reported by western blot assays based on variations in the molecular weight of the EBV- associated nuclear antigen proteins (Fucus.J, Podda.M, 2005).
DIAGNOSIS USING ELISA
To detect antibodies against HIV/AIDS, HLTV-1 and HLTV-2, etc, the molecular technique used is the enzyme- linked immunosorbent assay (ELISA), using whole-virus preparations for antigen preparation or in combination with recombinant and synthetic peptide-based immunoassay systems. The development of ELISA-based antigen-capture systems to detect small quantities of HTLV-! And HTLV-2 core antigens has increased the sensitivity and accuracy of HTLV culture systems and largely replaced measurement of reverse transcriptase activity to verify cultures that express these highly cell-associated viruses (Zhu.T, 2005).
WESTERN BLOT ASSAY
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Western blotting has mostly found use in detecting immunogenic responses elicited by infectious agents, such as bacteria and viruses, where the presence of these agents is
difficult to detect with methods that aim to isolate and culture the infectious agent from patient samples. Other diagnostic uses for Western blot assays include detection of the presence of abnormal cellular proteins such as the prion protein. In general, Western blot assays do not provide truly quantitative information. In these applications, the technique is usually used as a secondary method to confirm initial results obtained with enzyme immunoassays (EIAs) or enzyme-linked immunosorbent assays (ELISAs) or provides additional information not obtainable with these procedures. Often, many different antigens can be assayed together, thus providing a wider picture of the antigenic response being studied. For example HIV-I Diagnostics, HIV-1 and HIV-2 are the two causative agents of autoimmune deficiency disease (AIDS). The patient develops antibodies against viral proteins. Detection of these antibodies with EIAs is used in the routine detection of HIV infection in patients and screening of blood donors. Western blotting is recommended for use as a secondary confirmatory test to further analyze samples that have repeatedly produced positive EIA results. Anti-HIV antibodies can be found in most body fluids, including serum,
saliva and urea. All of these can be used as samples for testing for the disease (Soundy.P & Harvey.B, 2005).
This is a technique for analyzing the expression of multiple genes in a cell or tissue sample, frequently to compare a normal sample with a virus infected or diseased one. Known as functional genomic analysis. The probe DNAs can be cDNAs or oligo nucleotides which are arrayed on a solid surface such as glass, plastic or a silicon chip. mRNA from the specimens to be compared is labelled with a flurophore such as cyanide 5(red) or cyanide 3(green), and after hybridization the relative intensities are visualized in a microarray scanner to identify which genes are upregulated or downregulated as a result of disease or infection. These analyses provide a great deal of information, but must be interpreted with causion. For example some probes may cross cross-hybridize to the wrong mRNA, yealding false-positives or false-negatives (Mahy.W.B 2009).
These are the some of the molecular techniques that are used to diagnose viral infections in patients, the hypothetical disease may be of viral infection, so any one of the above mentioned techniques can be used as diagnostic method for hypothetical disease.The development of curative therapies for the hypothetical disease is explained below.
Many serious diseases and disorders are caused by genes that have undergone a change. Gene therapy involves replacing abnormal genes with normal genes, to cure diseases. First the abnormal genes were removed from the patient, treated, and then returned to the patient. There are two forms of gene therapy they are somatic-cell gene therapy (the insertion of genes in to body cells) and germ-line gene therapy (insertion of genes into cells that produce sperm, egg or early embryos). In somatic-cell gene therapy, viruses are used to carry the therapeutic genes to the cells. Viruses are efficient at targeting cells, and they are modified for gene transfer so that they do not cause disease in patient. This technique is used to cure viral diseases like cancer, HIV/AIDS, etc (Kinsey.B, 2010)
DNA vaccinationis a technique for protecting anorganismagainst disease by injecting it withgenetically engineeredDNAto produce an immunological response. DNA vaccines are still experimental, this can be applied to a number of microbial diseases such as virus, bacteria, etc, as well as to severaltumourmodels. DNA vaccines have many advantages over accepted standard vaccines, this has ability to induce a wide range of immune response types. DNA vaccines have entered into a variety ofhuman clinical trials for vaccines against various infectiousdiseases and for curative therapies against cancer, and are in developmentfor therapies against autoimmune diseases and allergy. Theyalso have become a commonly used laboratory tool for a varietyof applications ranging from proteomics to understanding Agpresentation and cross-priming. Despite their rapid and widespreaddevelopment and the usage of the term "DNA vaccines,"however, the DNA vaccines in humansunderscores the challenges encountered in the efforts to translateefficacy in preclinical models into clinical realities (Donnelly, et al, 2005). The ability of DNA vaccine to include appreciable antiviral anti-bodies was subsequently confirmed by several other investigators with other genes from influenza, HIV, rabies virus, and hepatitis B virus. The DNA vaccine approach has been used extensively to include antibody responses against various viral diseases. The antibodies produced by DNA vaccination are predominantly of the IgG subtype with lower levels of serum IgA and IgM. The protein-based vaccines, the inherent antigenicity of the protein expressed by DNA vaccines in part determines the potency of induced immune responses. Increased antibody responses can be achieved by expressing antigens as secreted proteins, or as fusion proteins with other antigens or helper T-cell epitopes. The DNA vaccines can be successfully delivered via different routes, such as intramuscular, intravenous, oral, intraepidermal intrarectal, etc (Levine et al, 2004).
