Stem cells and basic fibroblast growth factor failed to improve tendon healing: an in vivo study using lentiviral gene transfer in a rat model.

BACKGROUND: The aim of this controlled study was to investigate the influence of mesenchymal stem cells (MSCs) and lentiviral (LV) expression of basic fibroblast growth factor (bFGF) on tendon remodeling in an in vivo rat model of an Achilles tendon defect. METHODS: In eighty-four male Lewis rats, complete 2.4-mm tendon defects were created and were either left untreated (the phosphate-buffered saline solution [PBS] group) or were treated with mesenchymal stem cells expressing enhanced green fluorescent protein (the MSC-LV-eGFP group) or with mesenchymal stem cells expressing basic fibroblast growth factor lentivirally (the MSC-LV-bFGF group). After fourteen and twenty-eight days, the tendons were harvested and analyzed biomechanically and immunohistologically. RESULTS: After fourteen days, both mesenchymal stem cell groups were slightly superior in biomechanical testing. However, only the PBS control group showed a significant increase in biomechanical results over time (fourteen versus twenty-eight days; p = 0.012). Biomechanical results were better after twenty-eight days for the control group than for both MSC groups. However, the difference was significant only with regard to the stiffness results in the comparison of the PBS control and the eGFP stem cell group (p = 0.024). Histologically, the MSC groups had no better results than the control group after fourteen and twenty-eight days. In immunohistology, only labeling for type-I procollagen was strongly increased in both MSC groups in comparison with the PBS control group (p = 0.0009 for the MSC-LV-bFGF group and p = 0.0041 for the MSC-LV-eGFP group at fourteen days, and p = 0.004 and p = 0.132, respectively, at twenty-eight days). There were no significant differences in the immunohistological results between the stem cell groups. CONCLUSIONS: The biomechanical and immunohistological results showed that mesenchymal stem cells in both groups had only partially positive effects on tendon remodeling in the initial stages; however, in later stages, stem cells had potentially negative effects on biomechanical results. The additional expression of bFGF in stem cells had negligible effects on tendon remodeling. CLINICAL RELEVANCE: Preliminary studies using stem cells are partially promising; however, there are no relevant clinical data showing that stem cells are of significant benefit. The present study should lead to a more critical evaluation and thoughtful use of stem cells in humans until more clinical data are available.

microRNA expressions in CD4+ and CD8+ T-cell subsets in autoimmune thyroid diseases.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) are the most common autoimmune thyroid diseases (AITD). MicroRNAs (miRNAs) critically control gene-expression and play an important role in regulating the immune response. The aim of this study was to prove significant variations of key immunoregulatory miRNAs in peripheral blood mononuclear cells (PBMCs) and in CD4+ and CD 8+ T-cells of AITD patients. Selected miRNAs were amplified by a semiquantitative SYBR Green PCR from PBMCs and purified CD4+ and CD 8+ T-cells of 59 patients with GD, HT, and healthy controls. Both GD and HT showed significantly decreased miRNA 200a_1 and miRNA 200a2* in CD4+-T-cells (mean relative expression 12,57 in HT vs. 19.40 in control group (CG), p=0.0002; 12,10 in GD vs. 19.40 in CG, p=0.0002) and in CD8+-T-cells (13.13 in HT vs. 18,12 in CG, p=0.02; 11.66 in GD vs. 18.12 in CG, p=0.0002). GD and HT showed significantly decreased miRNA 155_2 and miRNA 155*_1 in HT in CD8+-T-cells (10.69 in HT vs. 11.30 in CG, p=0.01; 10.40 in GD vs. 11.30 in CG, p=0.005). This study confirms significant variations of miRNA200a and miRNA155 in patients suffering from GD and HT in vivo in CD4+ T-cells and CD8+ T-cells. These data may help to better understand the gene regulations in the causative cells causing these autoimmune processes. They extend our very limited knowledge concerning miRNAs in thyroid diseases.

Lung B cells promote early pathogen dissemination and hasten death from inhalation anthrax.

