Detection methods of immunoglobulin IgG in CSF and serum.

INTRODUCTION: This study represents a new approach to the extended analysis of correlation of findings of oligoclonal bands on gels and the level of intrathecal synthesis of immunoglobulin G in the central nervous system. Previous studies have shown that there is no correlation at this level as well as the number of tape or finding does not correlate with the forecast effect of therapy or patient outcome. AIMS OF THE STUDY: To determine the correlation of level of immunoglobulins IgG in CSF with the number of oligoclonal bands on the gel. MATERIAL AND METHODS: The retrospective study based on data processed in Clinical Immunology Clinical Center University of Sarajevo. Patients were assumed of multiple sclerosis according to clinical findings and magnetic resonance imaging. All CSF and serum samples were processed by nephelometry, isoelectric focusing on the gel. Statistical analysis of results was also performed by using SPSS statistical analysis program. RESULTS: Analyses were performed on 254 samples of cerebrospinal fluid and serum of patients from neurological clinic, suspected of multiple sclerosis. We concluded that there is no correlation between the level of intrathecal synthesis obtained by Reibergram with the number of oligoclonal bands on gels. We think that the reason could be a small sample of patients analyzed and it leaves room for future analysis on a larger sample. DISCUSSION AND CONCLUSION: For most patients with established MS we found intrathecal humoral response, type two, and the number and arrangement of IgG bands generally does not change during the disease, because they reflect long-term non-specific immune stimulation rather than a specific immune response that during infectious disease changes (quantitatively and qualitatively).

Monitoring of disease biomarkers activity and immunophenotyping as important factors in SLE clinical management.

The highly specific biomarkers for monitoring of SLE disease activity are not yet defined up to date, due to existing of different clinical SLE phenotypes caused by individual genetic variation. Basically, numerous clinical complications follow SLE patients such as nephritis, atherosclerosis and cardial, CNS, gastrointestinal and ophthalmological complications, as well. Their monitoring in clinical SLE management can be evaluated by analysing of specific biochemical parameters and require permanent clinical observation. The presence of ANAs and anti-ds-DNAs are usual diagnostic SLE autoimmunity parameters, while SLE disease activity biomarkers are C3 and C4 level, anticardiolipin antibodies, anti-Sm/RNPs and, recently level of CD4 and CD8 lymphocytes. However, the number of TCR molecules on the T-cells surface at SLE patients is lower then in normal condition, and otherwise for these receptors CD molecules make specific connection. On the other hand, the T lymphocytes can be also, therapeutical targets at SLE patients, because of their clear direct involving in SLE pathogenesis. The SLE phenotypes are characterized by double CD negativity ( CD3 +/-, CD4-) caused by abnormal level of IL-2 and IL-17. T-lymphocytes have usually alpha-beta and gamma-delta TCR receptors, but for SLE patients is characteristic lower number gama-delta TCR molecules, detected in the peripheral blood specimens. Taking into account all of the facts, we investigated the level of specific usual SLE activity biomarkers (anti-ds-DNAs, C3, C4, anticardiolipin antibodies (beta-2-IgG, beta-2-IgM, ACA-G, ACA-M, CD4 and CD8 level) in serum specimens of SLE patients who underwent to the corresponding chemotherapy in combination with other biochemical and clinical parameters. Once again proved to be, that SLE biomarker monitoring, could be useful aproach for SLE activity disease and prediction organ damage, as well. In our investigation we used the following methods: immunofluorescence microscopy (IFA-ANA), and nephelometry, Hycor ELISA system and Flow cytometry, for precisely quantitative measurements. We determined correlation between C3 and C4 complement components level, CD3 (T-Ly), CD3+/HLA-DR and total HLA-DR with regard to SLE disease activity. Also, CD4 (Th), CD4:CD8 ratio, beta-2-G, beta-2-M not proved to be useful biomarkers in this sense, despite some results specific for some special SLE phenotypes. Anti-Sm/ RNPs proved to be better in SLE diagnostic process.

