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The phagocytic actives of B cells in fish have been proven in recent years. In this study, five positive hybridomas secreting monoclonal antibodies (MAbs) against largemouth bass IgM were produced. Indirect immunofluorescence assay (IFA) demonstrated that five MAbs could specifically recognize membrane-bound IgM (mIgM) molecule of largemouth bass. Indirect ELISA and Western blotting analysis showed that all the five MAbs had no cross-reactions with the other two teleost IgMs. Flow cytometry analysis (FCM) revealed that the percentages of largemouth bass mIgM+ lymphocytes in head kidney, peripheral blood and spleen were 51.66 ± 0.608%, 16.5 ± 1.235% and 42.92 ± 1.091%, respectively. In addition, the phagocytosis rates of mIgM + lymphocytes ingesting Nocardia seriolae from head kidney, peripheral blood and spleen were calculated to be 5.413 ± 0.274%, 16.6 ± 0.289% and 26.3 ± 0.296%, respectively. The qPCR results of sorted cells indicated that most inflammatory cytokines (IFNγ, IL-1β, IL-2, IL-12β, IL-34, IL-10) , chemokine (CXCL12), chemokines receptors (CXCR2, CXCR4) and genes (FcγRⅠa, NCF1, CFL, ARP2/3, CD45, Syk, MARCKS) related to FcγR-mediated phagocytic signaling pathway in phagocytic mIgM+ lymphocytes were up-regulated significantly (P < 0.05). Taken together, the results suggested that the MAb (MM06H) produced in this paper could be used as a tool to study mIgM+ lymphocytes of largemouth bass, and FcγR may participate in the phagocytosis of mIgM+ lymphocytes, which is helpful to further study the role of mIgM+ lymphocytes in innate immunity. • Monoclonal antibodies against Micropterus salmoides were produced. • The phagocytosis of mIgM+ lymphocytes from largemouth bass was validated. • 33 immune-related genes of sorted phagocytic mIgM+ lymphocytes were up-regulated. • FcγR-mediated phagocytosis pathway may participate in the phagocytosis of mIgM+ lymphocytes. [ABSTRACT FROM AUTHOR]

Abstract B cells in some fish were recently found to have potent phagocytic activities. Sea bass ( Lateolabrax japonicus ) as an important economical marine fish species, it could be used as an appropriate model to study the functions of B cells in phagocytosis. In the paper, three positive hybridomas designated as 1E11, 2H4 and 3F3 secreting monoclonal antibodies (MAbs) against sea bass immunoglobulin M (IgM) were produced and used as research tools. Indirect enzyme-linked immunosorbent assay showed that all the three MAbs had a high binding capacity with sea bass serum IgM. Western blotting analysis showed that all the three MAbs were specific for the heavy chain of sea bass IgM. Indirect immunofluorescence assay (IFA) analysis suggested that both MAbs 1E11 and 2H4 could recognize membrane-bound IgM (mIgM) molecule of sea bass. Specificity analysis showed that three MAbs had no cross-reactions with other six teleosts IgMs. Flow cytometric analysis exhibited that the percentages of sea bass mIgM + lymphocytes in peripheral blood, spleen and pronephros were 25.6%, 21.1%, and 17.5%, respectively. Moreover, we found that the mIgM + lymphocytes of sea bass could phagocytose fluorescence microspheres and Lactococcus lactis , but lower phagocytosis rates of L. lactis was observed. These results demonstrated that the MAbs produced in this paper could be used as tools to study secretory IgM and mIgM + lymphocytes of sea bass, and mIgM + lymphocytes might also play an important role in innate immunity of sea bass. Highlights • MAbs against IgM of sea bass are produced and characterized. • The produced MAbs are special for the sIgM and mIgM of sea bass. • The produced MAbs don't cross-react with other six teleosts' IgMs. • The mIgM + lymphocytes of sea bass possess phagocytic capacity. • The phagocytosis rates of L. lactis are lower than those of fluorescence microsphere. [ABSTRACT FROM AUTHOR]

