Perioperative Outcomes of Laparoscopic Radical Nephrectomy for Renal Mass in Patients on Dialysis or with Renal Transplant in Place Compared to Normal Controls.

Background: The risk of renal cell carcinoma (RCC) development in the native kidney of patients on dialysis or with a renal transplant is increased compared to the general population. This study examines perioperative outcomes of laparoscopic radical nephrectomy (LN) in dialysis patients or renal transplant patients compared to normal controls. Methods: Four hundred twelve consecutive LN were evaluated (July 2007 to October 2018). Patients were divided into three groups (control, dialysis, and transplant). Perioperative outcomes, including operating room time (OT), postoperative complications, hospital length of stay, and 90-day readmission rates, were evaluated for the three groups. Results: There were 62 patients in the dialysis group, 20 renal transplants, and 330 normal controls. Dialysis patients were younger (median: 58 years versus 67 years; P = .002) and predominantly male (73% versus 59%, P = .047). Dialysis patients compared to controls had shorter total OT (median: 133 versus 149; P = .022), more papillary RCC (27% versus 10%; P < .001), and fewer high grade tumors (73% [8/11] versus 94% [100/106]; P = .038). Renal transplant patients had a higher rate of 90-day readmission (20% versus 6%; P = .034) and more papillary RCC (30% versus 10%; P = .016) compared to controls. Conclusion: LN on dialysis patients does not alter expected perioperative outcomes compared to a large cohort of control LN. LN on renal transplant patients carries a higher 90-day readmission rate than control LN.

New evidence on α-synuclein and Tau binding to conformation and sequence specific GC* rich DNA: Relevance to neurological disorders.

BACKGROUND: Deoxyribonucleic acid (DNA) topology plays a critical role in maintaining the integrity of the genome and cellular functions. Although changes in DNA conformation and structural dynamics in the brain have been associated with various neurological disorders, its precise role in the pathogenesis is still unclear. Previous studies from our laboratory have shown that there is a conformational change in the genomic DNA of Parkinson's disease (PD) (B to altered B-DNA) and Alzheimer's disease brain (B to Z-DNA). However, there is limited information on the mechanism on DNA dynamics changes in brain. OBJECTIVE: In the present study, we have investigated the DNA conformation and sequence specific binding ability of α-Synuclein and Tau with reference to B-DNA and Z-DNA using oligonucleotide (CGCGCGCG)(2) as a novel model DNA system. This sequence is predominantly present in the promoter region of the genes of biological relevance. MATERIALS AND METHODS: Natively, (CGCGCGCG)(2) sequence exists in B-DNA conformation, but in the presence of high sodium concentration (4 M NaCl), the oligo converts into Z-DNA form. We used circular dichroism, melting temperature and fluorescence studies to understand protein-DNA interactions. RESULTS: CD studies indicated that both α-Synuclein and Tau bind to B-DNA conformation of (CGCGCGCG)(2) and induce altered B-form. Further, these proteins increased the melting temperature and decreased the number of EtBr molecules bound per base pair of DNA in B-form indicating that DNA stability is favored to alter B-DNA conformation, which could be an intermediate form favoring Z-DNA conformation. Moreover, both α-Synuclein and Tau also bound to disease-linked Z-DNA conformation of (CGCGCGCG)(2) and further stabilized the Z-conformation. CONCLUSIONS: The present study provides vital mechanistic information on Synuclein and Tau binding to DNA in a conformation-specific manner causing conformational transition. Furthermore, both the proteins stabilize Z-DNA conformation. These have altered minor and major groove patterns and thus may have significant biological implications in relevance to gene expression pattern in neurodegeneration. We discuss the implications of α-Synuclein/Tau binding to DNA and stabilizing the altered conformations of DNA in neuronal cell dysfunction.

[Glioblastoma multiforme--new hope due to modern therapeutical approaches]. / Glioblastoma multiforme--Neue Hoffnungen dank moderner Therapieansätze.

Glioblastoma multiforme (GBM) is the most frequently encountered malignant cerebral tumor. Despite significant improvements in the treatment of GBM, this disease remains associated with a high morbidity and mortality, with more than half of all affected patients dying within the first year after diagnosis. Typical symptoms include focal neurological symptoms, seizures, personality changes and neurocognitive symptoms. GBM can be identified by means of cerebral imaging modalities and subsequently confirmed histopathologically through biopsy or resection. At present, surgical resection followed by radiotherapy with concomitant chemotherapy with temozolomide and subsequent adjuvant chemotherapy with temozolomide is considered the standard therapy for patients with GBM. Currently, many interdisciplinary studies with glioblastoma patients are accomplished with the aim to further improve the prognosis of the affected patients.

