Mirjana Pavlovic

Skin lesions - an indicator of disease activity in systemic lupus erythematosus?

Cutaneous manifestations have great diagnostic value for systemic lupus erythematosus (SLE). In this study we tried to establish a correlation between lupus erythematosus LE-specific and LE-nonspecific cutaneous lesions and disease activity measured by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Sixty-six patients with SLE were evaluated. They were divided into three groups having: (1) only LE-specific lesions (38 or 58.46%); (2) only LE-nonspecific lesions (4 or 6.15%); and (3) both types of lesions (23 or 35.38%). Results were analyzed using the Student t-test. Patients with LE-nonspecific skin manifestations had significantly increased disease activity compared to those with only LE-specific lesions. The number of different skin lesion types also correlated with disease activity. It was significantly increased in a group with three different types of lesion, either specific or nonspecific. Patients with only one type of lesion had mild disease. An intermediate disease activity was found in the group with two different lesion types. Lupus-specific skin manifestations serve primarily as an important diagnostic clue. In conclusion, patients with LE-nonspecific lesions have significantly more active SLE than those with LE-specific lesions and may therefore require more intensive therapy and disease monitoring.

Clinical and molecular evidence for association of SLE with parvovirus B19.

In addition to genetic and environmental factors, viruses have been suspected as causes and/or contributors to human autoimmune diseases, although direct evidence for the association is generally lacking. Parvovirus B19, the cause of Fifth disease in childhood, and possible trigger in the spectrum of autoimmune diseases in adults, has emerged as one of the central viral candidates within the last few years. In this article we propose a possible model for parvovirus B19 association with systemic lupus erythematosus (SLE). The basis for our model is the secretion of hydrolyzing anti-ssDNA autoantibodies in 30—70% of cases with SLE, which in turn can either hydrolyze viral B19 ssDNA in blood and other fluids, or intranuclear, replicated viral ssDNA after re-activation and translocation of the virus into the nucleus of the host permissive cells. Both mechanisms contribute to perpetuation and maintenance of a ‘vicious cycle’ with concomitant flares in SLE, and involve inevitable TLR9 sensitization and genetic switch for anti-ssDNA autoantibody production from activated B cells in individuals prone to triggering. This model strongly suggests a major potential impact upon early prevention (vaccination) and targeted therapy of this subclass within the SLE spectrum of diseases. Incorporated in this new concept is an innovative idea for developing the tool for more precise (individualized) diagnosis, prevention, and therapy.

Pathogenic and Epiphenomenal Anti-DNA Antibodies in SLE

The discoveries of natural and the development of manufactured highly efficient catalytic antibodies (abzymes) opens the door to many practical applications. One of the most fascinating is the use of such antibodies in human therapy and prevention (vaccination), of cancer, AIDS, autoimmune diseases. A special entity of naturally occurring DNA hydrolytic anti-DNA antibodies is emerging within past decades linked to autoimmune and lymphoproliferative disorders, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjogren Syndrome (SS), B - Chronic lymphocytic leucosis (B-CLL), and Multiple Myeloma (MM). The origin of the antibodies is unknown. The underlying mechanisms of these activities are suggested to be penetration into the living cells and translocation in the nucleus, with recognition of the specific binding sites at particular (ss or ds) DNA. There are controversies in the literature whether hydrolysis is a sequence-specific event. The interplay between anti-DNA antibodies and DNA is not yet elucidated. This molecular “twist” also suggests that anti-DNA antibodies with DNA hydrolytic capacity could be the organisms immune response to a microbial attack, with microbial DNA, or specific genes within microbial DNA sequence, as a target for neutralization. The catalytic antibody-based approach can become a key tool in selective chemotherapeutic strategies.

CD34+ cells obtained from “good mobilizers” are more activated and exhibit lower ex vivo expansion efficiency than their counterparts from “poor mobilizers”

BACKGROUND: The classification of patients into “good” or “poor” mobilizers is based on CD34+ cell count in their peripheral blood (PB) after granulocyte–colony‐stimulating factor (G‐CSF) injection. We hypothesized that, apart from their mobilization from marrow to the blood, the response to G‐CSF of CD34+ cells also includes activation of proliferation, metabolic activity, and proliferative capacity.

Molecular form and kinetic properties of phosphate-dependent glutaminase in the mitochondria isolated from the kidneys of normal and acidotic rats

Abstract 1. 1. Investigation of the mechanism of renal ammoniagenesis revealed that the Km of phosphate-dependent glutaminase ( l -glutamine amidohydrolase, EC 3.5.1.2) for glutamine was significantly decreased, whereas V was increased if the mitochondria from both normal and acidotic rats were suspended in a hypotonic medium. These and other data indicate that the inner membrane regulates glutaminase activity and that in metabolic acidosis the rate of glutamine influx is increased. 2. 2. The study of molecular form and kinetic properties of glutaminase contained in the mitochondria showed that only in the case of acidotic Wistar rats the enzyme exhibits unusual stability if heated 10 min at 50°C. Furthermore, 3 min heating at 50°C resulted in a strong activation of the enzyme. This suggests that in acidosis the enzyme is in a different molecular form or in a different conformational state. 3. 3. The enzyme is equally sensitive to the inhibition by glutamate and NaCl in both kinds of mitochondria, whereas the value of the Hill coefficient for phosphate is 2. This indicates that glutaminase is in the form of a monomer. It is most probable that in vivo there is an equilibrium between monomer and dimer forms of the enzyme. 4. 4. Determination of the activation energy of glutaminase showed that in the case of normal and acidotic Wistar rats a break in the Arrhenius plot at 28°C and 24°C occurs. This difference results probably from changes in the enzyme microenvironment during adaptation to acidosis.