Graphical abstract A series of oxadiazole-based S1P 1 direct-acting agonists, with high affinity for S1P 1 receptor and favorable selectivity against S1P 3 receptor was designed, synthesized and evaluated in vitro and vivo. Highlights • Oxadiazole-based derivatives were designed and progressively modified as S1P 1 agonists. • The agonism activities of compounds for S1P 1 and S1P 3 receptor were evaluated. • Most compounds show excellent activity and selectivity, particularly for 16-3b and 16 - 3g. • In vivo pharmacological, pharmacokinetics and druggability profiles were studied. • The binding mode between 16-3b and the activated S1P 1 model was also reviewed. Abstract Agonism of S1P 1 receptor has been proven to be responsible for peripheral blood lymphopenia and elicts the identification of various S1P 1 modulators. In this paper we described a series of oxadiazole-based S1P 1 direct-acting agonists disubstituted on terminal benzene ring, with high potency for S1P 1 receptor and favorable selectivity against S1P 3 receptor. In addition, two representative agents named 16-3b and 16 - 3g demonstrated impressive efficacy in lymphocyte reduction along with reduced effect on heart rate when orally administered. Furthermore, these compounds have been shown to possess desired pharmacokinetic (PK) and physicochemical profiles. The binding mode between 16 - 3b and the activated S1P 1 model was also studied. [ABSTRACT FROM AUTHOR]

The local lymph node assay (LLNA) has provided a large dataset against which performance of non-animal approaches for prediction of skin sensitisation potential and potency can be assessed. However, a recent comparison of LLNA results with human data has argued that LLNA specificity is low, with many human non-sensitisers, particularly hydrophobic chemicals, being false positives. It has been suggested that such putative false positives result from hydrophobic chemicals causing cytotoxicity, which induces irritancy, in turn driving non-specific lymphocyte proliferation. This paper finds that the apparent reduced specificity of the LLNA largely reflects differences in definitions of the boundaries between weak skin sensitisers and non-sensitisers. A small number of LLNA false positives may be due to lymphocyte proliferation without skin sensitisation, but most alleged 'false' positives are in fact very weak sensitisers predictable from structure-activity considerations. The evidence does not support the hypothesis for hydrophobicity-induced false positives. Moreover, the mechanistic basis is untenable. Sound LLNA data, appropriately interpreted, remain a good measure of sensitisation potency, applicable across a wide hydrophilicity-hydrophobicity range. The standard data interpretation protocol enables detection of very low levels of sensitisation, irrespective of regulatory significance, but there is scope to interpret the data to give focus on regulatory significance. • The apparent low specificity of the LLNA against human data is misleading. • Most so-called LLNA false positives are in reality weak sensitizers. • LLNA and human data differences reflect differences in data interpretation. • The concept of hydrophobicity-induced false positives is refuted by the evidence. [ABSTRACT FROM AUTHOR]

Immunosuppressive domain (ISD) is a conserved region of transmembrane proteins (TM) in envelope gene ( env ) of retroviruses. in vitro and vivo, a synthetic peptide (CKS-17) that shows homology to ISD inhibits immune function. Evidence has shown that ISD suppresses lymphocyte proliferation and allows escape from immune effectors of the innate and adaptive arms in mouse immune system. Previously, we have developed a tool ISDTool 1.0 to identify ISD of human endogenous retrovirus (HERV). However, several other important retroviruses exist and no method is devoted to ISD prediction of them so far. In the paper, a computational model is proposed to identify ISD of six typical retroviruses from three species. The model combines the minimum Redundancy Maximum Relevance (mRMR) feature selection criterion with weighted extreme learning machine (WELM) to achieve high identification accuracies of 98.95%, 96.34% and 96.87% using self-consistency, 5-fold and 10-fold cross-validation, respectively. A software tool named ISDTool 2.0 has been developed to facilitate the application of the model and a large number of new putative ISDs of the six retroviruses were predicted. In addition, motifs of ISD in these retroviruses were analyzed and the evolutionary relationship was discussed. Datasets and the software involved in the paper are available at http://sourceforge.net/projects/isdtool/files/ISDTool-2.0/ . [ABSTRACT FROM AUTHOR]