Reibergram and oligoclonal bands in diagnosis of multiple sclerosis.

INTRODUCTION: In this study authors have analyzed the correlation between the IgG immunoglobulins in cerebrospinal fluid and the findings of oligoclonal bands on gel. Immunoglobulin IgG in cerebrospinal fluid (CSF) can be detected in neurological diseasses (infections and inflammatory neurological diseases and in demyelinating diseases, like multiple sclerosis (MS)). Quantitative IgG in CSF can be expressed by different formulae Reiber (Reiber and Felgenhauer 1987), Tourtellotte (Tourtellotte 1970), Schuller (Schuller and Sagar 1983) and IgG Index (Link and Tibbling 1977). In this study we used Reibergram. Qualitative CSF IgG can be measured by electrophoresis and isoelectric focusing (IEF). We used IEF for analysig CSF and seum because of its higher sensitivity. AIMS OF THE STUDY: To determine the correlation of immunoglobulins IgG positivity in CSF with the finding of oligoclonal bands on the gel. MATERIAL AND METHODS: The retrospective study based on data processed in OJ Clinical Immunology KCUS. Patients were suspicious of multiple sclerosis according to clinical findings and magnetic resonance imaging. All CSF and serum samples were processed by nephelometry, isoelectric focusing on the gel. Statistical analysis of intrathecal synthesis was also performed according to Reibergram. RESULTS: Analyses were performed on 76 samples of cerebrospinal fluid and serum of patients from neurological clinic, suspected of multiple sclerosis. We received following results: 42 samples tested had type 1.25 samples tested showed type 2.3 samples had type 3.5 samples had type 4.1 sample had a fifth type. When we compare these results with values obtained by intrathecal synthesis of which is determined by Reibergram we obtained the following values: 16 samples had intrathecal synthesis of 20%-60%, 9 samples had a negative value of intrathecal synthesis of 10% or less. DISCUSSION AND CONCLUSION: For most patients with established MS we found intrathecal humoral response, type two, and the number and arrangement of IgG bands generally does not change during the disease, because they reflect long-term non-specific immune stimulation rather than a specific immune response that during infectious disease changes (quantitatively and qualitatively).

Expression of NK (CD16+56+) and B cells (CD19) Receptor Molecules as a Reliable Clinical Response Biomarkers of SLE and RA Patients Under the Rituximab Treatment.

INTRODUCTION: Lately, the use of biological therapy in various autoimmune diseases is increasing. The ideal marker for monitoring the effects of modern therapy is still non-existent. AIM: To investigate early response biomarkers of SLE and RA patients under the rituximab treatment are in research phase and each new investigations offer new and original useful data. MATERIAL AND METHODS: Immunophenotyping of cells was carried out by a standard method of sample preparation. We investigated by flow cytometric analyses expression of NK and CD19+ cells at ten SLE and five RA patients before and after treatment with rituximab, in laboratory of Department of Clinical immunology in the Clinical Centre University of Sarajevo. RESULTS: In both cases, SLE and RA patients, reduced number of CD16+ parameter indicates lower cytotoxic activity of NK cells. Increased number of B cells indicates higher pathological activity leading to severe autoimmune disease allegation. CONCLUSION: Determining the proportion of NK and B will be useful diagnostic tool in therapeutic strategy, and also in monitoring of effect of biological therapy.

ELISA Test for Analyzing of Incidence of Type 1 Diabetes Autoantibodies (GAD and IA2) in Children and Adolescents.

