Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.     

Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene

The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.     

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.     

Monitoring flap for buried free tissue transfer: its importance and reliability

To improve the success rate of microsurgical flap transfers into a buried area, it is important to monitor the circulation of the flap during the early stage. A monitoring flap includes such advantages as simplicity, reliability, noninvasiveness, and the ability to continuously monitor the vascular status of various buried flaps. This article describes experiences related to the importance and reliability of a monitoring flap. A total of 109 flaps in 99 patients were treated with buried free flaps, including a monitoring flap, between 1990 and 1999. Forty-nine patients received a tubed free radial forearm flap with a skin-monitoring flap, and six received a free jejunal flap with a jejunal segment monitoring flap for the reconstruction of the esophagus. Vascularized fibular grafts with a skin monitoring flap or peroneus longus muscle monitoring flap were used for reconstructing the mandible in six patients and for treating osteonecrosis of the femoral head in 48 flaps in 38 patients. Monitoring flap abnormalities were indicated in 14 flaps; therefore, immediate revisions were performed on the pedicle of the monitoring flap and microanastomosis site. Among these 14 flaps, nine showed true thrombosis and five showed false-positive thrombosis. Among the nine flaps that showed true thrombosis, five were salvaged and four were finally lost. The false-positive thrombosis in the five flaps was attributed to torsion or tension of the perforator of the monitoring flap in three flaps, an unclear determination in one flap because the monitoring flap size was too small, and damage to the perforator in the last flap. The total thrombosis rate was 8.3 percent (nine of 109), and the failure rate of the free tissue transfer was 3.7 percent (four of 109). The overall sensitivity of the monitoring flap was 100 percent, the predictive value of a positive test was 64 percent (nine of 14), and false-positive results occurred in 36 percent (five of 14). The salvage rate was 55.6 percent. To improve the reliability of a monitoring flap, it is recommended that the size of the flap be larger than 1 x 2 cm to assess the arterial status, and that a perforator with the appropriate caliber be selected. When a monitoring flap is fixed to a previous incision line or a newly created wound, any torsion or tension of the perforator should be avoided. In conclusion, the current results suggest that a monitoring flap is a simple, extremely useful, and reliable method for assessing the vascular status of a buried free flap.     

Neurovascular musculus obliquus internus abdominis flap free transfer for facial reanimation in a single stage

A study of the anatomy and transplantation of the musculus obliquus internus abdominis with a neurovascular pedicle transfer for facial reanimation in one stage is presented. Eleven adult cadavers (22 face sides) were dissected to observe the shape, thickness, innervation, and blood supply of the musculus obliquus internus abdominis. The blood supply of this muscle primarily comes from the musculus obliquus internus abdominis branch of the deep circumflex iliac artery (diameter, 1.3 +/- 0.2 mm), but it can also come from the eleventh intercostal artery (diameter, 1.14 +/- 0.3 mm) and the infracostal artery (diameter, 1.5 +/- 0.2 mm). The branch of the deep circumflex iliac artery and its vena comitans, or the infracostal artery and its vena comitans, could be anastomosed for muscle transplantation. The innervation of the musculus obliquus internus abdominis comes from the tenth and eleventh intercostal nerves (length, 12.7 +/- 1.5 cm) and the infracostal nerve (length, 12.9 +/- 1.3 cm). The eleventh intercostal nerve and the infracostal nerve were selected for anastomosis of muscle transplantation. From November of 1995 to November of 1999, 14 patients with long established facial paralysis were treated with transplantation of a musculus obliquus internus abdominis flap in one stage and were followed for 10 months to 6 years. In 13 patients, the dynamic functions of the transplanted muscles were restored, the obliqueness of the mouth and philtrum while static was corrected, and the facial muscle activities while smiling were harmonized. The eyelids of the paralyzed side could be closed postoperatively, indicating that the function of the orbicularis oculi of the paralyzed side was restored. The single-stage transplantation of a free musculus obliquus internus abdominis flap with one vascular, multi-nerve pedicle is a new method for facial reanimation in the treatment of long established facial paralysis. Because of the simplicity of the procedure and the completeness of the functional reanimation of the paralyzed facial muscles, compared with the results of other free muscle flap transfers, it is an ideal procedure for facial reanimation.     