This paper proposes a general framework for probabilistic certification of cancer therapies. The certification is defined in terms of two key issues which are the tumor contraction and the lower admissible bound on the circulating lymphocytes which is viewed as indicator of the patient health. The certification is viewed as the ability to guarantee with a predefined high probability the success of the therapy over a finite horizon despite of the unavoidable high uncertainties affecting the dynamic model that is used to compute the optimal scheduling of drugs injection. The certification paradigm can be viewed as a tool for tuning the treatment parameters and protocols as well as for getting a rational use of limited or expensive drugs. The proposed framework is illustrated using the specific problem of combined immunotherapy/chemotherapy of cancer. [ABSTRACT FROM AUTHOR]

Epidemiological studies suggest that dietary consumption of plant polyphenols is related to a lower incidence of various cancers. Among these compounds catechins (present in green tea and other beverages) are considered to be potent inducers of apoptosis and cytotoxicity to cancer cells. Thus these compounds can be used as leads to synthesize novel anticancer drugs with greater bioavailability. In view of this in this paper we have examined the chemical basis of cytotoxicity of catechins by studying the structure–activity relationship between catechin (C), epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG). Using single cell alkaline gel electrophoresis (comet assay) we have established the relative efficiency of cellular DNA breakage as EGCG > EGC > EC > C. We also show that cellular DNA breakage is the result of mobilization of copper ions bound to chromatin and the generation of reactive oxygen species. Further the relative DNA binding affinity order was confirmed using molecular docking and thermodynamic studies by studying the interaction of catechins with calf thymus DNA. The results suggest that the synthesis of any novel anti cancer molecule based on the structure of catechins should have as many galloyl moieties as possible resulting in an increased number of hydroxyl groups that may facilitate the binding of the molecule to cellular DNA. [ABSTRACT FROM AUTHOR]

Abstract: Boolean networks have been successfully used in modelling gene regulatory networks. However, for large networks, analysis by simulation becomes unfeasible. In this paper we propose a reduction method for Boolean networks that decreases the size of the network, while preserving important dynamical properties and topological features. As a result, the reduced network can be used to infer properties about the original network and to gain a better understanding of the role of network topology on the dynamics. In particular, we use the reduction method to study steady states of Boolean networks and apply our results to models of Th-lymphocyte differentiation and the lac operon. [Copyright &y& Elsevier]

Abstract: In this paper we develop a new mathematical model of immunotherapy and cancer vaccination, focusing on the role of antigen presentation and co-stimulatory signaling pathways in cancer immunology. We investigate the effect of different cancer vaccination protocols on the well-documented phenomena of cancer dormancy and recurrence, and we provide a possible explanation of why adoptive (i.e. passive) immunotherapy protocols can sometimes actually promote tumour growth instead of inhibiting it (a phenomenon called immunostimulation), as opposed to active vaccination protocols based on tumour-antigen pulsed dendritic cells. Significantly, the results of our computational simulations suggest that elevated numbers of professional antigen presenting cells correlate well with prolonged time periods of cancer dormancy. [Copyright &y& Elsevier]

Abstract: Dendritic cells (DCs), in peripheral tissues, derive mostly from blood precursors that differentiate into DCs under the influence of the local microenvironment. Monocytes constitute the main known DC precursors in blood and their infiltration into tissues is up-regulated during inflammation. During this process, the local production of mediators, like prostaglandins (PGs), influence significantly DC differentiation and function. In the present paper we show that treatment of blood adherent mononuclear cells with 10μM indomethacin, a dose achieved in human therapeutic settings, causes monocytes’ progressive death but does not affect DCs viability or cell surface phenotype. This resistance of DCs was observed both for cells differentiated in vitro from blood monocytes and for a population with DCs characteristics already present in blood. This phenomenon could affect the local balance of antigen-presenting cells, influence the induction and pattern of immune responses developed under the treatment with non-steroidal anti-inflammatory drugs and, therefore, deserves further investigation. [Copyright &y& Elsevier]

