Correlation between levels of adipokines and inflammatory mediators with spirometric parameters in individuals with obesity and symptoms of asthma: Cross-sectional study.

INTRODUCTION: The adipose tissue secretes adipokines and influences the release of inflammatory mediators contributing to a state of low-grade systemic inflammation that may change lung function. OBJECTIVE: To correlate levels of adipokines and inflammatory mediators with lung function in individuals with obesity and bronchial asthma symptoms. MATERIALS AND METHODS: A cross-sectional study, including women with obesity (grade II and III) with symptoms and clinical diagnosis of asthma. Anthropometric measurements (weight, height, BMI), pulmonary function test (spirometry), asthma control test questionnaire, collection of systemic inflammatory markers (blood collection) and pulmonary markers (sputum collection) were collected and were analyzed: IL-6, IL-8, TNF-α, adiponectin, resistin, leptin and C-reactive protein (CRP). The patients were stratified into two groups according to asthma control. RESULTS: 80 women were analyzed and 40% had an ACT score greater than or equal to 18 and were classified as "controlled asthma". More than half of the patients of ACT<18 score obtaining measures of FEV1, PEF and FEF25-75% below and 80% of predicted. There was a significant and negative correlation between IL-6 in the sputum with FVC and FEF25-75% in the group ACT<18 and with FVC and FEV1 in the group ACT≥18. CONCLUSIONS: Therefore, we concluded that the increase of interleukin-6 in the sputum is related to worse pulmonary function even in patients with controlled asthma, especially in the translate airway permeability measures.

Modeling the interference between shear and longitudinal waves under high intensity focused ultrasound propagation in bone.

Magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) is a noninvasive thermal technique that enables rapid heating of a specific area in the human body. Its clinical relevance has been proven for the treatments of soft tissue tumors, like uterine fibroids, and for the treatments of solid tumors in bone. In MR-HIFU treatment, MR-thermometry is used to monitor the temperature evolution in soft tissue. However, this technique is currently unavailable for bone tissue. Computer models can play a key role in the accurate prediction and monitoring of temperature. Here, we present a computer ray tracing model that calculates the heat production density in the focal region. This model accounts for both the propagation of shear waves and the interference between longitudinal and shear waves. The model was first compared with a finite element approach which solves the Helmholtz equation in soft tissue and the frequency-domain wave equation in bone. To obtain the temperature evolution in the focal region, the heat equation was solved using the heat production density generated by the raytracer as a heat source. Then, we investigated the role of the interaction between shear and longitudinal waves in terms of dissipated power and temperature output. The results of our model were in agreement with the results obtained by solving the Helmholtz equation and the frequency-domain wave equation, both in soft tissue and bone. Our results suggest that it is imperative to include both shear waves and their interference with longitudinal waves in the model when simulating high intensity focused ultrasound propagation in solids. In fact, when modeling HIFU treatments, omitting the interference between shear and longitudinal waves leads to an over-estimation of the temperature increase in the tissues.

Modelling the temperature evolution of bone under high intensity focused ultrasound.

Magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) has been clinically shown to be effective for palliative pain management in patients suffering from skeletal metastasis. The underlying mechanism is supposed to be periosteal denervation caused by ablative temperatures reached through ultrasound heating of the cortex. The challenge is exact temperature control during sonication as MR-based thermometry approaches for bone tissue are currently not available. Thus, in contrast to the MR-HIFU ablation of soft tissue, a thermometry feedback to the HIFU is lacking, and the treatment of bone metastasis is entirely based on temperature information acquired in the soft tissue adjacent to the bone surface. However, heating of the adjacent tissue depends on the exact sonication protocol and requires extensive modelling to estimate the actual temperature of the cortex. Here we develop a computational model to calculate the spatial temperature evolution in bone and the adjacent tissue during sonication. First, a ray-tracing technique is used to compute the heat production in each spatial point serving as a source term for the second part, where the actual temperature is calculated as a function of space and time by solving the Pennes bio-heat equation. Importantly, our model includes shear waves that arise at the bone interface as well as all geometrical considerations of transducer and bone geometry. The model was compared with a theoretical approach based on the far field approximation and an MR-HIFU experiment using a bone phantom. Furthermore, we investigated the contribution of shear waves to the heat production and resulting temperatures in bone. The temperature evolution predicted by our model was in accordance with the far field approximation and agreed well with the experimental data obtained in phantoms. Our model allows the simulation of the HIFU treatments of bone metastasis in patients and can be extended to a planning tool prior to MR-HIFU treatments.

Chaperonin 10 of Mycobacterium tuberculosis induces a protective immune response to foot-and-mouth disease virus.

