Evaluation of Pretransplant Donor-Specific Alloantibodies With Different Crossmatch Techniques.

Donor-recipient crossmatching for kidney transplantation is an obligatory step for the transplant work-up process. Attention is currently put on single bead assay (SBA) that enables virtual crossmatching. In contrast, methods developed for complement binding capacity are still not routinely established. We compared modified, cytolytic flow cytometry crossmatch (cFC-XM) with complement-dependent serological crossmatch (CDC-XM), SBA, and flow cytometry crossmatch (FC-XM) to verify whether newly developed techniques may be beneficial for transplant immunological matching. Also, the cutoff value for donor-specific alloantibodies levels currently used for virtual crossmatch was verified. Serum from 22 sensitized patients was crossmatched with surrogate donors by CDC-XM, FC-XM, and cFC-XM, while anti-HLA antibodies were measured by SBA. In all cases, virtual crossmatch was positive at 5000 mean fluorescence intensity cutoff. Among 22 tested sera with donor-specific alloantibodies above 5000 mean fluorescence intensity, the positive CDC-XM result was noted only in 41% of patients (n = 9), but positive FC-XM was noted in 86% (n = 19), and further lytic antibodies (cFC-XM) were confirmed in 27% of cases (n = 6). Our results suggest that donor-recipient immunological matching for kidney transplantation requires different methods to verify the importance of alloantibodies, and improvement in laboratory investigation is needed. This is especially important for immunized patients that have many types of alloantibodies and virtual crossmatching used as a tool for deceased donor allocation should balance between the likelihood of transplantation and risk of positive crossmatch result.

Inhibition of FASN and ERα signalling during hyperglycaemia-induced matrix-specific EMT promotes breast cancer cell invasion via a caveolin-1-dependent mechanism.

Since disturbed metabolic conditions such as obesity and diabetes can be critical determinants of breast cancer progression and therapeutic failure, we aimed to determine the mechanism responsible for their pro-oncogenic effects. Using non-invasive, epithelial-like ERα-positive MCF-7 and T47D human breast cancer cells we found that hyperglycaemia induced epithelial to mesenchymal transition (EMT), a key programme responsible for the development of metastatic disease. This was demonstrated by loss of the epithelial marker E-cadherin together with increases in mesenchymal markers such as vimentin, fibronectin and the transcription factor SLUG, together with an enhancement of cell growth and invasion. These phenotypic changes were only observed with cells grown on fibronectin and not with those plated on collagen. Analyzing metabolic parameters, we found that hyperglycaemia-induced, matrix-specific EMT promoted the Warburg effect by upregulating glucose uptake, lactate release and specific glycolytic enzymes and transporters. We showed that silencing of fatty acid synthase (FASN) and the downstream ERα, which we showed previously to mediate hyperglycaemia-induced chemoresistance in these cells, resulted in suppression of cell growth: however, this also resulted in a dramatic enhancement of cell invasion and SLUG mRNA levels via a novel caveolin-1-dependent mechanism.

Insulin Receptor Isoform Variations in Prostate Cancer Cells.

Men who develop prostate cancer (PCa) increasingly have one of the co-morbidities associated with a Western lifestyle that are characterized by hyperinsulinemia, hyperglycemia and increased expression of insulin-like growth factors-I (IGF-I) and IGF-II. Each have been associated with poor prognosis and more aggressive cancers that exhibit increased metabolism and increased glucose uptake. The insulin receptor (IR) has two splice isoforms IR-A and IR-B: IR-A has a higher affinity for IGF-II comparable to that for insulin, whereas the IR-B isoform predominantly just binds to insulin. In this study, we assessed alterations in the IR-A and IR-B isoform ratio and associated changes in cell proliferation and migration of PCa cell lines following exposure to altered concentrations of glucose and treatment with IGF-II and insulin. We observed that where IR-B predominated insulin had a greater effect on migration than IGF-II and IGF-II was more effective when IR-A was the main isoform. With regard to proliferation IGF-II was more effective than insulin regardless of which isoform was dominant. We assessed the abundance of the IR isoforms both in vivo and in vitro and observed that the majority of the tissue samples and cell lines expressed more IR-A than IR-B. Alterations in the isoforms in response to changes in their hormonal milieu could have a profound impact on how malignant cells behave and play a role in promoting carcinogenesis. A greater understanding of the mechanisms underlying changes in alternative splicing of the IR may provide additional targets for future cancer therapies.

Hyperglycaemia-induced chemoresistance in breast cancer cells: role of the estrogen receptor.

