Decreased expression of membrane IL-5 receptor alpha on human eosinophils: II. IL-5 down-modulates its receptor via a proteinase-mediated process

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.     

The role of the MHC class II transactivator in class II expression and antigen presentation by astrocytes and in susceptibility to central nervous system autoimmune disease

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4(+) T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4(+) Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-gamma-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-gamma activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.     

Crystal structures of transhydrogenase domain I with and without bound NADH

Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding alpha1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 A resolution in the absence of dinucleotide and at 1.9 A resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH- vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.     

Autologous breast reconstruction with the extended latissimus dorsi flap

The extended latissimus dorsi myocutaneous flap can provide autogenous tissue replacement of breast volume without an implant. Nevertheless, experience with the extended latissimus dorsi flap for breast reconstruction is relatively limited. In this study, the authors evaluated their experience with the extended latissimus dorsi flap for breast reconstruction to better understand its indications, limitations, complications, and clinical outcomes. All patients who underwent breast reconstruction with extended latissimus dorsi flaps at the authors' institution between January of 1990 and December of 2000 were reviewed. During the study period, 75 extended latissimus dorsi flap breast reconstructions were performed in 67 patients. Bilateral breast reconstructions were performed in eight patients, and 59 patients underwent unilateral breast reconstruction. There were 45 immediate and 30 delayed reconstructions. Mean patient age was 51.5 years. Mean body mass index was 31.8 kg/m2. Flap complications developed in 21 of 75 flaps (28.0 percent), and donor-site complications developed in 29 of 75 donor sites (38.7 percent). Mastectomy skin flap necrosis (17.3 percent) and donor-site seroma (25.3 percent) were found to be the most common complications. There were no flap losses. Patients aged 65 years or older had higher odds of developing flap complications compared with those 45 years or younger (p = 0.03). Patients with size D reconstructed breasts had significantly higher odds of flap complications compared with those with size A or B reconstructed breasts (p = 0.05). Obesity (body mass index greater than or equal to 30 kg/m2) was associated with a 2.15-fold increase in the odds of developing donor-site complications compared with patients with a body mass index less than 30 kg/m2 (p = 0.01). No other studied factors had a significant relationship with flap or donor-site complications. In most patients, the extended latissimus dorsi flap alone, without an implant, can provide good to excellent autologous reconstruction of small to medium sized breasts. In selected patients, larger breasts may be reconstructed with the extended latissimus dorsi flap alone. This flap's main disadvantage is donor-site morbidity with prolonged drainage and risk of seroma. Patients who are obese are at higher risk of developing these donor-site complications. In conclusion, the extended latissimus dorsi flap is a reliable method for total autologous breast reconstruction in most patients and should be considered more often as a primary choice for breast reconstruction.     

Analysis of pharyngocutaneous fistula following free jejunal transfer for total laryngopharyngectomy

The development of a pharyngocutaneous fistula is the most common and troublesome complication in the early postoperative period following free jejunal transfer for total laryngopharyngectomy. However, many aspects of this complication remain unclear. In this study, the authors analyzed their experience with the pharyngocutaneous fistula formation following free jejunal transfers to evaluate its clinical behavior, determine the significance of the anastomotic technique used, and evaluate the role of preoperative radiation therapy on its formation and management. Of 168 patients who underwent free jejunal transfers following total laryngopharyngectomy at the authors' institution between July of 1988 and March of 2000, 23 patients (13.7 percent) with postoperative fistulas were identified. The mean onset of fistula formation was 16 days. Of the 23 fistulas, 13 (56.5 percent) occurred at the proximal and 10 (43.5 percent) at the distal anastomoses. Whereas the majority of the proximal fistulas (69.2 percent) developed near the mesenteric side of the jejunal flap, most of the distal fistulas (90 percent) were located anteriorly. The incidence of proximal fistula formation was higher in patients with a single-layer repair than in patients with a two-layer repair of a proximal anastomosis (80 percent versus 38.5 percent, p = 0.09). The incidence of fistula formation was greater in patients who received preoperative radiation therapy than in those who did not (16.3 percent versus 11.4 percent, p = 0.36). In addition, whereas a majority of fistulas (80 percent) occurred at the proximal anastomosis in patients who did not receive preoperative radiation therapy, most fistulas (61.5 percent) occurred at the distal anastomosis in patients who did receive radiation therapy (p = 0.09). The fistulas closed spontaneously in 15 patients (65 percent). On average, spontaneous closure occurred in 7.4 weeks. Proximal fistulas had a significantly higher rate of spontaneous closure compared with distal fistulas (85 percent versus 40 percent, p = 0.04). The rate of spontaneous fistula closure was higher in patients who had not received preoperative radiation therapy than in those who had (90 percent versus 46 percent, p = 0.07). Surgical closure of the fistula was required in five patients. The fistulas were not repaired in three patients because of recurrent tumor. Twenty patients (87 percent) resumed oral feeding after the closure of the fistula, with 17 (85 percent) of 20 patients tolerating a regular diet and three (15 percent) of 20 a liquid diet only.In conclusion, most fistulas occur at the proximal anastomosis and near the mesenteric side of the jejunal flap, and the use of a two-layer anastomotic technique seems to be associated with a lower incidence of fistula formation at the proximal suture line. Most fistulas close spontaneously, especially ones that occur proximally. Preoperative radiotherapy does seem to increase the risk of fistula formation, especially at the distal anastomotic site and make subsequent resolution of the fistulas more difficult. Most patients are able to resume oral feeding once the fistula is closed.     