Chaperonin 10 of M. tuberculosis conferred partial or total protection against generalized foot-and-mouth disease (FMD) in guinea-pigs challenged with O1 Lausanne FMD virus. Chaperonin 10-immunized animals mounted an antibody response to the protein, one epitope of which was found in the C-terminal half. A similar recognition pattern was observed in FMD-convalescent guinea-pigs, swine and cattle. Anti-chaperonin 10 sera showed antiviral activity against FMDV-infected BHK-21 cells. There was strong evidence that early after infection these cells actively secrete their histones and that antisera to the chaperonin recognize them. The same antisera reacted with purified histones in immunoblotting. Most important, exogenously added histones abrogated the anti-viral activity of the antiserum and an anti-histone monoclonal antibody had strong antiviral activity against FMDV-infected BHK-21 cells. These results are consistent with previous reports on displacement of histones from the nuclear compartment and immune recognition of self-histones after viral infections. On the whole, they indicate that M. tuberculosis chaperonin 10 enables the immune system to react against early abnormalities of virus-infected cells; this is accomplished by antibody cross-reacting with histones released during virus infection.

Mycobacterial Cpn10 promotes recognition of the mammalian homologue by a mycobacterium-specific antiserum.

Self-tolerance, a key feature of the immune system, is still a matter of intense debate. We give here evidence for a peculiar behavior of an antiserum against Mycobacterium tuberculosis chaperonin 10 (m-Cpn10), which could have implications for the mechanism of self-recognition by antibodies against non-self. We show that this antiserum can interact in terms of both inhibition of biological activity and physical association (immunoprecipitation), with the mammalian homologue of m-Cpn10, but only if the bacterial protein is present. Several lines of evidence led us to exclude that the two proteins physically associate to form heterocomplexes: (1) the behavior of the antiserum was not shared by a monoclonal antibody against m-Cpn10; (2) a matrix selective for human Cpn10 (h-Cpn10) did not co-purify m-Cpn10; (3) the distribution pattern in non-denaturing isoelectric focusing of labeled m-Cpn10 was not altered by the presence of the unlabeled h-Cpn10. We conclude therefore that the antiserum against M. tuberculosis Cpn10 also recognizes mammalian Cpn10, with an affinity/avidity regulated by the mycobacterial protein, or by the promotion of hetero-oligomerization. This emergence of self-recognition in the presence of M. tuberculosis Cpn10 could imply a breaking of self-tolerance in situations of infection or vaccination.

Diaminic carbonates, a new class of anti-inflammatory compounds: their biological characterization and mode of action.

Taking advantage of a standard assay on mouse LM cells (murine fibroblast-like cells), we found that several diaminic carbonates, a new class of organic compounds synthesized in our laboratories, were able to inhibit human tumor necrosis factor alpha (huTNFalpha)-induced cytotoxicity in a dose-dependent manner. Structure-function relationship studies indicated precise structural requirements for compounds being active as huTNFalpha inhibitors. ITF1779, one of the most active compounds in inhibiting huTNFalpha-induced cytotoxicity, was selected for further studies. In vitro experiments showed that ITF1779 inhibited not only huTNFalpha-induced cytotoxicity on LM cells but also another response of the same cells, interleukin-1-induced interleukin-6 production. Receptor-binding studies performed under nonequilibrium conditions and morphologic evidence of vacuole formation in cells treated with high concentrations of ITF1779 showed that the effects were strikingly similar to those of chloroquine, a lysosomotropic agent. Consistent with a mechanism of action of diaminic carbonates closely matching that of chloroquine are some structural similarities between the two classes of compounds, in particular their both being diprotic weak bases. Moreover, ITF1779 was shown to be active in vivo because it afforded protection against lipopolysaccharide-induced shock in mice, a systemic inflammatory response crucially dependent on tumor necrosis factoralpha production.

Evidence for GroES acting as a transcriptional regulator.

Cochaperonins (cpn10) assist chaperonins (cpn60) in promoting folding and assembly of other proteins. Upon expression of Mycobacterium tuberculosis cpn10 in Escherichia coli we have purified a polypeptide which, through amino acid sequencing, was identified as the endogenous E. coli 10K-S protein. Subsequent studies showed that its expression was specifically upregulated upon cloning of different members of the cpn10 family, including GroES, the E. coli cpn10. Pulse-chase experiments demonstrated that 10K-S is but one of several proteins whose expression is modulated upon cloning of cpn10. Up-regulation of 10K-S was also observed after exposure of normal cells, but not of groES- mutants, to elevated temperatures (42 degrees C). This allowed us to define 10K-S as a heat-shock protein (hsp) whose expression is dependent on the production of another hsp, GroES. Northern blot experiments showed that enhanced expression of 10K-S was consequent to increased accumulation of transcripts and that groES- mutants were devoid even of baseline levels of transcripts both at 37 degrees C and after temperature upshift. These results show that GroES, in addition to its established role in assisting protein folding may act as a transcriptional regulator and is likely to play an important role in modulating gene expression particularly in those conditions, like the stress response, in which its production is greatly enhanced.