Abstract: To evaluate the role of interleukin 14 alpha (IL-14a) in Sjögren''s syndrome (SS), we evaluated the expression of IL-14a in the peripheral blood lymphocytes (PBL) of patients with primary and secondary SS and normal controls by quantitative RT-PCR. In addition, transgenic IL-14a mice were analyzed from 6 weeks of age to death for both histological and immunological features of Sjögren''s disease. Patients with both primary and secondary Sjögren''s syndrome expressed IL-14a at statistically higher levels in their peripheral blood compared to normal controls matched for age, sex and ethnic group. Transgenic mice in which IL-14a expression was increased constitutively were previously demonstrated to develop hypergammaglobulinemia, autoantibodies, infiltration of the parotid glands with lymphocytes, mild immune-complex mediated renal disease and large B cell lymphoma. In this paper we expand these observations to demonstrate that these mice develop all the clinical and immunological features of primary Sjögren''s disease in the same relative time frame as patients with primary Sjögren''s disease: stage 1—early hypergammaglobulinemia and autoantibody production, stage 2—decreased salivary gland function with early lymphocytic infiltration of the submandibular glands only, but antibody deposition in the submandibular and parotid glands, stage 3—lymphocytic infiltration of the submandibular, parotid and lacrimal glands with B and T lymphocytes and plasma cells along with interstitial lung disease and mild renal disease, and stage 4—large B cell lymphoma. Thus IL-14a is important in the pathophysiology of Sjögren''s disease. The IL-14a transgenic mouse is a novel animal model that can be utilized to understand the pathophysiology of Sjögren''s disease. [Copyright &y& Elsevier]

Abstract: Cytotoxic T lymphocytes (CTLs) play a dominant role in the pathogenesis of autoimmune diabetes, commonly denoted Type 1 Diabetes (T1D). These CTLs (notably T cells) recognize and kill insulin-secreting pancreatic cells, reducing their number by . The resulting reduction of insulin secretion causes the defective regulation of glucose metabolism, leading to the characteristic symptoms of diabetes. Recognition of cells as targets by CTLs depends on the interactions between MHC-peptide complexes on the surface of cells and receptors (TCRs) on T cells. Those CTLs with high affinity TCRs (also called high avidity T cells) cause most of the harm, while those with low affinity TCRs (also called low avidity T cells) play a more mysterious role. Recent experimental evidence suggests that low avidity T cells accumulate as memory T cells during the disease and may be protective in NOD mice (a strain prone to developing T1D), delaying disease progression. It has been hypothesized that such low avidity T cells afford disease protection either by crowding the islets of Langerhans, where cells reside, or by killing antigen presenting cells (APCs). In this paper, we explore the hypothesized mechanisms for this protective effect in the context of a series of models for (1) the interactions of low and high avidity T cells, (2) the effect of APCs and (3) the feedback from cell killing to autoantigen-induced T cell proliferation. We analyze properties of these models, noting consistency of predictions with observed behaviour. We then use the models to examine the influence of various treatment strategies on the progression of the disease. The model reveals that progressive accumulation of memory low avidity autoreactive T cells during disease progression makes treatments aimed at expanding these protective T cell types more effective close to, or at the onset of clinical disease. It also provides evidence for the hypothesis that low avidity T cells kill APCs (rather than the alternate hypothesis that they crowd the islets). [Copyright &y& Elsevier]