Proinflammatory cytokine profile in Vibrio vulnificus septicemic patients' sera

Vibrio vulnificus causes a fulminant and frequently fatal septicemia in susceptible hosts. The present study was designed to evaluate the proinflammatory cytokine profile in V. vulnificus septicemia patients' sera and the effect of doxycycline therapy on the levels of proinflammatory cytokines. Levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were measured in the sera of V. vulnificus septicemic patients and normal healthy volunteers using colorimetric sandwich ELISA. The mean values of TNF-alpha, IL-1beta and IL-6 in the sera of V. vulnificus patients (n=33) increased by 210-, 232- and 40-fold in comparison with those of normal healthy volunteers (n=5), but only the IL-6 level showed a statistically significant difference (P<0.05) between the two groups. Sera from the cases for which doxycycline treatment histories were obvious were designated 'before-treatment' (TX). All the others were included in the after-TX group. In the before-TX group (n=5), the levels of TNF-alpha and IL-1beta significantly increased (P<0.05) in comparison with the after-TX group (n=5). IL-6 levels in the two groups showed no difference. In conclusion, the levels of the well known proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 increased in the V. vulnificus septicemic patients' sera, and the levels of TNF-alpha and IL-1beta decreased significantly after doxycycline treatment. These data indicate that proinflammatory cytokines might play a critical role in V. vulnificus septicemia like in other endotoxemic shocks. The use of doxycycline as an effective bactericidal agent and as an effective modulator of proinflammatory cytokines is supported.     

Identification of a new segment involved in cagA 3' region variation of Helicobacter pylori

The cagA 3' region shows marked variation among Helicobacter pylori strains. Two segments of 102 bp and 57 bp are reportedly responsible for this variation. We analysed the cagA 3' region in 70 H. pylori strains using polymerase chain reaction and sequencing. We found that another segment, namely beta segment, was also involved in the variation of this region. The beta segment was 105 bp long and located between the aforementioned two segments. Six genotypes were identified based on the structure of the cagA 3' region. No relationship was found between these genotypes and the clinical outcomes or vacA genotypes. The numbers of tyrosine phosphorylation sites within the cagA 3' region varied among strains, but this was not related to the cagA genotypes. Our data suggest that the cagA 3' region is significantly variable. It appears that the variation of the cagA 3' region might contribute to the modification of virulence.     

Virus-cell fusion inhibitory activity of novel analogue peptides based on the HP (2-20) derived from N-terminus of Helicobacter pylori Ribosomal Protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antibacterial sequence derived from N-terminus of Helicobacter pylori Ribosomal Protein L1 (RPL1). It has a broad-spectrum microbicidal activity in vitro that is thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that HP (2-20) might be possessed virus-cell fusion inhibitory activity. To develop the novel virus-cell fusion inhibitory peptides, several analogues with amino acid substitution were designed to increase or decrease only net hydrophobic region. In particular, substitution of Gln and Asp for hydrophobic amino acid, Trp at position 17 and 19 of HP (2-20) (Anal 3) caused a dramatic increase in virus-cell fusion inhibitory activity without hemolytic effect.     

Crystallization and preliminary x-ray crystallographic studies of trichosanthin delta C7

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has various pharmacological properties. It is possible to reduce the antigenicity of TCS by deleting up to seven C-terminal residues of TCS (TCS-C7) with minimal effect on its activity. TCS-C7 has been crystallized and the crystal diffracted to 1.8 A. It belongs to space group P2(1), with unit-cell parameters a=71.6A, b=74.4A, c=87.6A, beta=97.0 degrees. It is given that there are four molecules per asymmetric unit.     

Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, we examine the specific functions of genes required for antenna development. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle, with Distal-less is required to establish antenna fate. Here we test which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. We report that the antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. We conclude that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. We also report for the first time phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna. We find that dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. We therefore propose that there are significant genetic similarities between the auditory organs of humans and flies.