Volume changes of rat kidney mitochondria, transport of glutamine and its inhibition by mersalyl

Abstract 1. 1. The use of different —SH reagents (mersalyl, NEM, pCMB and DTNB) showed that the glutamine carrier and phosphate-dependent glutaminase (PGD) of rat kidney mitochondria are two different proteins and that activity of the enzyme is not necessary for the transport of glutamine. 2. 2. However, it is interesting that there is a very similar sensitivity of these two proteins to the thiol blocking agents. 3. 3. Further investigation showed that volume changes of the mitochondria have a significant influence on glutamine transport and the mechanism of its inhibition by mersalyl. 4. 4. It is suggested that swelling of the mitochondria leads to unmasking of the functional —SH groups of glutamine carrier resulting in the stimulation of its activity and a higher sensitivity to the inhibition by mersalyl.

Stem cells in the arrangement of bone marrow repopulation and regenerative medicine.

Hematopoiesis is a permanent and complex event in which a spectrum of different mature blood cells from a small population of toti/pluri/multipotent stem cells (SCs) are produced through a variety of proliferative and differentiative processes. Hematopoietic SCs are defined as the cells with extensive self–renewal and proliferative potential, together with their ability to differentiate into all blood–cell lineages. Many studies have demonstrated that a multifactorious network of interactive cytokines and other blood–derived mediators regulate the survival, maturation, and proliferation of SCs .

Strategies in Ebola virus disease (EVD) diagnostics at the point of care

Abstract Ebola virus disease (EVD) is a devastating, highly infectious illness with a high mortality rate. The disease is endemic to regions of Central and West Africa, where there is limited laboratory infrastructure and trained staff. The recent 2014 West African EVD outbreak has been unprecedented in case numbers and fatalities, and has proven that such regional outbreaks can become a potential threat to global public health, as it became the source for the subsequent transmission events in Spain and the USA. The urgent need for rapid and affordable means of detecting Ebola is crucial to control the spread of EVD and prevent devastating fatalities. Current diagnostic techniques include molecular diagnostics and other serological and antigen detection assays; which can be time-consuming, laboratory-based, often require trained personnel and specialized equipment. In this review, we discuss the various Ebola detection techniques currently in use, and highlight the potential future directions pertinent to the development and adoption of novel point-of-care diagnostic tools. Finally, a case is made for the need to develop novel microfluidic technologies and versatile rapid detection platforms for early detection of EVD.

Highly Specific Novel Method for Isolation and Purification of Lupus Anti‐DNA Antibody via Oligo‐(dT) Magnetic Beads

Abstract:  A novel method for isolation and purification of anti‐ssDNA antibodies from human sera is developed. The process involves: antibody purification based on their affinity for single‐stranded sequence of thymidines and removal of remaining components via protein G coated magnetic beads, with high affinity for only IgG subclass. The high degree of purity and molecular weights of healthy versus lupus anti‐ssDNA antibodies were confirmed by SDS‐PAGE and silver staining. Western blot confirmed IgG isotype. This novel technique allows for diagnostic purposes, structural and functional analysis of anti‐DNA antibodies, and studies of their role in autoimmune diseases.

Autologous transplant in the treatment of severe aplastic anemia – A case report

The initial use of immunosuppressive therapy (IST) in severe aplastic anemia (sAA) or reapplication of IST-centered methods following disease relapse is successful only in well-selected patients. The potential treatment by autologous stem cell (SC) transplant in sAA is still an innovative/pioneering therapeutic approach. To our best knowledge, this is the second published case of autologous SC transplant in sAA. The aim of this work was to optimize mobilization and timing for SC harvesting - using our own controlled-rate cryopreservation - with higher CD34(+)/CD90(+) subset yield and recovery in order to obtain complete and long-term hematopoietic reconstitution following autologous SC transplant. We report a 35 year-old sAA male patient who initially underwent IST using rabbit ATG and Cyclosporine A (CsA). He was supportive transfusion dependent for the whole period of IST-phase. After the second IST-cycle, polymorphonuclear (PMN) cell count increase (>2.0 × 10(9)/L) was observed, when SC mobilization, two large volume leukapheresis procedures and following autologous transplant were performed. The yields of harvested CD34(+) and CD34(+)/CD90(+) cells were 5.75 × 10(6)/kgbm and 1.7 × 10(6)/kgbm, respectively. The quantity of applied CD34(+) and CD34(+)/CD90(+) cells in autologous SC transplant were 5.45 × 10(6)/kgbm (7-AAD(CD34)(+)(viability)=95.42%) and 1.63 × 10(6)/kgbm (7-AAD(CD34)(+)(/CD90)(+)(viability)=95.42%), respectively. Hematopoietic reconstitution registered due to second month after autologous SC transplant and he is 24 months in complete medullar, hematological and clinical remission, with normal cytogenetic status - applying only continuous CsA therapy. The results obtained strongly confirm that in sAA, with no allogeneic SC donor, autologous transplant can result in a successful clinical outcome. We suggest that CD34(+)/CD90(+) subset count in peripheral blood and/or cell-harvest could be more valuable predictive factor than total CD34(+) quantity of optimized collection-timing and superior treatment efficacy of autologous SC transplant in sAA.