Repair of massive ventral hernias with "quilted" mesh.

INTRODUCTION: Prosthetic reinforcement is a critical component of hernia repair. For massive defects, mesh overlap is often limited by the dimensions of commercially available implants. In scenarios where larger mesh prosthetics are required for adequate reinforcement, it may be necessary to join several pieces of mesh together using non-absorbable suture. Here, we report our outcomes for abdominal wall reconstructions in which "quilted" mesh was utilized for fascial reinforcement. METHODS: Patients undergoing open incisional hernia repair utilizing posterior component separation and transversus abdominis muscle release, with use of quilted synthetic mesh placed in the retromuscular position, were reviewed. Main outcome measures included patient, hernia, and operative characteristics and post-operative outcomes, including surgical site occurrence (SSO), surgical site infection (SSI), and recurrence. RESULTS: Thirty-two patients (mean age 55.7 ± 9.3, BMI 38.3 ± 5.8 kg/m(2)) underwent open ventral hernia repair with "quilted" mesh placed in the retromuscular position. The mean defect area was 760.1 ± 311.0 cm(2) with a mean width of 24.7 ± 6.4 cm. Quilted meshes consisted of two-piece (69 %), three-piece (19 %) and four-piece (12 %) configurations. Wound morbidity consisted of eight (25 %) SSOs, including four (13 %) SSIs, all of which resolved without mesh excision. With mean follow-up of 9.0 ± 13.6 months, there were two (6.3 %) lateral recurrences, both unassociated with mesh-to-mesh suture line failure. CONCLUSIONS: Massive ventral hernias that require giant mesh prosthetics, currently not commercially available, may be successfully repaired using multiple mesh pieces sewn together in a quilt-like fashion. Such retromuscular repairs are durable, without added morbidity due to the mesh-to-mesh suture line. However, additional operative time is required for quilting the mesh together, prompting strong calls for manufacturing of larger mesh prosthetics.

Generation of a recloned transgenic cat expressing red fluorescence protein.

Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean=21+/-7.7 embryos/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The presence of the RFP gene in the mRNA and genomic DNA of the Re-RFP TG cat was confirmed by polymerase chain reaction analyses, and red fluorescence was detected in its internal organs and placental tissue samples. Analysis of nine feline-specific microsatellite loci confirmed that the Re-RFP TG cat was genetically identical to the donor cat. To test whether results such as normality of offspring and a low cloning success were due to epigenetic modifications, global methylation of placenta from the two first cloned RFP TG cats (77.08% and 82.29%) and the Re-RFP TG cat (76.38%) were compared by bisulfite mutagenesis sequencing analysis. In conclusion, although cloning efficiency was low, we demonstrated the successful use of a cloned first RFP TG cat as a donor cat to produce a Re-RFP TG cat. These results may facilitate future developments in biomedical models for human therapeutic applications.

A novel factor isolated from Actinobacillus actinomycetemcomitans stimulates mouse B cells and human peripheral blood mononuclear cells.

A novel immunostimulating factor (ISTF) of Actinobacillus actinomycetemcomitans ATCC 29522 was isolated and characterized as inducing proliferation of mouse B cells and human peripheral blood mononuclear cells. This factor was isolated from the bacterial culture medium and purified by size exclusion chromatography, dye-ligand affinity chromatography, immunoaffinity chromatography using monoclonal antibodies, and preparative electrophoresis. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified ISTF migrated as a single band corresponding to a molecular mass of 13 kDa. ISTF was a proteinaceous material distinct from lipopolysaccharide; it directly induced the proliferation of B lymphocytes but had no effect on the proliferation of T lymphocytes, even in the presence of antigen-presenting cells. A B-lymphocyte-mitogenic activity of ISTF was also shown by flow cytometric analysis of responding cell subpopulations. Immunoblot analysis revealed that ISTF was a component of the outer membranes of bacteria, could exist as a soluble form, and was released by growing and/or lysed bacteria. These results suggest that ISTF produced by A. actinomycetemcomitans may play an important role in immunopathologic changes associated with A. actinomycetemcomitans infections.

Molecular mechanism of the impairment in activation signal transduction in CD4(+) T cells from old mice.