Breast cancer patients with diabetes respond less well to chemotherapy; in keeping with this we determined previously that hyperglycaemia-induced chemoresistance in estrogen receptor (ERα) positive breast cancer cells and showed that this was mediated by fatty acid synthase (FASN). More recent evidence suggests that the effect of metabolic syndrome and diabetes is not the same for all subtypes of breast cancer with inferior disease-free survival and worse overall survival only found in women with ERα positive breast cancer and not for other subtypes. Here we examined the impact of hyperglycaemia on ERα negative breast cancer cells and further investigated the mechanism underlying chemoresistance in ERα with a view to identifying strategies to alleviate hyperglycaemia-induced chemoresistance. We found that hyperglycaemia-induced chemoresistance was only observed in ERα breast cancer cells and was dependent upon the expression of ERα as chemoresistance was negated when the ERα was silenced. Hyperglycaemia-induced an increase in activation and nuclear localisation of the ERα that was downstream of FASN and dependent on the activation of MAPK. We found that fulvestrant successfully negated the hyperglycaemia-induced chemoresistance, whereas tamoxifen had no effect. In summary our data suggests that the ERα may be a predictive marker of poor response to chemotherapy in breast cancer patients with diabetes. It further indicates that anti-estrogens could be an effective adjuvant to chemotherapy in such patients and indicates the importance for the personalised management of breast cancer patients with diabetes highlighting the need for clinical trials of tailored chemotherapy for diabetic patients diagnosed with ERα positive breast cancers.

Modified flow cytometry crossmatch detecting alloantibody-related cytotoxicity as a way to distinguish lytic antibodies from harmless in allosensitised kidney recipients.

The serological complement-dependent cytotoxicity crossmatch (CDC-XM) permits routine identification of anti-donor alloantibodies in the sera of allotransplant recipients. However, in a small group of recipients, antibodies below the threshold of detection may still be responsible for hyperacute rejection. For the same reason, approximately 20% of recipients develop acute rejection episodes. The flow cytometry crossmatch (FCXM) was designed to address these problems, but because of the presence of clinically insignificant antibodies (linked, non-lytic), the FCXM appears to be too sensitive yielding false-positive results. We compared FCXM with its modified version assessing cell viability (cytolytic flow cytometry crossmatch; cFCXM) using sera from previously sensitised kidney recipients. The presence of alloantibodies was detected using the Luminex platform. The cFCXM proved to be of greater sensitivity than CDC-XM, which was additionally confirmed with bead-based Luminex techniques. The cFCXM was also superior to FCXM because it distinguished lytic from non-lytic antibodies. The cFCXM was superior to assess donor specificity, sensitivity, and detection of clinically relevant lytic antibodies.

Algorithm to manage highly sensitized kidney transplant recipients in Poland.

OBJECTIVE: Programs for immunized transplant recipients are essential to achieve graft survivals comparable to those of non-immunized recipients. The threshold in Poland is a PRA by the complement-dependent cytotoxicity (CDC) method greater than 50%, which includes approximately 3.8% of the patients. At the same time the United Network for Organ Sharing there recipients represent approximately 16% of the waiting list in the United Network for Organ Sharing (UNOS). The underestimation of the immunized group in Poland may be due to differences in laboratory techniques to assess alloantibodies. MATERIALS AND METHODS: This study investigated 55 potential recipients with a PRA by CDC>50%. We used the following algorithm to assess their immunization: Luminex screening test for an HLA antibody; specificity assessed with Luminex Single Antigen, vPRA (evaluation of immunization of the patient); and analysis of acceptable HLA incompatibilities (HLAMatchmaker). RESULTS: All recipients were positive class I anti-HLA antibodies and 94.5% were positive for class II. For the groups of subjects with PRA-CDC from 50% to 79% versus those greater than 80%, the average values of PRA-CDC were 62.2% and 89.5%, respectively. The virtual PRA results for these groups were 95.7% and 97.2%, respectively. In addition, anti-HLA-Cw, anti-DQ and anti-DP antibodies were detected in 77%, 84%, and 51% of recipients, respectively. Immunized recipients reported to the next transplant were characterized by the antibodies against mismatch only in 68%. For all potential recipients, additional acceptable non-compliance was determined with HLAMatchmaker: 152 specificity for locus A and 252 for locus B. CONCLUSIONS: Evaluation of immunization status of recipient candidates should be routinely performed using tests to assess class and specificity as well as level of alloantibodies to enable determination of a safe potential donor. As a routine test, PRA-CDC underestimates the number of highly immunized patients. Exclusion from the list of patients with repeated non-compliance is a simplification, which reduces their chance for transplantation.

Nitrogen-15 fractionation in the thermal decomposition of nitrous oxide of natural isotopic composition.

