Immunogenicity of Class I HLA but not preformed low MFI donor specific antibodies correlates with outcomes after first renal transplantation.

BACKGROUND: The role of low levels of circulating donor specific antibodies (DSA) producing negative flow cytometry cross match is not completely defined. The purpose of this study was to examine the clinical significance of preexisting low levels of class I DSAs in flow cytometry cross match (FC CM) negative first kidney transplant recipients (KTRs). METHODS: All of the KTRs (n=41) had low levels of anti-class I antibodies only. The kidney transplant outcome was evaluated by the development of a deleterious effect (DE) in recipients in the study cohort (Group 1: DE+, Group 2: DE-). Positivity for DE was determined based on the following criteria: biopsy proven transplant glomerulopathy (TG), de novo development of DSAs, increasing MFI values for preexisting DSAs, and the development of biopsy proven AMR. Anti-HLA antibodies were tested using single antigen Luminex technology. The HLAMatchmaker computer algorithm was used for the immunogenicity analysis of antibody verified (AbVer) mismatched eplets (MME) at the HLA-A and B loci. RESULTS: The results of this study showed that the number of AbVer MME is larger (P=0.03) in the group of KTR who developed DE. We also demonstrated that the number of AbVer MME is a strong predictor of post-transplant DE. These results indicate that persistent weakly reactive DSA is not a significant risk factor for the development of post-transplant DE and that recipients with such antibodies can be successfully transplanted. CONCLUSIONS: Immunogenicity of AbVer MME at HLA-A and B loci is strong predictor of post-transplant increases of the MFI values of preexisting or de novo developed DSA in the FC CM negative first KTR. Avoiding of transplants with more than eleven Class I AbVer MMEs may be the optimal approach to reduce the risk of kidney graft failure.

Engineering a waste management enzyme to overcome cancer resistance to apoptosis: adding DNase1 to the anti-cancer toolbox.

Cancer treatment is often complicated by resistance to conventional anti-cancer treatment and to more recently developed immunotherapy and gene therapy. These therapeutic modalities aim at activating death pathways within cancer cells. Attempts to activate the apoptotic death pathway, by overexpressing proapoptotic signals, are compromised by cancer defense mechanisms, which disrupt the apoptotic-signaling cascade downstream of the overexpressed component. Here, we describe a therapeutic option of triggering apoptosis without activating the apoptotic-signaling cascade or using the native apoptosis executioner nuclease. We have engineered Deoxyribonuclease-1 (DNase1), a waste-management enzyme, by deleting its signal peptide, adding a nuclear localization signal, and mutating its actin-binding site. Apoptosis studies and colony-forming assay for assessing cell viability were conducted in apoptosis-resistant Mel-Juso human melanoma cells. The modified DNase1 reduced cell viability by 77% relative to controls. It also induced typical microscopic features of cellular apoptosis, such as Terminal Transferase dUTP Nick-End Labeling-positive cells and DNA fragmentation. Quantification of apoptosis by Laser scanning cytometry demonstrated high-killing efficiency of 70-100%. The results suggest that this modified DNase1 can efficiently eliminate apoptosis-resistant cancer cells through apoptosis. Coupled to different tissue-specific gene expression elements, this recombinant DNase1 may serve as a platform for eliminating a variety of cancer types.

Subtypes of immunoglobulin (Ig)-G antibodies against donor class II HLA and cross-match results in three kidney transplant candidates.

Preexisting donor-specific antibodies (DSA) play a critical role in the success of solid-organ transplantation. Cross-match (CM) between donor lymphocytes and recipient serum is a pivotal methodology for detecting these antibodies. Luminex platform based solid-phase methodology for anti-human leukocyte antigen (HLA) antibody analysis has revolutionized the approach to antibody detection and HLA specificity identification. In this study, we have reported three cases of successful living donor kidney transplantations performed against strongly positive B lymphocyte flow cytometry (FC) CM owing to highly reactive DSA directed to HLA class II. IgG solid-phase subtype analysis showed that more than 50% of these antibodies were represented by non-complement binding IgG2/IgG4 subtypes. These findings account for antibody mediated rejection (AMR) free long-term post-transplant course in these patients despite, the high level of DSA. Thus, we conclude that routine application of single HLA-coated beads (SAB) IgG subtype assay may provide new insights regarding transplantation or desensitization of patients presenting with negative B-cell complement dependent cytotoxic (CDC) and positive FC CM.