It is well known that IL-2 production of CD4(+) T cells from old mice (old T cells) is impaired. In this study, we have examined TCR complex zeta chain expression of old T cells and their TCR downstream signal transduction pathways stimulated with anti-CD3. Activation of protein tyrosine kinases, Fyn and ZAP-70, and turnover of inositol phosphates stimulated with anti-CD3 were severely impaired in old T cells, although levels of these proteins were comparable to those in young T cells. Increase in intracellular Ca2+ concentration in old T cells was also impaired. Old T cells starting the Ca(2+) oscillation by the anti-CD3 stimulation were severely decreased in number and the oscillation waves were broader in shape. T cells with zeta-FcvarepsilonRgamma heterodimer in the TCR-CD3 complex were increased in proportion in old T cells with a concomitant decrease in the T cells with zeta-zeta homodimer. The density of the TCR-CD3 complex on old T cells was confirmed to be comparable to that on young T cells. The impairment in TCR downstream signal transduction pathways and the increase in zeta-FcvarepsilonRgamma heterodimer in the TCR-CD3 complex were confirmed to be the situation in Th1 clones established from old mice. These results indicate that old T cells are impaired in response to TCR stimulation, because T cells with the TCR-CD3 complex containing the zeta-FcvarepsilonRgamma heterodimer are increased in proportion in old T cells.

The first human case of Trichinella spiralis infection in Korea.

Three cases of human infection by Trichinella spiralis were first confirmed by detecting encysted larvae in the biopsied muscle in December 1997, in Korea. The patients were one 35- and two 39-year-old males residing in Kochang-gun, Kyongsangnam-do. They had a common past history of eating raw liver, spleen, blood and muscle of a badger, Meles meles melanogenys, and complained of high fever, facial and periorbital edema, and myalgia. Hematologic and biochemical examinations revealed leukocytosis and eosinophilia, and highly elevated levels of GOT, GPT, LDH and CPK. In the gastrocnemius muscle of a patient, roundly coiled nematode larvae were detected. The larvae measured 0.775-1.050 (av. 0.908) mm in length, and 0.026-0.042 (av. 0.035) mm in maximum width. The specific IgG antibody levels in three patients' sera were significantly higher when compared with those of normal controls. The patients were treated with flubendazole and albendazole for 15-30 days, and discharged at 13-34 days post-admission. From the above findings, it was confirmed that T. spiralis is present in Korea, and the badger plays a role of as the natural host.

Radioprotective effects of two traditional Chinese medicine prescriptions: si-wu-tang and si-jun-zi-tang.

We performed this study to determine the effect of Si-Wu-Tang, a basic prescription of traditional Oriental medicine as a blood-building decoction (Chinese medical concept: Bu-Xie), Si-Jun-Zi-Tang, a basic prescription as an energy tonic (Chinese medical concept: Bu-Qi) and its major ingredients on jejunal crypt survival, endogenous spleen colony formation, and apoptosis in jejunal crypt cells of mice irradiated with high and low dose of gamma-irradiation. Si-Wu-Tang administration before irradiation protected the jejunal crypts (p < 0.0005), increased the formation of endogenous spleen colonies (p < 0.05) and reduced the frequency of radiation-induced apoptosis (p < 0.05). In an experiment on the effect of ingredients of Si-Wu-Tang, the result indicated that extract of Danggui and Baishaoyao might have a major radioprotective effect. The radioprotective effect of Si-Jun-Zi-Tang and its ingredients were not as significant as that of Si-Wu-Tang. Although the mechanisms of this inhibitory effect remain to be elucidated, these results indicate that Si-Wu-Tang might be a useful radioprotector, especially since it is a relatively nontoxic natural product. Further studies are needed to characterize better the protective nature of Si-Wu-Tang extract and its ingredients.

[Comparison of virulence by Acanthamoeba strains in a murine model of acquired immunodeficiency syndrome].

The pathogenic potential of Acanthamoeba strains was evaluated by experimental infection of murine AIDS (MAIDS) model. C57BL/6 mice were induced to immunocompromized state by intraperitoneal injection of LP-BM5 MuLV and revealed the typical splenomegaly and lymphatic enlargement of axillar and inguinal regions on necropsy 4 weeks after viral infection. Although there was no significant difference in the mortality rate of MAIDS mouse according to the culture temperature, it was very different in the mortality rate from strain to strain of Acanthamoeba. A. healyi OC-3A strain isolated from the brain of a GAE patient showed the highest mortality rate and A. culbertsoni A-1 strain from tissue culture was the second. KA/S3 and KA/S2 strains isolated from soil revealed very low virulence. The mice infected by intranasal inoculation of Acanthamoeba showed relatively chronic course than intravenous inoculation. The gross findings of lungs and brains from infected mice were variable among mice. On the microscopic observations, the lungs showed much more severe inflammation and necrosis than the brains microscopically. This MAIDS model would be useful to study the opportunistic protozoan infections of AIDS patients. In the light of these results, the pathogenic potential and the virulence of Acanthamoeba may be determined genetically.