Abstract: Acute exercise is known for causing considerable changes in leukocyte counts and function. In this paper we report that differentiated changes in T-lymphocyte distribution occur in lymphoid and non-lymphoid organs depending on the type and the intensity of exercise. Using fluorescent cell tracking we observed a release of T-cells from the spleen while lung, bone marrow and Peyer’s patches served as target organs. The number of T-cells in the blood rose after intensive running while lymphopenia occurred after swimming exercise. Changes in number of labelled T-cells were neither found in the lymph nodes nor in the thymus regardless of exercise protocol. Following an α- or β-blockade, the exercise-induced release of T-cells from the spleen and the accumulation of T-cells in the lung were inhibited while the enhancement of T-cells in the Peyer’s patches was not affected. The administration of epinephrine partially mimicked the effects of exercise and resulted in a release of T-cells from both, the spleen and the liver, as well as in an increase of circulating blood T-cells. In conclusion, exercise induces a substantial re-distribution of T-cells within lymphoid and non-lymphoid organs. The migrating properties of T-cells could be partially explained by adrenergic mechanisms associated with exercise while the involvement of certain homing receptors remains to be shown. Our results suggest that the accumulation of T-cells in both, lung and Peyer’s patches, may enhance the immune vigilance in these compartments which serve as the body’s major defence barriers. [Copyright &y& Elsevier]

Abstract: In this paper, we focus our attention on the relevance of HLA-G in cancer in the light of our recent advances on the expression and immunological function of HLA-G. Regarding HLA-G function, we recently showed that in addition to its direct inhibitory effects on T, APC and NK function, HLA-G induces suppressor cells via two distinct processes: (i) either by cell differentiation of naïve T cells into lasting suppressor T cells or (ii) by rapid transfer of HLA-G from APC or tumor cells to T or NK cells converting them into temporary HLA-G-positive suppressor cells. Regarding HLA-G expression, we described that tumor-microenvironment factors such as hypoxia, IDO and, TNF-α regulate the expression of HLA-G by tumor cells in a way that favors tumor escape from NK lysis. These findings reinforce the role of HLA-G as one mechanism of tumor-driven immune evasion and provide potential targets for testing novel anticancer treatment strategies. [Copyright &y& Elsevier]

Abstract: Liver X receptors (LXRα and LXRβ) are nuclear transcription factors that inhibit transcription of genes of inflammation while inducing HMGCoA reductase. In this paper we demonstrate increased mRNA levels of LXRβ in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis patients with respect to other neurological patients and healthy controls (HC) (p <0.01). Agonist-induced activation of LXRs partially counteracts the anti-CD3+ anti-CD28-induced proliferation of T cells (p <0.01) and secretion of IFNγ (p <0.001) from PBMCs of MS patients as well as of HC. Secretion of IL-4 is not affected. Our findings suggest that regulation of cholesterol metabolism not strictly related to inhibition of HMGCoA reductase can modulate activity of lymphocytes in MS. [Copyright &y& Elsevier]

Abstract: Plant-derived dietary material contains several classes of polyphenols such as flavonoids, curcuminoids, stilbenes and hydroxycinnamic acids. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing cellular DNA degradation in the presence of transition metal ions such as copper. Earlier we have shown that the stilbene resveratrol is able to mobilize endogenous copper ions leading to oxidative breakage of cellular DNA. In this paper, we show that caffeic acid (a hydroxycinnamic acid), which is a major constituent of coffee, is also capable of DNA breakage in human peripheral lymphocytes. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that caffeic acid generates oxidative stress in lymphocytes, which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action. [Copyright &y& Elsevier]