Analysis of the repertoire of cattle CD4(+) T cells reactive with bovine viral diarrhoea virus.

Cell-mediated immunity and CD4(+) cells in particular are important for the resolution of acute infection with non-cytopathic bovine viral diarrhoea virus (BVDV). CD4(+) T cells were shown to recognise virus-infected and non-infectious-protein-pulsed APCs, whereas CD8(+) T cells recognised only virus-infected APCs. T cell recognition was strain cross-reactive and MHC-restricted. Using native and recombinant antigens, we identified the structural glycoprotein E2 and the non-structural protein NS3 as dominant CD4(+) T cell determinants. The repertoire of CD4(+) T cell responses to E2 and NS3 was examined using inbred, homozygous cattle and overlapping synthetic peptides. The repertoire was biased toward conserved regions of NS3 and excluded the hypervariable regions of E2. The number of peptides that were recognised varied between animals but patterns could be distinguished in those animals that shared the same DRB3(*) allele. Of particular interest were: (i) a determinant that was recognised in the context of both DRB3(*) alleles (i.e. DRB3(*)2002 and DRB3(*)0701), (ii) two determinants that were juxtaposed to B cell sites, and (iii) a determinant that had structural analogy with a NS3 epitope previously described for the closely related hepatitis C virus. The minimum stimulatory sequence of the latter, NS3(397-414), was located to residues NS3(400-410).

Single amino acid differences are sufficient for CD4(+) T-cell recognition of a heterologous virus by cattle persistently infected with bovine viral diarrhea virus.

Cattle that are persistently infected (PI) with one strain of bovine viral diarrhea virus (BVDV) can resolve infection with a second, antigenically heterologous strain but not the homologous strain. Since CD4(+) T cells are thought to be critical for the resolution of acute BVDV infection (Howard et al., 1992, Vet. Immunol. Immunopathol. 32, 303-314), we have examined the recognition of a heterologous virus (NADL) by CD4(+) T cells from Pe515-PI animals. The immune response of non-PI control cattle challenged with NADL or Pe515ncp was strain cross-reactive, whereas Pe515-PI animals responded to NADL only. The immune repertoire of both groups included NS3, which differs by approximately 1% (9/683) amino acids between these two viruses. Lymphoproliferative responses to proteins and synthetic peptides corresponding to three nonconservative differences in NS3 demonstrated that CD4(+) T cells from non-PI control animals responded well to proteins but poorly to the peptides from both viruses. In contrast, PI animals were responsive to heterologous proteins and peptides but nonresponsive to the homologous equivalents. A single amino acid difference between the two sequences was sufficient to allow responsiveness.

CD4(+) T-cell responses to bovine viral diarrhoea virus in cattle.

Friesian calves were infected with one of three isolates of bovine viral diarrhoea virus (BVDV) and used to establish parameters for an in vitro model of BVDV-reactive T-cell responses in cattle. The study assessed virus clearance, seroconversion, maturation of lymphoproliferative responses (both during and following disease resolution) and the antigen-specificity of CD4(+) T cells from recovered animals. Seroconversion and virus-specific lymphoproliferation were not detected until viraemia had resolved. Interestingly, lymphoproliferation was detected earlier in the animals infected with cytopathic viruses than in those infected with noncytopathic virus despite broadly similar rates of virus clearance and seroconversion for both biotypes. CD4(+) and CD8(+) T cells were induced to proliferate by virus-infected stimulator cells whereas only CD4(+) T cells responded to non-infectious antigens. Lymphoproliferation was strain cross-reactive and MHC-restricted. Induction of T-cell proliferation by recombinant proteins identified the major envelope proteins E(rns) and E2 and the nonstructural (NS) 2-3 protein as T-cell determinants. In addition, the capsid (C) and/or the amino-terminal proteinase, N(pro) were identified as T-cell determinants from the responses of short-term T-cell lines. Thus, in this model, the CD4(+) T-cell repertoire induce by acute BVDV infection includes at least the major envelope proteins, NS2-3, and capsid and/or N(pro).

Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen.

In this study we have examined the recognition of VP0, VP1, VP2, VP3 and P3Dpol by PBMC and CD4+ T-cells from infected, vaccinated-challenged, and multiply-vaccinated (O1, A24, C1 or ASIA1) cattle using recombinant proteins of an O1 serotype virus. The structural protein VP1 was recognised in an homotypic context whereas VP2, VP3, VP4 and P3Dpol were also recognised by T-cells from animals exposed to heterotypic viruses. Only the non-structural protein P3Dpol was consistently recognised by T-cells from the majority of animals examined and heterotypic recognition correlated with the presence of serologically detectable P3Dpol in purified virus. Thus, P3Dpol is a major cross-reactive immunodeterminant of FMDV, eliciting heterotypic T-cell responses and, therefore, with possible potential for inclusion in a subunit vaccine.