Evaluation of a standard immunofluorescence assay and a new flow cytometric method for the detection of autoreactive antibodies in dogs with tumours.

The major purpose of the presented study was to develop and to evaluate a flow cytometry-based assay (IIFC) for the determination of autoreactive antibodies in sera from canine cancer patients. A blinded study demonstrated the poor reproducibility of the standard, slide-based and microscopically evaluated indirect immunofluorescence test (IIF), especially with sera displaying a cytoplasmic reactivity. In the IIFC, the intra assay coefficient of variance ranged between 5% and 11%, the inter assay variance between 8% and 25%. The IIFC resulted in significantly less positive results among canine cancer patients (16%) than the IIF (40%). The latter results were due to low titered sera indicating that the standard assay may lead to a high proportion of false positive results. The limitation of the IIFC is that no conclusions can be made about the sub cellular localization of the fluorescence. However, this cytometry-based assay makes a more objective and standardized detection of canine autoreactive antibodies possible.

Analysis of microsatellite instability and hypermutation of immunoglobulin variable genes in Werner syndrome.

Werner syndrome (WS) is a human premature aging syndrome, which is associated with high frequencies of neoplasia and genetic instability. We have examined the occurrence of microsatellite instability, which may result from defective mismatch repair, in lymphoblastoid cell lines derived from nine WS patients. Instability was measured at the D2S123 locus by gel analysis of PCR products. Three WS cell lines had 4-13% altered alleles, compared with 0% in the other six lines. The increased frequency of microsatellite instability could not readily be associated with overt cancer or any other known clinical condition in the three patients. To examine whether the WS defect affected the humoral immune system, we measured the hypermutation of immunoglobulin variable genes in peripheral blood cells from the WS patient who donated the cell line with the highest frequency of microsatellite instability. The frequency and pattern of mutation was similar to that from normal individuals, suggesting that the Werner protein is not involved in generating hypermutation.

Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes.

The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.

Impact of age on hypermutation of immunoglobulin variable genes in humans.

Chronological aging is associated with an accumulation of DNA mutations that results in cancer formation. The effect of aging on spontaneous mutations in humans is difficult to study because mutations are infrequent in the overall genome and tumors are relatively rare. In contrast, somatic mutations in immunoglobulin variable genes are abundant and can be studied in peripheral blood lymphocytes. To determine if aging alters the frequency and pattern of hypermutation, we sequenced 331 cDNA clones with rearranged V(H)6 genes and compared 452 mutations from young humans to 570 mutations from old humans. There were more mutated clones in the young population compared to the old population. Among the mutated clones, the frequency, location, and types of substitutions were similar between the young and the old groups. However, the ratio of replacement-to-silent mutations was much higher in the complementarity-determining regions of heavy chains from old people, which indicates that their B cells had been selected by antigen. Among individuals, there was variability in the frequency of tandem mutations, which we have observed in mice defective for the PMS2 mismatch repair protein. Microsatellite variability in DNA, which is caused by impaired mismatch repair, was then measured, and there was a strong correlation between the frequency of tandem mutations and microsatellite alterations. The data suggest that individuals vary in their mismatch repair capacity, which can affect the mutational spectra in their antibodies.

A potential role for interleukin-8 in the metastatic phenotype of breast carcinoma cells.

This study shows a strong correlation between the metastatic potentials of breast carcinoma cell lines and their ectopic expression of interleukin-8 (IL-8). Correlations exist for both constitutive and induced levels of IL-8 released. A correlation was also observed between cell morphology, metastatic potential, and IL-8 profile. Metastatic lines are fusiform in appearance, whereas, nonmetastatic lines are epithelioid. The metastatic potential of two breast carcinoma lines was examined using an orthotopic model of spontaneous metastasis. Metastatic cells formed rapidly growing, poorly differentiated primary tumors that metastasized. Nonmetastatic cells formed rapidly growing differentiated primary tumors that did not produce detectable metastases. Comparison of IL-8 expression by the parental cells and cell cultures developed from primary and metastatic tumors, demonstrates that IL-8 released by cultured cells from the primary tumor is higher than that of the parental cells, and IL-8 released by cultured cells derived from the metastatic lung tumors is greater than that released by cultured cells derived from the primary tumor. These data demonstrate a strong correlation between the metastatic phenotype of a cell and its IL-8 expression, suggesting a role for IL-8 in promoting the metastatic potential of breast tumor cells.

