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With the advent of a new disease outbreak hitting the human population, molecular biologists have been charged with the task to elucidate the molecular pathology of a new disease (referred to as 'Disease-A' from now on) using approaches of molecular genetics, genomics and proteomics to identify, diagnose and suggest therapeutic methods to develop a suitable cure. Patients affected with Disease-A are presented with flu-like symptoms followed by anaemia, acute bone pain and lethargy. Within 4-6 weeks, patients are presented with high fevers and weight loss; and after 6-12 months, patients die from opportunistic infections caused by a severely impaired immune response. Clinical reports also revealed additional information, with patients having an elevated white cell count and highly metastatic lymphoma of T-cell origin. Disease-A is distributed evenly throughout the population with no particular race or ethnic group except for individuals of Afro-Caribbean decent which are less affected. Disease-A is also rare in homosexual males with a marked peak incidence between ages 18-25.
The first symptomatic emergence of Disease-A, flu, anaemia, acute bone pain and lethargy can be likened to a number of blood related disorders like sickle cell anaemia, Leukemia, Pernicious anaemia, Hypercalcemia and a host of others. High fevers and weight loss also suggests Disease-A is blood related. Patients dying after 6-12 months of the disease owing to opportunistic infections caused by a severely impaired immune response suggest that Disease-A compromises the patient's immune system. This can also be likened to a pathogen or virus infection like Human Immune Deficiency syndrome (HIV), Severe Combined Immunodeficiency (SCID), Human Cytomegalovirus, X-linked Agammaglobulinemia etc. The fast rate at which the disease progresses to fatality also suggests that the disease is acute in nature. Patients infected with Disease-A also show to have an elevated white cell count which highly suggests that it is a blood disease and being a highly metastatic lymphoma of T-cell origin shows the disease is also cancerous (of lymphatic origin). This makes Disease-A to be inclined towards an acute leukemia. The marked peak of incidence, 18-25 years, and disease rarity in non-sexually active children suggests the disease is common to sexually driven individuals and is also sexually transmitted. With an even distribution of Disease-A amongst bi-sexual and heterosexual communities, it goes on to support the hypothesis of the disease being sexually transmitted and caused by an infection, most likely a viral infection. With the above suggestions, Disease-A looks to be a blood related disorder induced by a viral infection which causes an immunodeficiency of lymphoid cells via the changes to T-cell transmissible by sex or exchange of body fluids.
Therefore the suggested mechanism of Disease-A is as follows. Patients get infected with the virus vertically from mother to child by breast feeding, horizontally by exchange of body fluid, or by blood transfusion. This is in line with viral diseases. The virus attacks the T-cell lymphocytes precursors of the immune system and incorporates its DNA into the host's genome to facilitate its replication. This results in the arrest and transformation of T-cell lymphoblast which escapes apoptosis, constantly divides and in turn increasing the white cell count most of which will be leukemic. The virus induces a mutation in the T-Cell leading to loss-of-function. The infected cells, now immortalised, increases in population, invade the bone marrow and crowd the normal blood cells, reducing their population. This brings about the symptoms of anaemia, Flu, lethargy and bone pain; all which are associated with blood disorders. As the disease progresses with time, the immune system is severely compromised and gives way for opportunistic infections like Pneumocystis pneumonia, Strongyloidiasis, Staphylococcus aureus, Pseudomonas aeruginosa and a variety of fungi, bacterial and viral infections etc., of which patients die if not treated. However, half of the patients with the initial emergent symptoms recover which suggests a form of resistance to the virus infection encoded within their genome.
A number of different techniques can be employed to test the hypothesis. A complete blood count (CBC) can be used to test for blood abnormalities or infections. In this case it will test for anaemia and elevated white blood count. The CBC findings will be confirmed using peripheral blood smear or blood films. A reduction in red blood cells and elevated white cell count will suggest anaemia and leukemia respectively. Bone marrow samples can also be collected to find the presence of leukemic cells by flow cytometry. Abnormal T-cells can be detected using PCR-based tests (Inverse PCR) on lymphoid cells to screen for the presence of the provirus DNA within the host genomic DNA. Blood serum or plasma can be screened for tumour markers via marker selection and the prediction can be validated using Real Time Polymerase Chain Reaction and an antibody-based assay to monitor the viral load. Chest X-ray can be used to search for opportunistic infections common to blood disorders like pneumonia, bone lesions and other related infections. Blood cultures should be obtained to also detect fever-causing infection like bacteria, fungi or virus. Genomes of patients who recovered should also be screened to check for natural variations capable of conferring resistance to the virus infection. This can be done by pyrosequencing or searching for allelic markers within a population of Disease-A affected patients and those who recovered without any ill-effects.