Sampling of mucosal antigens regulates immune responses but may also promote dissemination of mucosal pathogens. Lung dendritic cells (LDCs) capture antigens and traffic them to lung-draining lymph nodes (LDLNs) dependent on the chemokine receptor CCR7 (chemokine (C-C motif) receptor 7). LDCs also capture lung pathogens such as Bacillus anthracis (BA). However, we show here that the initial traffic of BA spores from lungs to LDLNs is largely independent of LDCs and CCR7, occurring instead in association with B cells. BA spores rapidly bound B cells in lungs and cultured mouse and human B cells. Binding was independent of the B-cell receptor (BCR). B cells instilled in the lungs trafficked to LDLNs and BA spore traffic to LDLNs was impaired by B-cell deficiency. Depletion of B cells also delayed death of mice receiving a lethal BA infection. These results suggest that mucosal B cells traffic BA, and possibly other antigens, from lungs to LDLNs.

Comparative study of bipolar eletrocoagulation versus argon plasma coagulation for rectal bleeding due to chronic radiation coloproctopathy.

BACKGROUND AND STUDY AIM: Chronic radiation coloproctopathy (CRCP) is a well-recognized complication of radiotherapy, with rectal bleeding the most common presentation. It is frequently refractory to conservative management, but the optimal endoscopic treatment of bleeding secondary to CRCP is still controversial. The efficacy and safety of bipolar eletrocoagulation (BEC) and argon plasma coagulation (APC) in the management of bleeding from CRCP were evaluated and compared. PATIENTS AND METHODS: 30 patients (mean age 67.4 years) with active and chronic bleeding from telangiectasias, were randomly allocated to BEC or APC and stratified by severity of CRCP according to clinical severity and endoscopic findings (Saunders score). Success was defined as eradication of all telangiectasias, and therapeutic failure as need for more than seven sessions or for other treatment. Complications were categorized as minor (e.g. fever, anal or abdominal pain) or major (hemorrhagic). RESULTS: Both treatments were equally effective for the treatment of CRCP rectal bleeding. Only one failure was observed in each group (P = 1.000). There was no significant difference between the two groups regarding number of sessions, minor or major complications, or relapse. However, overall complication rate was significantly higher in the BEC group (P = 0.003). CONCLUSIONS: BEC and APC are both effective for the therapy of bleeding telangiectasias from CRCP. There are probably no major differences between them. Although APC seemed safer than BEC in this investigation, further studies, involving a much larger population, are needed to assess the complication rates and determine the best management option.

The incidence of bacteraemia after argon plasma coagulation in patients with chronic radiation proctocolitis.

AIM: Argon plasma coagulation (APC) is considered a safe treatment for haemorrhagic chronic radiation proctocolitis (CRPC), but bacteraemia is a rare complication. The study aimed to evaluate the frequency of bacteraemia after APC. METHOD: A prospective study of 21 patients who underwent APC (30 procedures) for CRPC was carried out. Blood cultures (Bactec(®) ) were obtained before and 30 min after the procedure (60 samples total). Patients were monitored for 48 h after the procedure to detect signs of infection. RESULTS: None of the 21 patients had fever or any sign suggestive of infection after any of the 30 sessions. All baseline blood cultures were negative and two (7%) of the 30-min blood cultures were positive (Staphylococcus hominis n = 1; Streptococcus bovis and Rhodotorula sp n = 1). The first was likely to be a contaminant and the second patient had no evidence of any other colonic disease (neoplasia or polyps) beside CRPC. CONCLUSION: APC is a low-risk procedure regarding bacteraemia and does not warrant prophylactic antibiotic administration.

Productive syntax abilities in Huntington's and Parkinson's diseases.

This study examined syntactic changes in the spoken discourse of patients with Huntington's (HD) or Parkinson's disease (PD) and explored possible relationships between their syntactic changes and concomitant cognitive and motoric symptoms. Patient and control groups participated in a conversational discourse activity and completed a battery of standardized speech and cognitive tests. The HD group used shorter and fewer grammatically complete utterances than their healthy, age-matched peers, whereas there were no significant syntactic differences between PD patients and their healthy, age-matched peers or between PD and HD patients. Productive syntax abilities in HD and PD were meaningfully related to both neuropsychological and motor speech changes. These findings indicate that patients with subcortical disease, at least those with HD, may present with language production deficits and that these deficits are most likely the product of not only motor speech limitations (i.e., dysarthria) but also underlying cognitive impairments.

Development of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice.

We developed a competitive index assay for murine listeriosis that tests the virulence of Listeria monocytogenes strains in different organs and at various times postinoculation. Studies presented here demonstrate the reproducibility of this assay during primary and secondary infection of inbred and outbred mice. We verified the validity of this assay by performing competitive index analysis of a well-characterized strain of L. monocytogenes lacking the actA gene. In addition, we found that while L. monocytogenes strains unable to recruit vasodilator-stimulated phosphoprotein (VASP) to their surface exhibit a 10-fold virulence attenuation in the livers of naive animals, they display a 50-fold survival defect in the liver during secondary listeriosis.