Abstract: The immune system has evolved elaborate mechanisms to respond to diverse antigens while minimizing the risk for autoimmune reactivity. During lymphopenia, however, some mechanisms that normally serve to maintain host tolerance are temporarily suspended. Peripheral T cells proliferate in response to self-antigens in lymphopenic hosts, but proliferation toward these same antigens is prevented when T cell numbers are normal. This process, termed homeostatic peripheral expansion, augments peripheral T cell number and limits repertoire skewing during recovery from lymphopenia and also predisposes lymphopenic hosts to autoimmune disease. This paper reviews murine and human settings in which autoimmunity occurs in the context of lymphopenia. We propose a two-hit model, in which lymphopenia plus another insult is sufficient to induce autoimmune disease. Among the secondary insults that appear sufficient to induce autoimmunity during lymphopenia are overproduction of IL-21 as occurs in the NOD.SCID mouse, depletion of Tregs as demonstrated in murine colitis and gastritis models, and tissue inflammation as seen in HIV infected patients who develop immune reconstitution inflammatory syndrome (IRIS). Delineating critical cofactors which result in autoimmune disease during lymphopenia can provide insight into the pathophysiology of naturally occurring autoimmune diseases as well as generating testable hypothesis for inducing tumor-specific autoimmunity in lymphopenic hosts with cancer. [Copyright &y& Elsevier]

Abstract: In this paper, the circadian pattern of Clock and genes mediated by the Clock was investigated in peripheral lymphocytes of rats. Circadian rhythms of Clock are found under the regimes of constant darkness (DD) and 12-h light–12-h dark (LD12:12h), with the peak phase at CT7 and ZT21, respectively. Ten differential cDNA fragments were identified to be mediated by the Clock, including three known genes (catalase, myelin proteolipid protein, and histone acetylase), four known expressed sequence tags (ESTs), and three novel ESTs. Experiment of the RNA interference revealed that these ESTs were down-regulated by the Clock gene and three of them were identified as clock-controlled genes. Understanding of clock-mediated genes may lead to a new direction in drug design for control of circadian rhythms. [Copyright &y& Elsevier]

Abstract: Signaling pathways such as the pre-TCR and Wnt pathways regulate α/β T cell differentiation in thymus. Mice lacking an essential component of the pre-TCR exhibit arrest at the (CD4−CD8−) (CD44−CD25+) stage (DN3) of thymocyte development, and introduction of p53 deficiency into those mice abrogates this arrest, resulting in transition to the (CD4+CD8+) double-positive (DP) stage. This paper examines the effect of inactivation of p53 on thymocyte development in Bcl11b−/− mice that exhibit arrest at the DN3 or (CD4−CD8+) immature single-positive (ISP) stage. No DP thymocytes were detected in thymocytes of adoptive transfer experiments in scid mice that were derived from p53−/−Bcl11b−/− precursors but ISP thymocytes increased in the proportion and in the cell number approximately three times higher than those from Bcl11b−/− precursors. Consistently, the level of apoptosis decreased to the level of wild-type precursors. These results suggest that inactivation of p53 is sufficient for DN3 thymocytes to differentiate into the ISP, but not to DP, stage of thymocyte development in Bcl11b−/− mice. This provides evidence for a novel p53-mediated checkpoint that regulates the transition from the DN3 to ISP stage of thymocyte development. [Copyright &y& Elsevier]

Haploidentical stem cell transplantation has became a clinical reality in the last 10 years as it provides the chance of transplant for about 50% of patients with hematological malignancies who do not have a matched related or unrelated donor. Proper graft preparation for this type of transplant is crucial and this paper analyses our work over the past decade in the search for the optimal graft processing procedure moving from E-rosetting and soybean agglutination, through a combination of negative or positive selection of hematopoietic stem cells to the current method of one-step positive selection. In preparing a graft for haploidentical transplant, three essential requisites must be met. It must contain (1) a megadose (>10 × 106 × kg recipient b.w.) of hematopoietic stem cells to overcome the HLA histocompatibility barrier; (2) very few T-lymphocytes (CD3+ cells <3 × 104/kg recipient b.w.) to prevent severe acute and chronic graft-versus-host disease (GvHD); (3) very few B-lymphocytes to prevent Epstein–Barr virus-related lymphoproliferative disorders.With current graft processing technologies based on positive selection of hematopoietic stem cells, these requirements can be met. A 70–80% hematopoietic stem cell recovery ensures the target megadose is achieved in over 70% of cases with a T-cell depletion of more than 4 logs and a B-cell depletion of over 3 logs.Progress in graft processing has ensured primary, sustained engraftment rates of over 90% and has significantly reduced the incidence of severe acute GvHD and EBV-related lymphoproliferative disorders.Modern time-saving automated graft processing devices ensure reproducibility, reliability, and biological safety, which make widespread application of the haploidentical transplant currently feasible. [Copyright &y& Elsevier]

