Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity.

The processing of asparagine-linked oligosaccharides on the alpha-chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N-acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.

Influence of photodynamic therapy on peripheral immune cell populations and cytokine concentrations in head and neck cancer.

BACKGROUND: Photodynamic therapy (PDT) represents a palliative treatment option for a selected group of patients with head and neck squamous cell carcinoma (HNSCC). PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. However, the systemic impact on peripheral immune cells has not been described so far. METHODS: HNSCC patients (n=9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before, during and after PDT. Age-matched healthy donors served as control group (NC, n=15). The frequency and absolute number of T- and B-lymphocytes, CD4+CD39+ regulatory T-cells (Treg) and NK-cells were measured by 10-color flow cytometry. Serum concentrations of T cell related cytokine panel, including HMGB1, IL-6, IL-10 and perforin were measured by bead array and ELISA. RESULTS: In heavily pretreated HNSCC patients, the number and frequency of Treg and NK-cells were increased as compared to NC. PDT induced a further increase of the frequency of Treg and NK-cells in the peripheral blood. Additionally, the serum concentrations of HMGB1, IL-6 and IL-10 showed a significant elevation after treatment with simultaneously decreased perforin levels. CONCLUSION: Although PDT is a local treatment regimen, a systemic inflammatory response with altered peripheral immune cell populations and cytokine concentrations is visible. The increased Treg and NK cell numbers after PDT support the hypothesis that PDT may successfully be combined with NK cell or T cell activating immune checkpoint modulators in HNSCC patients to improve HNSCC specific immunity.

Beta-thiopropionyl cytochromes c modified at lysyl residues: preparation and characterization of the monosubstituted horse cytochromes c.

beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the derivatives modified at lysines 72, 73, and 79 which are discussed separately. The electron transfer activity of the beta-thiopropionyl cytochromes c with bovine heart cytochrome-c oxidase was decreased to extents dependent on the position of the modification. Aminoethylation, a secondary modification which reverses the charge change, restored the electron transfer rate to that observed with the unmodified cytochrome c, irrespective of the location of the primary modification. These results afford a direct experimental demonstration that alterations in kinetics with physiological electron transfer partners resulting from modifications which cause a change of the charge of surface side chains are solely due to the electrostatic effects. Of the many chemically modified cytochromes c prepared to date, the singly substituted beta-thiopropionyl cytochromes c are likely to be particularly useful as the thiol allows covalent linkage of any sulfhydryl-reactive reagent to a well-defined location on the protein surface by a simple procedure, even when the secondary modifier is relatively unstable, a crucial advantage not otherwise readily achieved.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

Large de novo renal cell carcinoma in a 10-year-old transplanted kidney: successful organ-preserving therapy.

BACKGROUND: A recent review of the Cincinnati Transplant Tumor Registry recorded 24 de novo renal cell carcinomas developing in renal allografts. However, late development of these tumors after transplantation is very rare. Only four reports exist regarding conservative surgery on kidney transplant tumors. METHODS: This is a report on a case of a large 6-cm de novo renal cell carcinoma in a 10-year-old transplanted kidney. Optimal therapy by transplant nephrectomy or tumor enucleation was discussed. RESULTS: Partial resections or enucleations of renal cell carcinoma are still less than ideal in carcinomas larger than 3 cm considering the higher risk of local recurrence. But the recipient in this case had done so well and had had such a high quality of life after transplantation that partial nephrectomy as therapy of choice was selected. Now the patient is 2 years tumor free. CONCLUSION: The case report demonstrates that in certain select cases of large tumors, organ-preserving surgery could be an alternative approach in combining complete tumor removal with preservation of graft function.

[Contrast enhanced MR angiography in the preoperative evaluation of living kidney donors]. / Die kontrastverstärkte MR-Angiographie in der präoperativen Diagnostik von Lebendnierenspendern.

PURPOSE: To compare Gadolinium-enhanced MR angiography with conventional DSA in the preoperative evaluation of living kidney donors. MATERIAL AND METHODS: 27 potential living kidney donors were examined with contrast-enhanced MR angiography after conventional angiography. The MR angiograms were evaluated for the number of renal arteries, the presence of early arterial branching and vascular pathologies by two independent readers. The results were compared with those of selective conventional angiography and intraoperative findings. RESULTS: Conventional angiography detected 14 accessory renal arteries. Reader A detected 13 of 14 accessory arteries with no false positive result (sensitivity 93%, specificity 100%). Reader B detected 11 of 14 accessory vessels with one false positive finding (sensitivity 79%, specificity 98%). Early arterial branching was detected by both readers in 9 of 12 vessels with no false positive result (sensitivity 75%, specificity 100%). None of the patients had additional vascular pathology. DISCUSSION: Gadolinium-enhanced MR angiography is a non-invasive alternative to conventional angiography in the preoperative evaluation of living kidney donors. In order to achieve high accuracy in detecting accessory renal arteries and early arterial branching extensive experience with the method and the specific preoperative needs is required. Selective conventional angiography is still superior in detecting very small accessory vessels and early arterial branching.