Different cytotoxic activity and intracellular fate of an anti-CD5-momordin immunotoxin in normal compared to tumour cells.

We investigated the different sensitivity of peripheral blood mononuclear cells (PBMC) and human T cell leukaemias (Jurkat and CEM) to an anti-CD5-momordin immunotoxin. In a short-term assay, the immunotoxin displayed different cytotoxic activity on normal and tumour cells: for leukaemic cell lines an incubation time of 72 h was necessary for the immunotoxin to reach the IC50 of 41-53 pM, compared to the 1 h sufficient for 6 pM immunotoxin to inhibit 50% of PBMC protein synthesis. In a long-term clonogenic assay (15 days), the immunotoxin demonstrated a comparable efficacy of clonogenic cell killing for both cell types. We investigated the immunotoxin internalization pathway by a flow-cytometric method and our data seem to indicate that the molecules meet a different intracellular fate in the two cell populations. It may be assumed that the low cytotoxic activity of immunotoxins on tumour cells, detected in the short-term assay, is due to inefficient delivery to their cytoplasmatic target, while a longer exposure of the cells to the immunotoxin promotes adequate intracellular distribution.

Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10.

We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post-translational acetylation is not necessary for its activity.

Anti-CD30 immunotoxins with native and recombinant dianthin 30.

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein from Dianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30+ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus), L428 and L540 (both from Hodgkin's lymphoma). IC50 values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30+ cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC50 higher than nM).

Heterologous expression, purification, activity and conformational studies of different forms of dianthin 30.

Dianthin 30, a ribosome inactivating protein (RIP) from Dianthus caryophyllus, has been expressed in Escherichia coli. Heterologous expression of a deletion mutant dianthin 30 delta 255-270 resulted in the production of a protein identical to carnation mature dianthin 30, including the absence at the carboxy-terminal of a putative 16 amino acid long pro-signal peptide. The production of a form of dianthin 30, which includes the pro-signal, is described as well. Both dianthin 30 delta 255-270 and dianthin 30 expressed in E. coli are mainly localized (90%) in the soluble fraction. Dianthin 30 delta 255-270 and dianthin 30 have been purified to homogeneity and were shown to inhibit protein synthesis in vitro with an IC50 of 8 and of 11 ng/ml, respectively. Secondary structure analysis, carried out by circular dichroism spectroscopy, indicated that the naturally occurring and the recombinant forms of dianthin 30 and dianthin 30 delta 255-270 possess the same secondary structure composition, accounting for an alpha + beta type architecture. RIPs as immunotoxins in clinical trial and as mitotoxins in experimental models have been extremely efficacious. In addition, growing evidence indicates their effective use as antiviral agents, including in HIV-1 infection. These data indicate the ability to produce either chemically linked or recombinant fusion proteins with dianthin 30 and cell-binding ligands for production of new reagents for clinical and experimental use.

Cochaperonins are histone-binding proteins.

Cochaperonins (cpn10) assist chaperonins (cpn60) in mediating folding of polypeptide substrates in an ATP-dependent reaction. Moreover, they have been shown to be secretory products of living cells and to perform discrete biological activities without the need to interact with cpn60. Here, we have investigated the possible existence of cellular cpn10 binding sites that could mediate such activities. For this purpose, we performed binding studies with iodinated cpn10 on whole cells and on electrophoretically separated eukaryotic cell lysates. The former studies yielded negative results, whereas in the latter binding to several proteins was detected. These proteins were identified as being histones. Binding was observed to all core histones (H2A, H2B, H3 and H4) and, although weaker, to the linker histone H1 as well. These results show that cpn10 are histone-binding proteins.

Identification and cloning of human chaperonin 10 homologue.

We have identified a heat-shock-inducible 10 kDa protein in the human hepatoma cell line HepG2. The total RNA extracted from the heat-shocked cells was amplified by reverse transcription PCR (polymerase chain reaction) using 21 5' and 18 3' oligonucleotides of rat cpn10 (chaperonin10) cDNA as primers. Sequencing of the above PCR fragment showed a very high homology between human, bovine and rat cpn10 cDNA. The predicted amino acid sequence revealed a 100% identity with the bovine homologue.

Production and characterization of monoclonal antibodies against the ribosome inactivating proteins dianthin32 and momochin.