Costimulatory effect of IL-12 on the activation of naive, memory CD4+ T cells, and Th1 clone.

In the present experiments, costimulatory effects of interleukin 12 (IL-12) and B7-2 on interleukin 2 receptor alpha chain (IL-2R alpha) expression and IL-2-dependent proliferation of naive and memory CD4+ T cells were studied in comparison with the effect of these molecules on Th1 clones. The naive and memory T cells were negatively sorted on the basis of the expression of CD44 and CD45RB, respectively, to diminish the effect of these antibodies on the activation of these cells. When these three T cell populations were stimulated with allogeneic B cells, the addition of IL-12 elicited IL-2R alpha expression and proliferation of all these T cell populations. With anti-B7-2 being included in culture, these responses of naive T cells were abrogated, whereas those of memory T cells and Th1 clones were affected only marginally. When they were stimulated with immobilized anti-CD3, the inclusion of either IL-12 or Chinese hamster ovary cells expressing B7-2 (B7-2CHO) induced IL-2R alpha expression and enhanced IL-2-dependent proliferation of memory T cells, whereas these responses of naive T cells were induced by the inclusion of B7-2CHO and enhanced by the addition of IL-12, although the addition of IL-12 alone did not affect the responses. The responses of Th1 clones were markedly enhanced by IL-12, but not by B7-2CHO, and the enhancement of the proliferation was augmented further by the addition of both IL-12 and B7-2CHO. In IFN-gamma production B7-2 costimulation was required for the enhancing effect of IL-12 on the responses of naive T cells. IL-2 production of all T cell populations was not affected by IL-12 even in the presence of B7-2CHO. These results indicate that IL-12 provides the costimulation for IL-2R alpha expression and IL-2-dependent proliferation of memory T cells and Th1 clones stimulated with TCR ligation, whereas naive T cells require B7-2 costimulation for their response to IL-12. Possible mechanisms of B7-2 costimulation in the response of T cells to IL-12 is discussed.

A polypeptide encoded within the murine AIDS defective virus stimulates primary proliferation of CD8+ T-cells.

The murine AIDS (MAIDS) is a retrovirus-induced disease that shows severe immunodeficiency with abnormal lymphoproliferation in susceptible strains of mice. To clarify the antigenicity of gag gene products of the LP-BM5 defective virus, which is known as the causative virus of MAIDS, we expressed and purified the gag p12 gene product (P12) by using a baculovirus expression vector system. The P12 protein strongly stimulated the proliferation of normal C57BL/6 (B6) lymph node T-cells in vitro. Furthermore, a 25-mer synthetic polypeptide within the P12 sequence gave rise to the similar or even higher activation of T-cells. The phenotype of responding T-cells was found to be CD8+ CD44low, indicating that naive CD8+ T-cells respond against a peptide encoded within a MAIDS defective virus gag p12 gene. Finally, the expression of T-cell receptor (TcR) V beta on the responding CD8+ T-cells was analyzed. Although CD8+ T-cells with the particular V beta chains were expanded in response to the 25-mer peptide, this polypeptide does not seem to be a superantigen, since this response is MHC class I-restricted and the V beta preference is not striking. The presentation pathway of this highly antigenic polypeptide will be discussed.

MHC class I presentation of an exogenous polypeptide antigen encoded by the murine AIDS defective virus.

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12-25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2b) CD8+ T cells in vitro. The presentation of P12-25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12-25 polypeptide. In fact, when P12-25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2Db)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8+ T cell immunity in vivo.

Abnormality in the early signal transduction pathway is responsible for the impaired proliferative response and low K+ current in a T-cell clone by stimulation with anti-CD3 antibody.

Two T-cell clones were established from young and old C57BL/6 mice, respectively. The proliferative response to anti-CD3 stimulation was significantly greater in the young (YT5) than in the old (OT13) T cell clone. However, a similar high response was observed in both T-cell clones upon stimulation with phorbol myristate acetate (PMA) and ionomycin (INM). The calcium-dependent K+ current (IK(Ca)) was recorded with the patch-clamp method in these T-cell clones. With anti-CD3 stimulation, the amplitude of the outward K+ current was significantly lower in OT13 than in YT5 cells. With stimulation with PMA and INM, however, no significant difference in IK(Ca) was obtained between the two types of cells. The level of proliferative response of T-cells to mitogens was well reflected by the amplitude of IK(Ca). Some abnormality in the early pathway of signal transduction, which led to the intracellular Ca2+ influx, appears to be responsible for the impaired proliferation of the old T-cell clone.