INTRODUCTION: Anti GAD (antibodies on glutamic acid decarboxylase) and anti-IA2 antibodies (against tyrosine phosphatase), today, have their place and importance in diagnosis and prognosis of Type 1 diabetes. Huge number of patients with diabetes mellitus type 1 have these antibodies. Insulin antibodies are of critical importance in diagnosis of diabetes mellitus type 1 for pediatric population. MATERIALS AND METHODS: During 2014, the samples of 80 patients from Clinical Center University Sarajevo (CCUS) Pediatrics clinic's, Endocrinology department were analyzed on anti-GAD and IA2 antibodies. The samples of serums of all patients were analyzed with ELISA tests using Anti GAD ELISA (IgG) kites from EUROIMMUN company. These are quantitative in vitro tests for human antibodies against decarboxylase of glutamine acid (GAD) and IA2, in serum or EDTA plasm. RESULTS: During the period of one year, in CCUS's Organizational unit, Institute for Clinical Immunology, 80 samples of patients with anti GAD and IA2 antibodies were analyzed. Out of total number of samples, 41 were male patients, or 51% and 39 female, or 49%. The youngest patient was born in 2012, and the oldest in 1993. Age average was represented by the patients born in 2001. Share of positive results for IA2 antibodies and GAD antibodies was 37% for IA2 antibodies, and 63% for GAD antibodies. DISCUSSION: During an autoimmune - mediated Diabetes mellitus type 1 leads to T-cell mediated destruction of beta cells of pancreatic islets, reduced production of insulin and glucose metabolism. Studies have shown that these bodies are the most intense single marker for identifying persons with increased risk for diabetes development.

Financial aspects and the future of the pharmaceutical industry in the United States of america.

INTRODUCTION: The U.S. pharmaceutical industry is defined by the U.S. Census Bureau as "companies engaged in researching, developing, manufacturing and marketing of medicines and biological for human or veterinary use". Besides its main role in improving human health, the US pharmaceutical industry represents one of the most critical, key decision makers' lobbying prone and competitive sectors in the economy. The cost in the environment of very limited government price regulation remains one of the major problems fuelling aggregate health care cost inflation. Pharmaceuticals have created huge benefits for public health and economic productivity by the means of saving lives, increasing life expectancy, reducing illness related suffering, preventing surgeries and decreasing hospital stays. PURPOSE: The goal of this review paper is to show the present conditions and future trends of the pharmaceutical industry in the U.S. METHODOLOGY: THIS PAPER REPRESENTS A THOROUGH LITERATURE REVIEW OF THE MULTIFACETED SOURCES INCLUDING: studies, books, peer reviewed journals, U.S. government sources (i.e. U.S. Census Bureau, U.S. Bureau of Economic Analysis, etc.). DISCUSSION: In the thirty years pharmaceutical companies have consistently developed and launched new medicines, bringing hope to sick or - at risk patients. They also usually provide above the average financial returns for its shareholders. U.S. pharmaceutical companies had as their goal to discover blockbuster drugs. Blockbuster drugs are generally defined as drugs that solve medical problems common to hundreds of millions of people and, at the same time generate large sales increases and profits for the pharmaceutical companies. The main approach of these companies includes huge investments in research and development (R&D), innovation, marketing and sales. The trend analysis shows that for the most part the era of blockbuster drugs is nearing an end. CONCLUSION: Numerous blockbuster drugs will be coming off patent in the next few years, opening the way to generics and eliminating a major source of the industry's profits. Still, there is plenty of room for improvement in the medications people take while there is no shortage of human suffering to alleviate. It is doubtful whether big pharmaceutical firms will be able to pursue these goals within the old model of developing exclusive new drugs that can be sold further in the future. In the past, medicines for the ailments that were never before addressed, like anti-cholesterol or anti-depression drugs were developed. Currently, and in the future, it is expected that only blockbuster modifications will be developed. This phenomenon is expected to create market saturation, which will significantly reduce profits. The business model that drove the major drug makers' success is not working anymore. Pharmaceutical companies must create new ways and to bring new ideas. The survivors will be those that market strategies supported by innovative approaches and winning capabilities.

The incidence of ANA and ETI-dsDNA detected by enzyme immunoassays and indirect immunofluorescence assay (IFA).