Role of LFA-1/ICAM-1, CLA/E-selectin and VLA-4/VCAM-1 pathways in recruiting leukocytes to the various regions of the chronic leg ulcer.

The center, edge and distant regions of the venous leg ulcer differ in inflammatory cell composition, suggesting that these represent different developmental stages. Our goal was to determine which recruitment pathways contribute to the differences in leukocyte composition between the various ulcer regions. The multiple region biopsy approach, which enables to study the different development phases of the ulcer at one time-point, was employed to immunohistochemically identify the vascular adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and their counter-ligands on extravasated leukocyte cutaneous lymphocyte-associated antigen (CLA), lymphocyte function-associated antigen (LFA-1) and very late activation antigen-4 (VLA-4), respectively. E-selectin expression was highest at the ulcer edge, while ICAM-1 was highest at the ulcer center. VCAM-1 expression was minor at all ulcer regions. CLA stained up to 80% of the epidermal Langerhans' cells, 62% of the T cells, and only 9% of the macrophages. LFA-1 did not stain Langerhans' cells, stained up to 89% of the T cells and up to 11% of the macrophages. VLA-4 stained up to 30% of the T cells and 71% of the macrophages. In conclusion, the results indicate that Langerhans' cells, T cells and macrophage are each recruited by more than one adhesion-molecule pathway to any of the chronic venous leg ulcer regions.

A case for cellulosic membrane hemodialyzers.

Regenerated cellulose membranes have been held responsible for a variety of ill effects in dialysis patients. Many of these effects (hypotension, changes in well-being) have not been supported by studies, while other effects were more likely due to ethylene oxide. The lower cost of dialyzers composed of these membranes may allow implementation of otherwise cost-prohibitive lifesaving therapy in some countries.

Immunohistochemical characterization of the cutaneous cellular infiltrate in different areas of chronic leg ulcers.

Current understanding of the immunological mechanisms involved in the pathogenesis of venous leg ulcers is insufficient. In this study the cellular composition of skin biopsies taken from the center, the edge, and 2 cm distant from the edge of venous leg ulcers was characterized quantitatively by immunohistochemical staining. In the epidermis the mean numbers of Langerhans cells (CD1a+) were four times lower at the edge of the ulcer compared to clinically intact epidermis 2 cm distant from the edge. In the dermis a statistically significant increase in the mean numbers of macrophages (CD68+) and neutrophils (NP57+) from the distant area towards the center of the ulcer was observed. No significant differences were observed in the distribution of T cells nor in the ratio of CD4+/CD8+ T-cell subsets between the different regions of the ulcer. About 30% of T lymphocytes were CD8+ in all microenvironments. The center and the edge of the ulcer were dominated by macrophages comprising 63% and 53% of the cells respectively, while T lymphocytes dominated the distant area. The area 2 cm distant from the edge was also heavily infiltrated by macrophages and neutrophils. B cells (CD22+) and NK cells (CD56+) were relatively rare in all areas, comprising less than 3% of the dermal infiltrate. In conclusion, local microenvironments each with a different cellular composition can be defined within venous leg ulcers.

[Course over several decades of echinococcosis with peritoneal and pulmonary spread]. / Jahrzehntelanger Verlauf einer Echinokokkose mit peritonealer und pulmonaler Aussaat.

Report on a 67-year-old man with echinococcus-granulosus-cysts in liver, lungs, spleen and mesentery since at least 31 years. In 1969 and 1974 surgical intervention was performed. In 1983 suppurative colliquation and spontaneous depletion by a fistula of a cyst of the liver, output of the cysts of the right lung by coughing. In 1986 spontaneous resorption of a cyst of the left lobe of the liver. In 1987 measurable shrinking of a cyst of the left lower lobe of the lung. Two until today existing cysts (spleen and upper part of the abdomen in the midline) are in supervision by sonography.

Effect of Moloney murine leukemia virus on the carcinogenicity of 3-methylcholanthrene in normal rat kidney cells.