Recognition of foot-and-mouth disease virus and its capsid protein VP1 by bovine peripheral T lymphocytes.

The role of T cells in immunity to foot-and-mouth disease virus is still poorly defined compared to that of the humoral response. In this paper we describe a systematic, longitudinal study on the cellular recognition of FMDV and its subunit protein VP1 by bovine peripheral blood T lymphocytes. Multiple vaccination with a single virus serotype induced a serotype cross-reactive proliferative T cell repertoire that varied in magnitude between individual animals and with the serotype of the vaccine used. Primary proliferative T cell responses of vaccinated and acutely infected cattle were weak relative to the magnitude of responses determined for the same animals after boosting. In contrast, the level of circulating antibody produced after both primary and secondary exposure to virus was good. Phenotypic analysis of lymphocytes from vaccinated or infected cattle showed a small increase in CD8+ T cells after infection compared to vaccination. However, in general the profiles of circulating lymphocytes elicited were similar. Thus, we were not able to use proliferative or phenotypic analyses to distinguish between vaccinated and convalescent cattle. T cell recognition of VP1 by multiply-vaccinated cattle was serotype-specific implying that the cross-reactive responses observed with whole virus may be attributed to proteins other than VP1. In contrast to other studies, immunization with recombinant VP1 induced only low levels of neutralizing antibody and failed to elicit profound proliferative responses or protection ever after two immunizations.

MHC class II restricted recognition of FMDV peptides by bovine T cells.

A putative synthetic vaccine for foot-and-mouth disease (FMDV15) has proved less successful in a host species, cattle, than predicted by results in a small-animal model. Possible reasons for this include non-recognition by T cells influenced by major histocompatibility complex (MHC)-linked immune response gene control. It is now possible to type for human leucocyte antigen (HLA) DR-like bovine MHC (BoLA) class II polymorphisms with a one-dimensional isoelectric focusing (IEF) technique. Using this method 14 unrelated cattle were selected with eight different BoLA class II IEF types. After immunization with FMDV15, 13 cattle generated a T-cell response to FMDV15. However, the fine specificity and magnitude of the response was related to BoLA class II type. The non-response by one animal and low response by two other animals were associated with two of the BoLA class II types. Response to the region 149-158 was immunodominant and animals which did not respond to this region had low responses to the whole peptide. Using FMDV-specific T-cell lines five BoLA class II types associated with responder animals were able to present FMDV15 in an MHC class II-restricted fashion, indicating that this peptide is capable of binding to different MHC class II molecules and may account for the broad response observed. The restriction patterns of the lines indicated that the IEF method does not distinguish all functional polymorphisms. At least two of the IEF-defined types could each be split into two distinct specificities and revealed that the three sets of animals with identical IEF types in fact expressed distinct restriction elements.

A T cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle.

Synthetic peptides representing regions of the VP1 protein of foot-and-mouth disease virus strain 01 Kaufbeuren were screened for their ability to stimulate proliferation of PBMC from virus vaccinated cattle. Sites were identified at residue 21-40 (peptide FMDV32) and in the region C-terminal to residue 161. Cells responding to FMDV32 were MHC class II-restricted, CD4+ and secreted IL-2. Thus, this region is defined as a Th site. Of 19 virus vaccinated Friesian cattle, 89% (17/19) responded to purified virus while 37% (7/19; 41% of virus responders) also responded to FMDV32 suggesting that this site is immunodominant for the cattle used. Furthermore, immunisation of FMDV32 responder and non-responder cattle with a related peptide, FMDV5 (FMDV32 co-linearly synthesized with the 141-160 VP1 B cell site), induced neutralizing antibody and a virus-specific T cell population in the FMDV32-responder but not the non-responder animals.

Heterotypic recognition of foot-and-mouth disease virus by cattle lymphocytes.

Lymphoproliferation against foot-and-mouth disease (FMD) virus was examined using peripheral blood mononuclear cells from vaccinated cattle. Ten weeks after revaccination the optimum conditions for proliferation were obtained with 1 microgram/ml of purified virus after 5 to 6 days in culture. This contrasted with the response at 20 months post-revaccination, when the response required less antigen and showed a peak response after 3 to 4 days in culture. Proliferation was specific for FMD virus, but was cross-reactive between serotypically distinct strains of the virus. The proliferative response to isolated virus proteins (VP) involved all three major capsid proteins (VP1, -2 and -3), although the proliferation of lymphocytes from heterotypically vaccinated cattle was due to VP3. Furthermore, the response induced by purified virus, chemically fixed virus and subunit virus particles was indistinguishable and thus it is likely that processing was required for the induction of proliferation. Together these data strongly suggest that FMD virus-induced lymphoproliferation is T cell-mediated and that VP3 may contain dominant, cross-reactive sequences.