Female BALB/c mice were immunized with either dianthin32 or momochin, type 1 ribosome-inactivating proteins (RIPs) derived from Dianthus charyophyllus and Momordica cochinchinensis, respectively. Five anti-dianthin32 and 6 anti-momochin secreting hybridomas were obtained by somatic fusion of lymphocytes with myeloma cell line NS0. The monoclonal antibodies (MAbs) produced were highly specific, as demonstrated by cross-reactivity assays performed with taxonomically related and unrelated type 1 RIPs, and recognized different epitopes of the antigen. The affinity constant of anti-RIPs MAbs ranged between 10(8) M-1 and 10(10) M-1.

Increased p21ras-specific Guanine-nucleotide exchange causes tumor-formation in nude-mice.

RAS activation state is set by GTPase Activating Proteins (GAP) and Guanine Nucleotide Releasing Proteins (GNRP). The latter were discovered in yeast as the products of the CDC25 and SDC25 genes; two protein families with homologous catalytic domains but different structural organization exist also in mammals. We show that the C-terminal, catalytic domain of a mouse homologue of CDC25 transactivates the ras-responsive fos promoter in vivo. The increased p21ras-specific guanine nucleotide releasing activity of fibroblasts expressing CDC25Mm catalytic domain correlates with tumor induction in nude mice, suggesting that deregulation of these proteins may be important in tumor development.

Delivery of saporin to human B-cell lymphoma using bispecific antibody: targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing.

A panel of bispecific F(ab')2 antibodies (BsAb) have been constructed for delivering the ribosome-inactivating protein saporin to human B cell lymphoma. Each derivative was prepared with specificity for saporin and CD19, CD22, CD37, or immunoglobulin. In vitro studies measuring inhibition of [3H]leucine uptake by cultured Daudi and Raji cells demonstrated that, despite all BsAb capturing saporin on the cell surface, BsAb targeting through CD22 were far more cytotoxic than those functioning via CD19, CD37, or surface immunoglobulin. This exceptional activity of the CD22-specific BsAb appears to derive from its ability to deliver and accumulate saporin inside the target cells. Further studies showed that four CD22-specific BsAb all performed with equal potency and were able to increase saporin toxicity (50% inhibitory concentration) up to 1000-fold, from 2 x 10(-7) M to 2 x 10(-10) M. Pairs of anti-CD22 BsAb which recognized different nonblocking epitopes on the saporin molecule were able to bind saporin more avidly to the target cell and, as a consequence, increased cytotoxicity by at least an additional 10-fold, resulting in 50% inhibitory concentration for protein synthesis of 2 x 10(-11) M. These results suggest that selected combinations of BsAb which bind cooperatively to a toxin and the cell surface may provide an efficient way of delivering toxins to unwanted cells in patients.

Expression and activity of pre-dianthin 30 and dianthin 30.

Dianthin 30 is a ribosome inactivating protein (RIP 1) found in different tissues of the carnation (Dianthus caryophyllus). Recently we have isolated and sequenced a cDNA clone from a lambda gt11 expression library [Legname et al. (1991) Biochim. Biophys. Acta 1090, 119-122]. Here we describe specific PCR amplifications of either the full length pre-dianthin 30 or dianthin 30, the mature polypeptide lacking the 23 amino acid signal peptide. In vitro expression of both proteins in reticulocyte lysate generated products of the expected molecular weight. Moreover, the activity of both proteins has been evaluated confirming the characteristics of the natural product. A first attempt to produce recombinant dianthin 30 in Escherichia coli is described.

In vitro and in vivo properties of an anti-CD5-momordin immunotoxin on normal and neoplastic T lymphocytes.

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified from Momordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC50 = 1 - 10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC50 = 10 pM). Moreover, the in vitro performance of the immunotoxin compared favorably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model of nu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%, P < 0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.

Initial experience in treating human lymphoma with a combination of bispecific antibody and saporin.

Results are presented showing the use of bispecific F(ab')2 antibodies (bsAbs) in the delivery of saporin for the treatment of 2 human B-cell malignancies. BsAbs delivering saporin through CD22, but not through CD19, were effective at inhibiting the uptake of [3H]leucine by Daudi and Raji cells. Furthermore, a combination of 2 anti-CD22 bsAbs, selected to bind simultaneously to saporin, bound saporin 20 times more avidly and inhibited protein synthesis far more efficiently than any single bsAb. In the first patient, with end-stage chronic lymphocytic leukaemia (CLL), treatment with 10 mg of saporin complexed to 100 mg of anti-CD19 bsAb over 43 days showed no therapeutic effect. In contrast, the second patient, with end-stage non-Hodgkin's lymphoma (NHL), given 5 mg of saporin complexed with a pair (50 mg) of anti-CD22 bsAbs over 15 days showed a marked clinical response, including complete clearance of tumour from the blood, clearance of ascites and shrinkage of tumour masses. Neither patient experienced any toxic side-effects, either during or after treatment. However, the second patient developed a strong anti-mouse Fab (HAMA) response 28 days after the treatment started. No anti-saporin response could be detected.