While the SLE (Systemic Lupus Erythematosus) specificity of ANA is low, that of anti-dsDNA autoantibodies is high. The DNA used in the assay must be double stranded: autoantibodies to single-stranded (ss) DNA exist in many diseases and specific to none. The prevalence (70%) of anti-dsDNA autoantibodies is much higherin SLE, giving a higher diagnostic sensitivity than the similarly disease-specific anti-Sm autoantibodies (30%). Anti-dsDNA autoantibodies are usually detected by very analytically sensitive techniques, such as ELISA (Enzyme Linked Immunosorbent Assay). Within SLE, ds-DNA autoantibodies tend to associate with the presence of glomerulonephritis. Their levels are used to monitor disease activity. We suggest the use of ds-DNA to find the difference between SLE patientswith benign variants and classical syndrome of severe skin and renal disease.

Clinical management of patients with systemic lupus erythematosus (SLE) with different C1q-CIC and C3 concentrations.

Over the third of SLE (Systemic Lupus Erythematosus) patients have a high level auto-antibodies-antigen complex that contains some complement proteins, especially C1q as the trigger protein in the classical complement activation pathway. So, the SLE, as an autoimmune disease, is certainly related to disorders caused by activation of complement system, that finally leads to tissue damage. It may also be caused by hereditary deficiency (complement genes mutations). In such case, some components of the complement system might be inactivated. There are mutations that cause disorders in each of three complement system activation pathways (classical, alternative and lectin).The serum samples of SLE patients show the presence of specific autoantibodies for some complement components. Today, for clinical management of SLE patients, determination of level of C1q-CIC and C3 complement component in serum specimens have great diagnostic and therapeutic importance. During the year 2000, we analyzed a numerous serum samples from patients suspected to autoimmune diseases (SLE especially). The samples were collected from several clinics in the Clinical Center of University of Sarajevo, mostly from Clinic of Infectious Diseases, Pediatrics, Internal Medicine and Gastroenterohepatology Clinic. Primary samples went through screening for the presence of ANA using ANA-IFA method and further characterization of ANA positive samples was carried out using IFA-ANA titration, ELISA and nephelometry.

ELISA subtypization of anti-ENA autoantibodies in clinical management of autoimmune diseases in Bosnia and Herzegovina.

The basis of autoimmune diseases such as SLE (Systemic Lupus Eritematodes), Sjogren's syndrome, scleroderma, dermatomyositis and polymiositis is the creation of auto-antibodies to the following specific extractable nuclear antigens (ENA):Jo-1, Ssl-70, SS-A, SS-B, Sm and Sm/RNPs. Some of these antigens are in fact enzymes (Jo-1-histidil-tRNA synthetase, Scl-70-topoisomerase) which are inhibited by specific autoantibodies--this leads to disturbance in the metabolism of DNA and protein biosynthesis. During 2009, we analyzed total of 87 serum samples of patients suspected for autoimmune disorder using ANA-IFA and ELISA-ENA-6 methods. After establishing IFA-ANA positivity (83.9%), all serum specimens; ANA positive and negative, were subtypized by ELISA ENA-6 test. Analysis showed the highest incidence of anti-SS-A (56%), and incidence of anti-SS-B (29.8%), anti-Sm/ RNP (11.5%), anti-Jo-1 (2.3%) and anti-Scl-70 (1,1%) auto-antibodies. Also, 78.5% of IFA-ANA negative serum specimens showed high level of positivity (212.50 and 277.0 IU/ml) to SS-A (78.5%) and SS-B (21.4%) antigenes using ELISA-ENA-6 subtypization. Following these results, we conclude that it is necessary to introduce Western blot confirmation testing. After comparing with other clinical findings, we diagnosed the following autoimmune diseases: SLE, Sjogren's syndrome and dermatomiosytis.

Transplantation of organs: one of the greatest achievements in history of medicine.

The history of transplantation is a scientific journey describing the medical community's effort to understand how the human body works. Humans have long realized the possibilities which transplantation of organs and tissue provides. Throughout history people have always been intrigued by the possibilities of the transplantation of organs and tissues. In the 6th Century BC Indian surgeons described how to reconstruct facial wounds by transplanting skin from one place on the body to the other. During the middle age there were many references in historical medical literature of attempted blood transfusions as well as the transplantation of teeth. A skin transplant and a corneal transplant were reported in medical journals dating as far back as 1880. These early attempts were usually unsuccessful. Early in the twentieth century transplantation started to offer the promise of restored health and life. One of the exceptional medical advances of the twentieth century, organ transplantation has become a routine treatment for patients with organ failure which was a goal.