Normal rat kidney (NRK) cell were found to be resistant to neoplastic transformation by diverse carcinogenic chemicals. To study chemical-retroviral co-carcinogenesis in this cells they were infected with a low multiplicity of Moloney murine leukemia virus (M-MLV). Using a single cell cloning procedure, a virus-producing clone was isolated from the infected cells, which was shown to carry only one integrated M-MLV provirus per cell. It was found that this single provirus was sufficient to render the clone susceptible to transformation by 3-methylcholanthrene (3-MC). However this clone responded differently to the carcinogen at different passages after infection. When exposed to 3-MC at a low passage postinfection (passage 5), cell transformation was evident only after 11 subsequent subcultures. On the other hand when it was chemically treated at a high passage postinfection (passage 29), cell transformation could clearly be detected already at the next subculture after the chemical treatment. It is suggested that an M-MLV-mediated cumulative effect is necessary to complement the action of the carcinogen in order to complete the carcinogenic process in these cells. This cumulative viral effect appeared to be associated with a change in the control of the virus expression, since 3-MC was found to stimulate virus replication in this clone also only at the high passage postinfection. Indeed virus release by cells of isolated transformed foci, produced by the chemical-M-MLV co-carcinogenesis, was extremely higher than by untransformed cells.

[Is Monroe's point the site of choice for the establishment of pneumoperitoneum in laparoscopy?]. / Ist der Monroe'sche Punkt zur Anlage des Pneumoperitoneum bei der Laparoskopie der Ort der Wahl?

On the occasion of a case of death during performance of pneumoperitoneum before peritoneoscopy the presence of adhesions in the left lower quadrant of the abdomen in 850 peritoneoscopied patients was investigated. In 91,55% of the evaluated results the left lower quadrant of the abdomen was free from adhesions. Thus, the Monroe-point is even further on the place of choice in order to make the pneumoperitoneum. The Verres-needles are preferable to all other types of needles.

Characteristics of women forensic patients in a federal hospital.

Increased criminal activity among women has prompted greater interest in the mentally ill female offender. An analysis of 72 women forensic inpatients at St. Elizabeths Hospital in Washington, D.C., indicated the typical patient was black, unmarried, in her mid-30s, poorly educated, and diagnosed as schizophrenic. The authors examine some relationships between race, type of crime, drug use, institutional history, and age and the utility of such variables as predictors of adjustment. A comparison of the primary sample with a cohort of 72 patients admitted ten years earlier showed that admissions related to public-order and technical offenses such as prostitution, parole violations, and drug violations decreased from 50 to 12 per cent, while admissions related to crimes of violence rose by 17 per cent.

[Diabetic gastroparesis]. / Diabetische Gastroparese.

It is reported on a 61-year-old patient in whom since 1959 has been existing a diabetes mellitus in need of insulin. 1973 in the endoscopic control of roentgenological findings of a stomach suspect to tumour by means of the Wolf-Schindler gastroscope the suspicion to a gastric carcinoma was expressed. In May 1975 he was again admitted to hospital on account of a hypoglycemic shock. Roentgenologically large filling defects in the stomach were found, gastroscopically macroscopically no clearly explained whitish yellow masses (Histology: remains of food, chronic superficial gastritis, extended settlement of fungi). Only after several days of food carency and daily gastric lavages gastroscopically normal findings could be made. Together with the roentgenological findings of the stomach the diagnosis gastroparesis diabeticorum was made. Therapeutically an optimum stopping of diabetes is recommended and the application of metoclopramide. A surgical intervention is not advisable.

[Diagnosis of an organic disease in an accentuated personality]. / Verkennung einer organischen Erkrankung bei akzentuierter Persönlichkeit.

In a female patient with Dunbar-syndrome the cause of the complaints is not recognized during 18 years. Therefore, the symptomatology of the stenosis of the truncus coeliacus is briefly outlined. The patient reacts more and more neurotic on the misunderstanding of organically conditioned complaints, so that at last a consilium of experienced specialists makes the diagnosis "functional intestinal disturbances, hypchondriac depressive syndrome in accentuated personality". Therefore functional disturbances are to be diagnosed only then (also in the psychopathologic behaviour of the patients!), when an organic disease is excluded. The difficulty to justify such a demand is great.