Vol. 53, No. 5, 2005

Min Li^{1, 2}, Lauren S. Becnel^{1, 3}, Wei Li⁴, William E. Fisher², Changyi Chen¹ and Qizhi Yao^{1, 3}, (¹Molecular Surgeon Research Center, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77021, USA, ²Elkins Pancreas Center, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77021, USA, ³Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77021, USA, ⁴School of Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, 250014, China)

Abstract. Pancreatic cancer is a devastating disease because of the lack of early detection markers and effective treatments. It is the fourth leading cause of cancer-related death in western countries, including the United States. The mechanisms of pancreatic cancer progression remain unknown. Transforming growth factor β (TGF-β), a multifunctional cytokine, regulates cell growth and differentiation in healthy tissues, yet fails to do so in pancreatic cancer. Alterations of the TGF-β and TGF-β receptor/Smad signal transduction pathway have been implicated in pancreatic cancer. Furthermore, both the TGF-β receptor and Smad proteins interact with a variety of cellular signal pathways, such as the somatostatin receptors (SSTRs), ERK1/2, and Wnt signal transduction cascades. This suggests that pancreatic cancer is a multi-gene-controlled malignancy and that effective treatments for pancreatic cancer should be aimed at multiple targets. In this review, we summarized the major signal intermediates involved in pancreatic cancer signal transduction pathways and specifically discussed how alterations in the regulatory functions of TGF-β and Smad proteins allow for pancreatic carcinogenesis.

Keywords: pancreatic cancer; TGF-β; SSTR.

Masaru Yamaguchi and Kazutaka Kasai, (Department of Orthodontics, Nihon University School of Dentistry at Matsudo, Japan)

Abstract. Orthodontic forces are known to produce mechanical damage and inflammatory reactions in the periodontium and dental pulp, as well as inflammatory mediators, e.g. prostaglandins, interleukin (IL)-1, IL-6, tumor necrosis factor α, and receptor activator of nuclear factor κB ligand, in the periodontal ligament (PDL) and dental pulp. We have studied the effects of aging on the production of inflammatory mediators in the PDL using in vitro and in vivo methods and found that aging of PDL tissues may be an important factor in the severity of periodontal disease through a higher production of inflammatory mediators in response to mechanical forces. Further, the levels of inflammatory mediators in gingival crevicular fluid, an osmotically mediated inflammatory exudate found in the gingival sulcus, have been shown to be significantly elevated during orthodontic treatment. In order to reduce inflammation, low-level laser therapy has been recently studied in vivo and in vitro by many investigators as a substitute for anti-inflammatory drugs. Clinical and experimental studies have shown that low-level laser irradiation reduces orthodontic post-adjustment inflammation. We believe that orthodontic forces (mechanical forces) may play an important role in periodontal inflammation and that low-level laser therapy may be useful for its inhibition.

Keywords: inflammation; mechanical forces; cytokines; orthodontics; periodontal disease.

Uday Kishore^{1, 2}, Andrés López Bernal³, Mohammed F. Kamran⁴, Shweta Saxena⁵, Mamta Singh⁶, P. Usha Sarma⁶, Taruna Madan⁶ and Trinad Chakraborty², (¹Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK, ²Institute of Medical Microbiology, Faculty of Medicine, Justus-Liebig-University, D-35392 Giessen, Germany, ³Clinical Science at South Bristol (Obstetrics & Gynecology), Level D, St. Michael’s Hospital, Bristol BS2 8EG, UK, ⁴Department of Infectious Diseases, Imperial College London, London W12 0NN, UK, ⁵Bhopal Memorial Hospital and Research Center, Bhopal 462038, MP, India, ⁶Institute of Genomics and Integrative Biology, Council for Scientific and Industrial Research, Delhi University Campus, Delhi 11007, India)

Abstract. Surfactant proteins A (SP-A) and D (SP-D) are lung surfactant-associated hydrophilic proteins that have been implicated in surfactant homeostasis and pulmonary innate immunity. They are collagen-containing C-type (calcium-dependent) lectins, called collectins, and are structurally similar to mannose-binding protein of the lectin pathway of the complement system. Being carbohydrate pattern-recognition molecules, they recognize a broad spectrum of pathogens and allergens via the lectin domain, with subsequent activation of immune cells via the collagen region, thus offering protection against infection and allergenic challenge. SP-A and SP-D have been shown to be involved in viral neutralization, clearance of bacteria, fungi, and apoptotic and necrotic cells, down-regulation of allergic reaction, and resolution of inflammation. Studies on single-nucleotide polymorphism, protein levels in broncho-alveolar lavage, and gene knock-out mice have clearly indicated an association between SP-A and SP-D and a range of pulmonary diseases. In addition, recent studies using murine models of allergy and infection have raised the possibility that the recombinant forms of SP-A and SP-D may have therapeutic potential in controlling pulmonary infection, inflammation, and allergies in humans.