Comparative study of interleukin 1ALFA and interleukin 6 concentrations in serum specimens detected by ELISA.

Interleukin 1 (IL-1) contains two proteins, which are the products of distinct genes, but which recognize the same cell surface receptors. In the liver, IL-1 initiates the acute phase response resulting in an increase in hepatic protein synthesis and decreased albumin production IL-1 also plays an important role in immune functions, having effects on macrophages/monocytes, T lymphocytes, B lymphocytes, NK cells, and LAK cells. Interleukin-6 (IL-6) is a cytokine that regulates immune responses. We analyzed total 160 serum specimens of patients from Clinical Center University of Sarajevo with different inflammatory diseases by ELISA method on interleukins: IL-1alfa and IL-6. Tests that we performed with IL-lalfa and IL-6 by ELISA method confirmed that serum specimens with IL-6 ELISA showed increased values of tested specimens, than the lowest standard and blank. We had average levels of IL-1alfa 3.7 pg/ml which was below the level of the lowest standard. All obtained results were in accordance with the results in IBL protocol for blank and lowest standard values, as well as the average levels of serum specimen values.

The incidence of antinuclear antibodies (ANA) detected by indirect immunofluorescence assay (IFA) method.

Human anti-nuclear antibodies (ANA) in Systemic Lupus Erythematosus (SLE) react specificaly with DNA, RNA, several proteins and ribonucleoproteins. Systemic Lupus Erythematosus is the classic type of polysystemic autoimmune disease. The high frequency of ANA is determined in these patients. Actually, all SLE patients are ANA positive. ANA testing by IFA (Indirect Immunofluorescence Assay) is an excellent screening tool for SLE cases, but it is not so highly specific test. Patients with connective tissue diseases, such as rheumatoid arthritis, scleroderma and dermatomyositis are also frequently positive. Results of IFA ANA have relative low specific degree, and for this reason the titration of these specimens to the end point is usually recommended. Indirect immunoflourescence is reference method for ANA testing. Common substrates are thin sections of rodent organs or various types of cell lines. The cell line substrates are preferable to organ sections, because these rapidly dividing cells have higher level for detection of certain clinically relevant antigens such as (e.g., centromere, SSA (Ro) and Scl-70). In this paper we present the results evaluation of ANA incidence, detected by IFA in serum specimens of corresponding clinical patients, during 2005 and 2006.

Optimisation of RT-PCR for detection of enteroviruses.

Enteroviruses are members of Enterovirus genus of Picornaviridae family. On the basis of their pathogenesis and host range, most human enteroviruses are classified into one of three groups (Coxsackie's viruses, echoviruses and polioviruses). Some unclassified human enteroviruses may cause bronchitis (type 68), acute hemorrhagic conjunctivitis (type 70), meningitis and paralysis (types 70 and 71) and hepatitis (type 72 or hepatitis A virus). Enteroviruses can be propagate in primary cultures of human monkey kidney cells and in some cell lines such as HeLa, Vero and WI-38. Virions are small (22-30 nm diameters) containing ss RNA, monopartite and have icosahedra symmetry. The fast, high sensitive and specific detection of enteroviruses today is achieved by using of PCR (Polymerase chain reaction) method. But, PCR is so much more than just mixing reagents in a tube and running a thermal cycler. In each laboratory with PCR facilities, it is necessary to find optimal PCR conditions (performing of PCR optimization experiments). In this paper we presented results of PCR optimization for enteroviruses by using of poliovirus type 1(Sabin). Optimal obtained PCR parameters were: 2,5 mM MgCl2, dNTPs dilution 10(-1) and annealing temperature 50 degrees C, after 30 amplification cycles in Perkin Elmer 2400 thermal cycler.