To diagnose Disease-A, several serological test can be adopted. Blood samples from the infected patients should be taken with the plasma screened to check the presence of viral antibodies using Enzyme-linked immunosorbent assay (ELISA). ELISA is an immunodiagnostic test used to detect virus antibodies in blood samples/ serum, allergies and diseases in humans and animals (Butler, 2000). ELISA utilizes enzyme-conjugated antibodies, with antigen or antibodies bound to a solid support. It reports the quantitative determination of antibodies and measures changes in enzyme activities involved in immune reactions (Engvall and Perlmann, 1972). This method can also be used to detect plant viruses (Clark and Adams, 1977). Results from ELISA can be confirmed by a simple western blot and hybridised to a cDNA probe to confirm the presence of viral proteins within the infected patient serum sample. This is necessary because ELISA can give a false positive result for the virus antibody. To confirm using western blotting, the serum sample will be positive for the viral antibody. Another serological test that can be used to diagnose Disease-A is by indirect immunoflourescence technique. This technique uses the specific binding of antibodies to antigen to target fluorescent dyes to specific targets in the cell. This method is preferred to direct immunofluorescence because of its specificity. This technique has been used to detect the percentage increase of leukocytes antibodies in patients with febrile transfusion reactions (Verheugt et al, 2008) and monoclonal antibodies produced against the Dengue virus (Henchal et al, 1982). In using this technique Disease-A will be diagnosed for the presence of the viral antibody in the infected patient blood serum.
To diagnose suggested leukemia, a bone marrow aspiration and biopsy (BMAB) can be used to confirm leukemic cells. This method is used to diagnose a number of blood-related and non-blood related disorders like chronic lymphocytic leukemia (CLL), Hodgkin and Non-Hodgkin lymphoma, hairy cell leukemia multiple myeloma, thrombocytosis, leucocytosis, anaemia, tuberculosis, Mycobacterium avium intracellulare (MAI) infections, histoplasmosis, leishmaniasis, and other fungal infections, however more suitable techniques are available to diagnose most of the disease mentioned. To diagnose Disease-A by BMAB, bone marrow samples are taken from infected patient and biopsied. The result of the biopsy should be positive for a highly metastatic lymphoma. A novel technique used to identify disease biomarkers from a population of diseased and normal patients can also be used as a diagnostic tool for Disease-A, called Surface-Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF MS) (Grizzle et al, 2003). This technique can be used to generate protein expression profiles from serum and clinical samples as well as biomarker identification for various cancers and diseases (Tolson et al, 2004). This powerful technique is also used to study protein-protein and protein-DNA interaction. Proteomics has been used to predict novel targets for cancer therapy and drug resistance (Hanash et al, 2002). It has been reportedly used in biomarker identification and target cancer therapy in renal cancer (Tolson et al, 2004), prostate cancer (Grizzle et al, 2003), acute leukemia (Hanash et al, 2002), B-cell chronic lymphocytic leukemia (Voss et al, 2001) etc. This technique can also be adopted to screen for biomarkers in Disease-A in order to design 'drugable' therapeutic targets.
The first step in curative therapy is to cure identified opportunistic infections with repeated drug treatment and antibiotics. Expression profiles using proteomics have been used to identify targeted therapy for some cancer types. This method will also be used to develop curative therapy for Disease-A. As described earlier, SELDI-TOF MS can be used to identify high levels of protein in diseased patient serum relative to non-diseased patient. In a disease similar to Disease A, i.e. acute lymphoid leukemia, high levels of a polyprotein, oncoprotein 18 (Op18), was seen in diseased lymphoid cells compared to those of healthy lymphoid cells. Therefore it is suggested that inhibiting Op18 expression may infectively inhibit leukemic cell proliferation (Melhem et al, 1997). Likewise, down regulating Op18 expression inhibited tumorigenecity of leukemic cells in an immunodeficient mouse model (Hanash et al, 2002). This approach can be used as a therapeutic drug target for Disease-A by using SELDI-TOF MS to identify high expression levels of protein with the diseased patients and control.