Keywords: lung; surfactant; innate immunity; pathogen; allergy; disease; pregnancy.

Catherine M. Greene and Noel G. McElvaney, (Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland)

Abstract. Toll-like receptors (TLRs) belong to a family of transmembrane proteins that can recognize and discriminate a diverse array of microbial antigens. Following their activation by specific ligands, TLRs initiate intracellular signaling cascades that culminate in the activation of transcription factors and ultimately lead to changes in pro-inflammatory gene expression. The TLR family constitutes an important component of the innate immune system and, although most commonly considered to be associated with immune cell responses, TLRs are also known to be functionally expressed on a variety of other cell types. Epithelial cells represent a significant component of the cellular content of the airways. These cells provide both a barrier to infection and an active defense mechanism against invading microbes. The expression and function of TLRs on airway epithelial cells has been an area of increasing interest in the recent past. This review will summarize advances in our understanding of the role of TLRs in airway epithelial cells.

Keywords: Toll-like receptors; airway epithelial cells; inflammatory lung disease.

Aneta Szczerkowska-Dobosz, (Dermatology Department, Medical University of Gdańsk, Poland)

Abstract. Psoriasis is a multifactoral and heterogenetically inherited disease. The role of hereditary transmission is supported by familial association, twin studies, and correlation with human leukocyte antigens (HLA). Numerous studies have proved that B13, B17, Cw6, and DR7 antigens are positively associated with psoriasis. Cw6 antigen has been repeatedly indicated to be the most significant marker for the risk prediction of the disease. On the basis of epidemiological studies and HLA analysis, a concept of two distinct disease patterns of psoriasis vulgaris was proposed. In type I psoriasis the disease has an early onset, strong correlation with Cw6, B13, B17, and DR7 antigens, and familiar inheritance. Type II psoriasis has a late onset, weak correlation with HLA antigens, and sporadic familiar occurrence. Both types seem to differ clinically. Moreover, some extended haplotypes were shown to be correlated with the disease, especially with the type I psoriasis. Although a psoriasis susceptibility gene(s) has not been yet identified, a number of candidate genes were studied, with evidence for a major locus located within the major histocompatibility complex (PSORS 1). Cw6 allele is the most extensively investigated candidate gene, but present evidence suggests that it is rather in strong linkage disequilibrium with the PSORS 1 gene than the susceptibility allele itself. This article reviews past and current data on the genetic background of psoriasis with special attention to its correlation with HLA antigens.

Keywords: Cw6; epidemiology; haplotypes; HLA; MHC; psoriasis.

Agnieszka Bonar , Magdalena Chmiela , Wiesława Rudnicka and Barbara Różalska, (Department of Immunology and Infectious Biology, Institute of Microbiology and Immunology, University of Łódź, Poland)

Abstract. Introduction:
Phagocytosis is the critical first step in the Mycobacterium (M.) tuberculosis-phagocyte interaction. The process involves microbial ligands and phagocyte surface receptors. It is known that serum mannose-binding lectin (MBL), an innate immune system component, may enhance the uptake of microbes by phagocytic cells and activate the complement system. Since phagocytes are the replicative environment for mycobacteria and, as we described earlier, tuberculosis patients differ from controls in serum MBL level, we asked whether MBL plays a role in promoting M. tuberculosis access to phagocytic cells.

Materials and Methods:
To estimate the influence of MBL on the phagocytic process, FITC-labeled Mycobacterium bovis BCG was used as a model bacterium. Neutrophils from healthy individuals were used as phagocytes. Phagocytosis was performed in the presence or absence of recombinant MBL (rMBL; 2 or 20 μg/ml). The activation of complement was determined by dot-blot immune assay with monoclonal antibodies against C5b-C9.

Results:
We showed that phagocytosis of the bacteria was more intensive in the presence of human rMBL. Both attachment and ingestion of mycobacteria were enhanced when MBL and active complement components (fresh serum) were present in the medium. The dot-blot method showed that the bacteria slightly activated complement by themselves. This effect was enhanced in the phagocyte-bacteria co-cultures containing rMBL.