Requirements for bone marrow-derived antigen-presenting cells in priming cytotoxic T cell responses to intracellular pathogens.

Bone marrow (BM)-derived antigen-presenting cells (APCs) are potent stimulators of T cell immune responses. We investigated the requirements for antigen presentation by these cells in priming cytotoxic T lymphocyte (CTL) responses to intracellular bacterial and viral pathogens. [Parent-->F(1)] radiation BM chimeras were constructed using C57BL/6 donors and (C57BL/6 x BALB/c)F(1) recipients. Infection of chimeric mice with either Listeria monocytogenes or vaccinia virus expressing the nucleoprotein (NP) antigen from lymphocytic choriomeningitis virus (LCMV) primed H2-D(b)-restricted, but not H2-K(d)-restricted CTL responses, demonstrating the requirement for BM-derived APCs for successful priming of CTL responses to these pathogens. Surprisingly, this did not hold true for chimeric mice infected with LCMV itself. LCMV-infected animals developed strong CTL responses specific for both H2-D(b)- and H2-L(d)-restricted NP epitopes. These findings indicate that in vivo priming of CTL responses to LCMV is remarkably insensitive to deficiencies in antigen presentation by professional BM-derived APCs.

H2-M3 restricted presentation of a Listeria-derived leader peptide.

Protective immunity to infection by many intracellular pathogens requires recognition by cytotoxic T lymphocytes (CTLs) of antigens presented on major histocompatibility complex (MHC) class I molecules. To be presented for recognition by pathogen-specific CTLs, these antigens must gain access to the host cell class I processing pathway. In the case of intracellular bacterial pathogens, the majority of bacterial proteins are retained within the bacterial membrane and therefore remain inaccessible to the host cell for antigen processing. We have isolated a CTL clone from a C57BL/6 mouse infected with the intracellular gram-positive bacterium Listeria monocytogenes (LM) and have identified the source of the antigen. Using a genomic expression library, we determined that the clone recognizes an antigenic N-formyl peptide presented by the nonpolymorphic murine MHC class Ib molecule, H2-M3. Several lengths of this peptide were able to sensitize cells for lysis by this CTL clone. The source of this antigenic peptide is a 23-amino acid polypeptide encoded at the start of a polycistronic region. Analysis of mRNA secondary structure of this region suggests that this polypeptide may be a leader peptide encoded by a transcriptional attenuator.

CTL responses to H2-M3-restricted Listeria epitopes.

Cytotoxic T cells (CTL) play a critical role in the murine immune response to Listeria monocytogenes (Listeria). Bacterial antigens are presented to Listeria-specific CTL by products of both conventional, polymorphic MHC class Ia and non-polymorphic MHC class Ib alleles. The H2-M3 class Ib gene product, M3, preferentially presents formylmethionine-initiating (fMet) peptides derived from the N termini of bacterial and mitochondrial proteins. Thus, M3 signals the presence of bacterial invaders to CTL effectors. Listeria-encoded fMet peptide epitopes for H2-M3-restricted CTL have recently been identified. These and other identified fMet peptides are predominantly comprised of hydrophobic residues and appear to be cleaved from membrane-bound proteins. The subcellular location and membrane topology of such proteins may be significant factors in their selection as target antigens for H2-M3-restricted CTL. Such rules may prove useful for prediction of candidate fMet peptide epitopes from other bacterial proteins and species. Studies using synthetic fMet peptides to stimulate CTL ex vivo are also discussed. These latter studies indicate that Listeria infection boosts H2-M3-restricted CTL responses. However, in contrast to MHC class Ia-restricted CTL responses, fMet peptide-specific CTL are observed in a large proportion of cultures from non-immunized, conventionally housed (non-SPF) mice. The CTL activity in these latter cultures may reflect priming in vivo on cross-reactive antigens, or may indicate that requirements for priming of H2-M3-restricted CTL are less stringent than for class Ia-restricted responses.

Primary and secondary immune responses to Listeria monocytogenes.