Biochemical and molecular characterization of a strain KA/S2 of Acanthamoeba castellanii isolated from Korean soil.

A strain, KA/S2, isolated from Korean soil and morphologically assigned to Acanthamoeba castellanii, was characterized by isoenzyme analysis, and total proteins profile, and mitochondrial (Mt) DNA restriction fragment length polymorphism (RFLP), and compared with four reference strains assigned to the species (the authenitic Castellani, Neff, Ma, and Chang strains). It was found that four isoenzyme, total proteins, and Mt DNA RFLP patterns by eight restriction endonucleases of the strain KA/S2 were identical with those of the Neff strain, isolated from soil of California, USA. The Chang strain was unique in its morphology and total protein patterns. Interstrain polymorphisms of isoenzyme profiles and Mt DNA RFLP patterns were observed among the Castellani, Neff, Ma, and Chang strains. Mt DNA RFLP was confirmed to be highly appropriate for the strain characterization and identification of Acanthamoeba spp.

Characterization of extrathymic T cells induced by recombinant murine interferon-beta in the peritoneal cavity of nude mice.

The effects of recombinant murine interferon-beta (rMuIFN-beta) on extrathymic lymphocyte formation in the peritoneal cavity of BALB/c athymic nude mice and tumor-bearing nude mice were examined for comparison with BALB/c normal euthymic mice. The 7 days of administration of 1 x 10(5) IU rMuIFN-beta caused the number of these cells to increase remarkably in the peritoneal cavity as a unique subset of asialoGM1+CD4+CD8-TcR alpha beta+ T cells. The asialoGM1+CD4+ T cells produced IL-2 and IFN-gamma in primary culture without stimulant but did not proliferate. Thus, extrathymic T cells can be induced easily in the peritoneal cavity in addition to the thymus for host defense systems.

Both cathepsin B and cathepsin D are necessary for processing of ovalbumin as well as for degradation of class II MHC invariant chain.

The effect of highly selective inhibitors of cathepsins on the processing of ovalbumin (OVA) and the presentation of an OVA-derived antigenic peptide (OVA323-339) by antigen presenting cells (APC) was investigated. Both CA-074 (a specific inhibitor of cathepsin B) and pepstatin A (a specific inhibitor of cathepsin D) showed an inhibitory effect on the IL-2 production from an OVA-specific, I-Ad-restricted helper T (Th) cell clone upon stimulation with OVA presented by the I-Ad-positive APC. In contrast, the presentation of the antigenic epitope, OVA323-339, to the same Th clone was not inhibited by either CA-074 or pepstatin A alone, nor even by the mixture of both inhibitors. When APC were treated with cathepsin inhibitor for 24 h, and then antigen and Th were added to the culture, the presentation of not only OVA but also an OVA-derived antigenic peptide was inhibited by either cathepsin inhibitor alone. In addition, the expression of invariant chain on APC was significantly augmented by the pretreatment of APC with either cathepsin inhibitor. Two main conclusions are drawn from these results. First, not only aspartyl protease, such as cathepsin D, but also thiol protease, such as cathepsin B, is involved in antigen processing by APC. Second, both cathepsin B and cathepsin D are necessary for degradation of the invariant chain (Ii) from the MHC class II alpha beta heterodimer in endosomes in order to express functional MHC class II molecules for binding antigenic peptides.

Different requirements of CD3 cross-linkage for the activation of memory and naive CD8+ T cells.

In the present experiments, the requirement of CD3 cross-linkage was studied in terms of activation of naive and Ag-primed CD8+ T cells in a murine system. The results showed that Ag-primed CD8+ T cells and both monoclonal and polyclonal T cell lines produced IL-2 by Fab anti-CD3 stimulation without cross-linkage in the absence of accessory cells. In contrast, naive T cells did not produce IL-2 in response to Fab anti-CD3 without participation of accessory cells; however, they produced IL-2 in the presence of accessory cells. These results indicate that the cross-linkage of CD3 molecules is not necessarily required for both naive and memory CD8+ T cells, but naive T cells, not memory T cells, require some interaction with accessory cells for IL-2 production in response to soluble monovalent anti-CD3.