Vaginal reconstruction after extended radical pelvic surgery for cancer: comparison of two techniques

An immediate partial or total vaginal reconstruction is frequently proposed in cases of exenterative or extended radical pelvic surgery for cancer treatment. One of the main complications after this reconstruction is the vagina obliteration caused by the healing process. This study compares the results of two different reconstructive techniques, particularly focusing on general complications and the risk of vaginal occlusion. A transversus rectus abdominis musculoperitoneal (TRAMP) composite flap has been performed in five cases, and an inverted inferior transverse rectus abdominis musculocutaneous flap (TRAM) has been used in another five cases. Recovery was uneventful in eight cases. One patient (case 5) developed an aortofemoral embolism requiring a bilateral transfemoral embolectomy and heparin administration. Another patient (case 9) experienced severe peritonitis because of the partial leak of the rectal anastomosis, and therefore a Mikulicz's colostomy was performed. Four patients who underwent the TRAMP flap developed a complete closure of the neovagina. In one patient with a TRAMP flap, a severe shortening (2 cm) of the neovagina occurred. Five patients out of five who underwent a reconstruction with a TRAM flap had a stable length of the neovagina (6 to 12 cm) and no shrinkage in diameter occurred, even though a vaginal stent was not used. The conventional inferior TRAM flap with a skin paddle seems to better maintain a stable length of the neovagina than the TRAMP composite flap with peritoneum.     

Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein

The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.     

Delineation of the HLA-DR region and the residues involved in the association with the cytoskeleton

Whereas the association of major histocompatibility complex (MHC) class II molecules with the cytoskeleton and their recruitment into lipid rafts play a critical role during cognate T/antigen-presenting cell interactions, MHC class II-induced signals, regions, and residues involved in their association and recruitment have not yet been fully deciphered. In this study, we show that oligomerization of HLA-DR molecules induces their association with the cytoskeleton and their recruitment into lipid rafts. The association of oligomerized HLA-DR molecules with the cytoskeleton and their recruitment into lipid rafts occur independently. Furthermore, the association with the cytoskeleton is HLA-DR-specific, since oligomerization of HLA-DP triggers its recruitment only into lipid rafts. HLA-DR molecules devoid of both alpha and beta cytoplasmic tails did not associate with the cytoskeleton, but their recruitment into lipid rafts was unimpeded. Deletion of either the alpha or beta cytoplasmic tail did not affect the association of HLA-DR with the cytoskeleton and/or recruitment into lipid rafts. HLA-DR molecules that were devoid of the alpha cytoplasmic chain and that had their beta cytoplasmic chain replaced with the HLA-DP beta chain or with a beta chain in which the residues at positions Gly(226)-His(227)-Ser(228) were substituted by alanine no longer associated with the cytoskeleton. They were, however, still recruited into lipid rafts. Together, these results support the involvement of different regions of the cytoplasmic tails in the association and the recruitment of HLA-DR into different compartments. The differential behavior of HLA-DP and -DR with respect to their association with the cytoskeleton may explain the previously described difference in their transduced signals.