Since the lymphoma was refractory to conventional chemotherapy and radiotherapy in Disease-A, another curative therapy can be done by using bone marrow transplantation. As reported by Borg et al. (2003), allogeneic bone marrow transplantation from a histocompatible Human T-cell lymphoblastic Virus 1 (HTLV-1) negative donor to a HTLV-1 positive patient showed complete remission and PCR revealed no evidence of the viral infection after 23 months. The result is similar for a leukemic disorder, Philadelphia chromosome-positive acute lymphoblastic leukemia, where the bone marrow transplantation proved effective as a therapeutic method for lymphoblastic leukemia (Barrett et al, 1992) which is also a similar disease to Disease-A. Therefore, bone marrow transplantation from a healthy patient to a Disease-A patient can prove effective as long as they are histocompatible. Other bone marrow transplantation techniques have also been reported which can be adopted for Disease-A i.e. using unrelated and related T-cell depleted bone marrow transplantation (Small et al, 1999). The use of allogeneic hematopoietic stem cell transplantation has been elucidated as an alternative to bone marrow transplantation. This involves the transfer of hematopoietic stem cells from the bone marrow or blood from a healthy individual to a diseased individual in the treatment of T-cell leukemia (Utsunomiya et al, 2001).
Another therapy that could be developed to cure Disease-A is gene therapy. It involves using DNA that encodes a functional gene to replace the mutated gene to provide treatment. This method though risky can be a form of curative therapy if used effectively. It uses viral vectors to convey the therapeutic material into cells. This method has been used successfully in the treatment of cancer according to Zhang et al. (1996). Viral vectors used for gene therapy include adeno-associated virus, adenovirus, lentivirus and the most ideally used being the retrovirus. Other gene therapies include small-interfering RNA gene knockdown and RNA enzyme-directed gene therapy (Altman, 1993).
Targeted drug therapy can also be used as curative therapy as in the Acute Lymphoblastic Leukemia which is similar to Disease-A. The treatment uses drugs designed to recognise and kill lymphocyte cells without harming stem cells. The monoclonal antibody, Alemtuzumab is used to target mature lymphocyte cells (Enblad et al, 2004). Alemtuzumab binds CD52, a protein found on mature lymphocytes and more on transformed lymphocytes, and becomes a target for destruction. Other type of targeted drug therapy include interferon alpha, topoisomerase inhibitor, zidovudine plus interferon alpha, arsenic trioxide plus interferon alpha, blockade of NF-KB and by monoclonal antibodies IL-2R receptors (Taylor and Matsuoka, 2005). In relation to being refractory, a nucleoside drug, Clofarabine, has been used as a curative therapy in leukemia (Jeha et al, 2004) which should also apply to Disease-A as the disease is suggested leukemic. In another study, a combination of Clofarabine and Cytarabine (ara-C) has been used for refractory acute leukemia (Faderl et al, 2005). Another nucleoside drug Nelarabine is also used for refractive T-cell malignancy (Kurtzberg et al, 2005) which is common to Disease-A.
Curative therapy can also be developed If natural variations are found within the population who recovered or who are rarely affected by Disease-A, in this case Afro-Caribbean's. This variation can be engineered into infected Disease-A patients to confer resistance to the disease. This method has been reported by Perez et al. (2008) in which a deletion in the coding region of a chemokine receptor, CCR5, confers resistance to HIV-1 infection in homozygous carriers. Some human populations carry a natural mutation of this receptor and are resistant to HIV-1 infection (Huang et al, 1996). This led to the use of engineered Zinc finger nucleases to induce a mutation in the CCR5 gene in infected individuals conferring resistance to HIV-1 CD4+ T cells (Perez et al, 2008). This can be exploited if natural variations are found in the non-affected Disease-A population.
In summary, individuals infected with the unknown Disease-A were present with flu-like symptoms followed by anaemia, acute bone pain, lethargy, high fevers and weight loss and after 6-12 months, died from opportunistic infections caused by a severely impaired immune response. Clinical and laboratory investigations revealed an elevate white cell count and a highly metastatic lymphoma of T-cell origin. Based on the symptoms and epidemiological studies it was suggested that Disease-A was a blood related disorder induced by a viral infection which causes an immunodeficiency of lymphoid cells via the changes to T-cell transmissible by sex. The hypothesis was tested by doing a complete blood count to ascertain infections causing the observed symptoms and a PCR-based test to monitor the viral load. A number of diagnostic methods were outlined including serology test, bone marrow biopsy and SELDI-TOF MS to ascertain leukemic cells and viral antibody. And finally, a number of curative therapies were highlighted; bone marrow transplantation, drug therapy and gene therapy, to name a few.