In this paper, we propose and analyse a mathematical model for chronic myelogenous leukemia (CML), a cancer of the blood. We model the interaction between naive T cells, effector T cells, and CML cancer cells in the body, using a system of ordinary differential equations which gives rates of change of the three cell populations. One of the difficulties in modeling CML is the scarcity of experimental data which can be used to estimate parameters values. To compensate for the resulting uncertainties, we use Latin hypercube sampling (LHS) on large ranges of possible parameter values in our analysis. A major goal of this work is the determination of parameters which play a critical role in remission or clearance of the cancer in the model. Our analysis examines 12 parameters, and identifies two of these, the growth and death rates of CML, as critical to the outcome of the system. Our results indicate that the most promising research avenues for treatments of CML should be those that affect these two significant parameters (CML growth and death rates), while altering the other parameters should have little effect on the outcome. [Copyright &y& Elsevier]

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (Bh) and rat (Br), as well as that of pro-granzyme K (Kh) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm Ah, Mh, Bm, and Cm from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (Kh), and granzyme B (Bh). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2′ binding subsites in all granzyme, which may be involved in the structure–function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of β-sheet structure with a little α-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme Bh. For example, in granzyme Mh, valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu → Phe), S3 (Ser → Gly), and S4 (Arg → Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme–inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis. [Copyright &y& Elsevier]

In the present paper we examined the effects of the aqueous extract from Rhizoma notopterygii (RN-ext) on picryl chloride-induced contact sensitivity (PCl-CS). The extract, administered during either induction or effector phase showed a significant inhibition on the ear swelling in mice with PCl-CS. By using the isolated spleen cells from the mice with PCl-CS, we demonstrated the inhibitory effects of RN-ext on matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. However, such inhibition was not found in those from normal mice. The inhibitory effects on MMP-2 and MMP-9 of RN-ext were also observed when it was administered in vivo. In addition, the extract significantly inhibited the migration of spleen cells from PCl-CS mice in transwell system without affecting the cell adhesion to fibronectin. These results suggest that RN-ext exerts its inhibitory activity on the contact sensitivity through decreasing the localization to the inflammation site via down-regulating MMP activities. [Copyright &y& Elsevier]

The broadly reactive cysteine protease dipeptidyl peptidase I (DPPI, cathepsin C) is thought to activate all progranzymes (zymogens of lymphocyte serine proteases) to form mature granzymes. We synthesized dipeptide 7-amino-4-methylcoumarin (AMC) substrates containing progranzyme activation sequences and showed that they were efficiently hydrolyzed by DPPI. However, DPPI will not hydrolyze Ile-Ile-AMC, the N-terminal dipeptide sequence found in mature granzymes. Introduction of the nonphysiological homophenylalanine (Hph) residue at P1 resulted in the best substrate Ala-Hph-AMC for DPPI (kcat/Km=9,000,000 M−1 s−1). The charged N-terminal amino group of the substrate was essential and replacement of the NH2 group with OH or NH(CH3) in Gly-Phe-AMC reduced the kcat/Km value by two to three orders of magnitude. A hydrazide azaglycine analog, NH2NHCO-Phe-AMC, was not hydrolyzed at pH 5.5, but underwent slow hydrolysis at lower pHs where the amino group is partially protonated. DPPI also failed to hydrolyze NH2COCH2-Phe-AMC, where the NH2 group is unprotonated. The results reported in this paper should be useful in the design of better DPPI inhibitors to block granzyme maturation and granzyme-dependent apoptosis. [Copyright &y& Elsevier]