Characterization of long-term cultured bovine CD4-positive and CD8-positive T-cell lines and clones.

Long-term cultured CD4+ or CD8+ bovine T-cell lines and clones were established. The CD8+ T-cell line and clones had a strong lectin-dependent cytotoxicity, whereas the CD4+ T-cell line did not. Both phenotype cell lines grew in an interleukin-2 (IL-2)-dependent manner and expressed 50,000-55,000 MW and 65,000-75,000 MW proteins associated with a putative IL-2 receptor (IL-2R), as demonstrated by the cross-linking of radioiodinated recombinant human IL-2 (rhIL-2). Additional molecules of 13,000 and 27,000 MW were also observed on CD8+ T cells. The binding of rhIL-2 was blocked by crude bovine IL-2, and Scatchard plot analysis of the binding data showed that both phenotype cells expressed two different affinity IL-2R that had equilibrium dissociation constants of 12-20 pm (3000-6000 sites/cell) and 146-490 pM (16,000-25,000 sites/well). Only IL-2 stimulated DNA synthesis in these cell lines, whereas mitochondrial enzymes activity, protein synthesis and protein secretion were enhanced by IL-2, mitogens and phorbol myristate acetate. The supernatant from mitogen-stimulated CD4+ cells was unable to enhance the DNA synthesis of either the CD4+ or CD8+ lines, whereas both freshly prepared Con A blasts and anti-immunoglobulin-treated bovine B cells showed elevated DNA synthesis under the same conditions.

T cell-dependent induction of antibody against foot-and-mouth disease virus in a mouse model.

Nude and normal BALB/c mice were primed by intravenous inoculation of purified, infectious foot-and-mouth disease virus (FMDV) type A24, strain Cruzeiro. Frequency estimation of antigen-specific antibody-secreting cells (ASC) and Thy 1+ T cells in the spleens of immunized mice identified that the IgM response was similar for both nude and normal mice, whereas substantial numbers of both IgG ASC and Thy 1+ cells were present in normal mice only. In contrast, nude and normal mouse sera both contained IgG although the nude mouse serum was deficient in IgG1. Antigen-specific antibody could not be induced in spleen cell cultures from C57BL/6 mice after depletion of T cells with monoclonal antibody plus complement. However, the antibody response could be reconstituted if either a source of exogenous lymphokines or T cells from primed but not unprimed mice were added. Similarly, polyclonal stimulation or unprimed T cells could restore the in vitro response and thus complemented the finding of a low frequency of helper T cells in unprimed mice. Taken together, these data identify that the induction of IgG in FMDV-immunized mice is T cell-dependent and regulated by lymphokines. Furthermore, in nude mice a site other than the spleen must be responsible for the synthesis of the observed serum IgG.

Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures.

Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus. Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells. The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced. For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response. The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures. We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus.

The detection and inhibition of proteolytic enzyme activity in concentrated preparations of inactivated foot-and-mouth disease virus.

Proteolytic enzyme activity was detected in a large number of concentrated preparations of inactivated foot-and-mouth disease virus. Several lines of evidence indicated that at least some of this activity could be attributed to BHK cells, although low levels of microbial contamination in many of our preparations could not be discounted and would certainly enhance the cellular proteolytic activity. From an experiment with different concentrations of trypsin, it was concluded that the proteolytic activities of virus concentrates were sufficient to cause significant degradation of VP1. It was also shown that Trasylol and ox serum were effective inhibitors of proteolytic enzymes in concentrated virus preparations stored at 4 degrees C for 8 months.

Observations and implications of proteolysis in preparations of foot-and-mouth disease virus.

The integrity of the VP1 protein of foot-and-mouth disease virus was assessed by polyacrylamide gel electrophoresis (following storage at 4 degrees C of conventional tissue culture preparations and concentrated preparations of the virus. There was little evidence of VP1 degradation in tissue culture filtrates whereas considerable degradation was observed throughout a range of different concentrates. The use of the proteolytic enzyme inhibitor 'Trasylol' appeared to inhibit VP1 degradation in some virus preparations. Vaccination experiments with guinea pigs indicate that cleavage of O BFS 1860 virus by a range of proteolytic enzymes was always associated with a lowered stimulation of neutralising antibody and total antibody. Experiments with six other strains treated with trypsin gave similar results to those obtained with O BFS 1860.