Recent studies have revealed the complexity of cytokine and cellular interactions required for resistance to primary Listeria monocytogenes infection and have illustrated that resistance to secondary infection may occur through multiple pathways. Analyses of Listeria epitope generation and the specificity of protective CD8(+) T cells have suggested that future research should focus on secreted protein antigens in specific resistance to infection and have increased our understanding of Listeria antigens presented by MHC class l-b molecules.

Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3.

Using expression cloning, we have identified an H2-M3-restricted epitope of the intracellular bacterial pathogen Listeria monocytogenes. Picomolar concentrations of an amino-terminal N-formylated hexapeptide, fMIGWII, targeted cells for lysis by CD8+ cytotoxic T cells, while the nonformylated peptide was approximately 100-fold less active. The sequence of the 185 aa protein source of this epitope predicts a transmembrane protein that retains its N terminus and assumes an N(out)-C(in) topology. This membrane orientation offers an explanation for the protection of the epitope from deformylases present in the bacterial cell and suggests an explanation for the ability of phagocytes to present H2-M3-restricted bacterial epitopes via a vacuolar TAP-independent mechanism.

Interleukin-2-induced lymphocyte infiltration of multiple organs is differentially suppressed by soluble tumor necrosis factor receptor.

Interleukin-2 (IL-2) mediates the regression of metastatic cancer, but clinical application is restricted by associated toxicities. Previous studies implicate tumor necrosis factor (TNF) as an important mediator of certain IL-2-induced toxicities. We hypothesized that soluble TNF receptor (sTNFr), a TNF antagonist, would alter lymphocyte trafficking into normal tissues and ameliorate IL-2-induced toxicity. Four groups of C57BL/6 mice were treated for 4 days with intraperitoneal injections of 100,000 IU IL-2 alone, 100,000 IU IL-2 and 30 micrograms sTNFr combined, 30 micrograms sTNFr alone, or equal volumes of saline. Animal activity was graded and blood obtained for SGPT and SGOT. At necropsy, organs were harvested for wet:dry ratios as a measurement of organ edema. The lung, liver, and thymus were examined histologically for lymphocytic infiltration and graded on a scale of 1 to 5. IL-2-treated groups had a statistically significant increase in organ edema, lymphocytic infiltration into the lung and liver, liver enzyme elevation, and pancytopenia when compared with controls. Soluble TNFr significantly suppressed IL-2-induced pulmonary lymphocytic infiltration and associated serum lymphopenia without significant alteration of other IL-2-induced effects. These data implicate TNF as a mediator of the pulmonary lymphocytic infiltration and of lymphopenia that accompanies IL-2 therapy and further suggest that alternative mechanisms are involved in other IL-2-induced deleterious effects.

Medium pH modulates matrix, mineral, and energy metabolism in cultured chick bones and osteoblast-like cells.

The effects of medium pH were tested on calvariae, tibiae, and osteoblast-like cells from chick embryos. Bones and isolated cells were incubated for 5 h or 2 days in Hepes-buffered medium at pH values ranging from 6.8 to 8.2. Osteoblast function was evaluated by lactate production, oxygen consumption, alkaline phosphatase activity (AlPase), Ca and inorganic phosphate (Pi) flux, proline hydroxylation, DNA content, and thymidine incorporation. As medium pH was increased, glycolysis, collagen synthesis, and AlPase increased, while Ca efflux decreased. No effect of pH was seen on mitochondrial activity, Pi efflux, or cell number or proliferation. The importance of glycolysis as an endogenous pH regulator was demonstrated by inhibition with iodoacetic acid or glucose restriction and by adding lactate to the medium. The results suggest that the pH of bone interstitial fluid may be regulated by glycolysis and that changes in pH of this compartment may have marked effects on osteoblast function.

Smokeless tobacco contains an inhibitor of prolyl hydroxylase activity.

The effects of smokeless tobacco extract (STE) and various constituents of STE on prolyl hydroxylase activity were determined using enzyme extracted from chick embryos. STE inhibited prolyl hydroxylase activity in a concentration-dependent manner, but nicotine and anabasine had essentially no effect. Enzymatic activity was inhibited by zinc, but not by the other inorganic elements in STE; however, the zinc concentration in STE was not high enough to produce the observed inhibition. The inhibition by STE was diminished by increasing concentrations of 2-oxoglutarate, but not by increasing concentrations of other cofactors. Thus, STE contains an inhibitor of prolyl hydroxylase which may be competitive with 2-oxoglutarate.

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP.

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity.

Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.