Conclusions:
It is possible that MBL may serve in vivo as one of the factors facilitating the entry of mycobacteria into phagocytes, pathogen spread, and the establishment of infection.

Keywords: tuberculosis; mycobacteria; phagocytes; collectins; mannose-binding lectin.

Anna Łaba¹, Alina Ostrowska², Dariusz Patrzałek³, Leszek Paradowski⁴ and Andrzej Lange^{1, 5}, (¹Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland, ²Tissue Transformation Technologies, Inc., Edison, NJ, USA, ³Department of Vascular Surgery, Medical Academy, Wrocław, Poland, ⁴Department of Gastroenterology, Medical Academy, Wrocław, Poland, ⁵Lower Silesian Center for Cellular Transplantation with the National Bone Marrow Donor Registry, Wrocław, Poland)

Abstract. Introduction:
The successful use of hepatocytes depends on a reliable demonstration of the functional and morphological integrity of isolated cells. Herein we investigated whether the isolation and cryopreservation of primary human hepatocytes can compromise cell viability and liver-specific characteristics.

Materials and Methods:
Hepatocytes were isolated from encapsulated human liver segments by a modified 2-step perfusion technique. Isolated cells were Percoll-purified, cryopreserved, and stored in liquid nitrogen for 1–12 months. For rapid assessment of fresh and cryopreserve/thawed hepatocyte yield and viability, the cells were stained with trypan blue or labeled with fluorochromes. For immunocytochemical analysis, the cells were labeled with monoclonal antibodies for the presence of the following antigens and chemokines: CD3, CD45Ro, CD45Ra, CD34, CD68, CD90, CD95, CD20, HLA-DR, Ki67, PCNA, Bcl-2, p53, CXCR3, CXCR4, and SDF-1. The cells were tested for several specific functions, such as ureagenesis, energy status, MTT activity, lactate dehydrogenase leakage, and total CYP450 content.

Results:
Assessment of both freshly isolated (Percoll-purified) and cryopreserved/thawed hepatocytes revealed a low constitutive level of contamination by non-parenchymal cells compared with crude (unpurified) preparations and tissue sections. All viable hepatocytes showed intact morphology and retained CYP450 protein, energy status, and urea synthesis.

Conclusions:
Modifications in hepatocyte preparations, such as depletion of dead, damaged, and nonparenchymal cells, improves cell purity, which can be adapted to further evaluation of hepatocyte immunogenicity. These data illustrate the importance and feasibility of human hepatocyte banking.

Keywords: hepatocyte isolation; human; cryopreservation; non-parenchymal cells.

Maria Kraj, Katarzyna Owczarska, Urszula Sokołowska, Piotr Centkowski, Ryszard Pogłód and Barbara Kruk, (Department of Hematology, Institute of Hematology and Blood Transfusion, Warsaw, Poland)

Abstract. Introduction:
Recent studies suggest that multiple myeloma (MM) triggers osteoclastogenesis by disrupting the balance between the receptor activator of NF-;kappa&B ligand (RANKL) and osteoprotegerin (OPG), its natural antagonist.

Materials and Methods:
Determinations of bone marrow (BM) and serum OPG and sRANKL concentrations were performed in 133 MM patients and 42 healthy subjects by the ELISA method using Osteoprotegerin ELISA and sRANKL ELISA kits.

Results:
MM patients had elevated serum levels of OPG compared with controls (p<0.0001) and OPG levels were higher in patients with renal failure and patients with hipercalcemia (p<0.001 and p=0.04, respectively). Serum OPG levels correlated with age, serum β₂-microglobulin, and BM OPG concentrations and did not correlate with the presence of osteolysis or with stage of disease. sRANKL serum levels in MM patients and in controls were not statistically different (p=0.42). In MM patients, serum OPG and sRANKL levels were similar at diagnosis and in the plateau phase of disease. There was a correlation between BM and serum sRANKL concentrations (p<0.001). Median values of the sRANKL/OPG ratio for BM and serum of MM patients were 0.14 and 0.11, respectively. The median value of the sRANKL/OPG ratio for the serum of controls was 0.11.

Conclusions:
In 20% of MM patients, serum OPG levels are elevated, and this may be a compensative reaction related to increased bone destruction. There is not statistically significant relationship between sRANKL serum and BM levels and the main clinical and laboratory parameters of the disease. Determination of BM and the serum sRANKL/OPG ratio seems to have no clinical value.

Keywords: multiple myeloma; osteoprotegerin; RANKL; osteolysis.