[Laser coagulation treatment of urothelial tumors of the upper urogenital tract with the Nd:-YAG laser]. / Laserkoagulationsbehandlung von Urotheltumoren des oberen Urogenitaltrakts mit dem Nd:-YAG Laser.

Six patients with upper urinary tract tumors were treated in our department with the Nd:-YAG laser between 1989 and 1993. The average follow-up was 23.8 months. The mean age of the patients was 65.7 years. The patients were selected for organ-conserving treatment because of bilateral synchronous tumors (2 cases), a solitary renal unit (2 cases), renal insufficiency with high surgical risk (1 case) and high surgical risk alone (1 case). Two patients were treated with open tumor excision and laser, two patients with ureteroscopic and percutaneous procedures one patient with percutaneous treatment and one patient with ureteroscopy. Two patients underwent nephroureterectomy; one of them died of metastatic bladder cancer 9 months later. Local relapses were observed in one patient who had been treated endoscopically. In a follow-up period of 6-36 months there were three patients who had no relapses. Three patients received intracavitary therapy. Kidney conserving procedures with the Nd:-YAG laser in patients with upper urinary tract tumors should be reserved for highly selected cases.

Characterization of MOPC 315 IgA oligosaccharide processing intermediates.

The structures of alpha 1,2-mannose containing partially processed asparagine-linked oligosaccharides on the alpha-chain of MOPC 315 IgA were characterized using specific glycosidases and acetolysis. Man6GlcNAc2, a substrate for a Golgi alpha 1,2-mannosidase, was found to be a single isomeric structure. Likewise, Man7-9GlcNAc2 were single isomers indicating an ordered sequence of removal of alpha 1,2-linked mannose residues on this murine immunoglobulin heavy chain.

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.

Aggressive treatment of the first acute rejection episode using first-line anti-lymphocytic preparation reduces further acute rejection episodes after human kidney transplantation.

The detrimental effect of acute rejection episodes on long-term outcome of renal allografts in cyclosporin-treated patients is well established, although has not been seen by all investigators. To analyse the possibility that aggressive treatment of the first episode may ameliorate this detrimental effect, we performed an open label, randomised prospective trial in cyclosporin-based, immunosuppressed recipients of postmortem renal allografts in order to compare two different treatment protocols during primary acute rejection episodes: (1) group 1 of 25 patients received 3 x 250 mg methylprednisolone (MP) i.v.; (2) group 2 of 25 patients received 7 x anti-thymocyte globulin (ATG)-Fresenius i.v. (4 mg/kg body weight). During a period of 4 years, the following clinical observations were made: (1) The incidence of an acute re-rejection episode was significantly reduced in the ATG-treated study group (16%) compared to the MP-treated study group (72%); (2) The severity of the first acute rejection episode (intensity of renal dysfunction measured in terms of 10-day creatinine area under curve) showed no significant difference between the groups (37 mg x 10-d/dl to 58 mg x 10-d/dl); and (3) The half-lives of allografts in both groups have not shown any significant differences so far. In conclusion, aggressive treatment of the first rejection episode of renal allografts with the use of ATG reduced the incidence of re-rejection episodes which, however, are not reflected so far by improvement of the 4-year survival rate of these allografts. Since it could be observed that re-rejection is an even worse predictor for chronic transplant failure, a better long-term outcome of renal allografts in ATG-treated patients may be expected during a longer observation period. The incidence of a third episode was also reduced in the ATG-treated group (0%) compared to the MP-treated group (12%).

Hyperimmunoglobulin prophylaxis, monitoring and preemptive ganciclovir treatment eliminate the risk of CMV infection to improve patient and renal allograft survival.

This study was designed to investigate whether the introduction of ganciclovir to clinical use for anti-CMV treatment changes the risk of CMV infection in renal transplant patients. A total of 1545 cases who had received cadaveric renal transplants were divided into two groups: group 1 (n = 721) was made up of patients who received their transplants within 6 years before the introduction (1991) of ganciclovir and group 2 (n = 824), of individuals transplanted thereafter. Patient and graft survival of CMV D+/R- patients was uni- and multivariately compared with non-CMV D+/R- patients. In CMV D+/R- patients in group 1, survival was significantly lower, and their relative risk for graft loss was 1.32-fold (P = 0.0483) that of non-CMV D+/R- patients. In group 2 patient and graft survival was identical regardless of whether the patients were at risk for CMV infection or not. The risk of CMV infection can be eliminated by hyperimmunoglobulin prophylaxis, CMV monitoring and preemptive ganciclovir treatment in renal transplant patients.