HUMANIZED MONOCLONAL ANTIBODIES
Monoclonal antibodies can be made easily and reproducibly in large quantities. Many of the side effects of the monoclonal antibodies in clinical use are due to foreign Ig- constant regions. Several approaches have been taken employing for fusion of human cells with animal myelomas or with human tumour cells of various kinds, and use of Epstein-Barr virus to immortalize antibody- producing cells. Production of populations of sensitized human cells to be fused presents another problem, since the donors cannot be immunized at will. One of the example is explained below, in vitro stimulation of lymphocytes with antigen followed by fusion of mouse myeloma cells has been used to generate a series of antibodies to varicella zoster. Another approach to production of monoclonal antibodies with human characteristics involves application of genetic engineering. The part of the antibody structure recognized as foreign by humans can be minimized by combining human constant regions with mouse variable regions by molecular genetic techniques. Antigen binding specificity is retained in some cases, and the humanized chimeric molecules have many of the advantages of human hybridomas (Paul.E,W, 2008).
IMMUNOTHERAPY FOR MALIGNANCIES
Virus associated tumors can be cured using this technique. For example EVB-associated monoclonal tumors is otherwise immunocomponent individuals are known to be mostly invisible for immunological point of view. In comparison lymphoproliferative diseases after hamatopotic stem cell transplantation usually arise as polyclonal proliferations of EBV-infected immortalized cells that are subjected to immune control. Immediate partial reconstruction of immune surveillance is the first action to lake. Without treatment benign polyclonal proliferations often progress to oligoclonality or monoclonality with greatly reduced changes for successful therapy (Fucus.J, Podda.M, 2005).
Some of the viral infection can treated by using inhibitors. These inhibitors are, Nucleoside-Analog Reverse Transcriptase Inhibitors (NRTI), this drug inhibit viral RNA- dependent DNA polymerase and incorporated in to DNA of the virus., Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), similar to NRTIs, NNRTIs are not incorporated into viral DNA, they inhibit HIV replication directly by binding non-competitively to reverse transcriptase. And finally Protease inhibitors, these are specific for the HIV- protease and competitively inhibit the enzyme, preventing the maturation of virions capable of infecting other cells.
The curative therapies like gene therapy, DNA vaccines, humanized monoclonal antibodies, etc, can be used in the treatment of hypothetical disease. The mentioned curative therapies are used to cure immunodeficiency viral infections in the patients, these curative therapies can be implemented to treat hypothetical disease because the hypothetical disease have similar symptoms of above mentioned diseases.
The study for the hypothetical disease states that, the disease may be due to viruses, Such as Human T-cell leukemia-lymphoma virus, Epstein-Barr virus, Hepatitis B virus and Human immunodifiency virus, etcetera. Any of these viruses may infect immune system of the patients. Leads to lymphocytosis, it is due to increased number of precursor cells that eventually grow in to mature white blood cells count (WBCs). So the level of white blood cell count increases in the blood and leads to metastatic lymphoma. The above mentioned diseases have similar symptoms of hypothetical disease. So the diagnostic methods such as, PCR, Western blot assay, ELISA, DNA microarray and curative therapies, such as Gene therapy, DNA vaccines, Humanized monoclonal antibodies and immunotherapy for malignancies, are used to treat similar symptoms diseases of hypothetical disease. Molecular genetics are very helpful to identify and understand the infections in genes, and it is useful in understanding the disease pathogenesis. So the newly upcoming diseases can be easily identified using molecular techniques and it can be cured by using appropriate therapies during the primary stage of the disease, so that we can minimise the risk of the diseases.
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