The 15N fractionation in the thermal decomposition of nitrous oxide (N2O) of natural isotopic composition has been investigated in quartz reaction vessel in the temperature interval 888-1073 K. The formulas relating the observed experimentally 15N fractionations with the primary 15N kinetic isotope effect, (k14/k15)p for 14N15N16O, and secondary 15N kinetic isotope effect, (k14/k15)s for 15N14N16O, have been derived. The experimentally estimated 15N kinetic isotope effects have been compared with the primary and secondary 15N kinetic isotope effects calculated with the absolute rate theory formulations applied to linear three atom molecules. A good agreement was found for the primary 15N kinetic isotope effect, (k14/k15)p, in the temperature interval 888-1007 K. But at 1073 K the decompositions of N2O, accompanied by NO (nitric oxide) formation proceed with a twice times smaller primary kinetic isotope effect, (k14/k15)p of 1.0251 +/- 0.0009, only, suggesting the nonlinear transition state structures with participation of the fourth external atom at high temperature decompositions of nitrous oxide. The nitrogen isotope effects determined in this study correlate well with nitrogen isotope fractionations observed in the natural biological, earth and atmospheric processes.

Carbon isotope fractionation in the decarboxylation of phenylpropiolic acid in hydrogen donating media.

13C kinetic isotope effect (KIE) in the decarboxylation of phenylpropiolic acid (PPA) in tetralin medium (Tn) has been determined at 409-432 K and found to be of magnitude similar to the 13C KIE observed in the decarboxylation of malonic acid where the rupture of the C-C bond is the rate determining step. 13C KIE equals 1.0318/at 136 degrees C in the decarboxylation of PPA in Tn medium. Intramolecular 13C KIE in the decarboxylation of malonic acid equals 1.0316 at this temperature. Thus it has been shown that the nearly "full" 13C KIE can be achieved by providing the excess hydrogen to Calpha of PPA (or to triple acetylene bond) using not only strong mineral acids as the source of protons but also by carrying out the decarboxylation in organic medium like tetralin. A mechanism of decarboxylation of PPA in Tn is suggested.

[Comparison study of some neutrophils indices in neonates and adults]. / Próba porównania wybranych wskazników granulocytarnych u noworodków oraz u osób doroslych.

The purpose of this study was to estimate the difference in a range of number values, function and metabolism of neutrophil granulocyte sampled from umbilical cord of healthy and full-term newborns and healthy adults. The following indices were measured: total neutrophils number, neutrophil adherence to fibre, nitrobluetetrazolium reduction ability, phagocytic activity, alkaline phosphatase activity and lysozyme activity in serum. Results of the study showed that neutrophils from healthy newborns, when compared with neutrophils from adults, demonstrated the increase of oxidation-reduction processes, the increase of alkaline phosphatase activity in these cells and lysozyme activity in serum. It was associated with the increase of count of neutrophils what resulted from the decrease of their adherence.

Humoral and cellular immunity rates in chemical plant workers employed in the production of liquid pesticides.

In order to assess the immunological system of the chemical plant workers certain rates of cellular and humoral immunity were estimated. The study group was composed of 19 males employed in the production of liquid pesticides, and 18 females performing ancillary jobs and handling closed containers. They were alternatively exposed to phosphoroorganic compounds and pyrethroides, and to chlorinated hydrocarbons, carbamates, nitrophenols and organic solvents, however exposure to the latter was lower. Chronic bronchitis was observed in 7 (37%) males and 4 (22%) females. Serum concentrations of immunoglobulins G, A and M, complement protein Cs, and circulating immune complexes were estimated. The peripheral blood leukocyte count and percentage, the granulocyte adherence and phagocytic activity, spontaneous NBT-dye reduction as well as cytochemical reactions to alkaline and acid phosphatase, beta-glucuronidase, myeloperoxidase and catalase of neutrophils were evaluated; the lymphocyte subpopulations CD3, CD4, CD8, CD16 were also estimated. As compared to controls, a significantly increased serum IgG concentration was found, together with elevated IgM in males and IgA in females. The leukocyte count in males was significantly higher. A considerable decrease in the percentage of neutrophils was accompanied by a significantly greater spontaneous NBT-dye reduction in both groups. Neutrophil adherence impairment was observed in males. Cytochemical reactions to beta-glucuronidase and catalase in both sexes, to alkaline and acid phosphatase in females, and to myeloperoxidases in males were significantly lowered, whereas the reaction to acid phosphatase in males was significantly enhanced. The percentages of lymphocytes CD3, CD4 and the CD4/CD8 ratio were significantly decreased.

[Study of nitroblue tetrazolium reduction by granulocytes in workers handling x-ray equipment]. / Badanie redukcji blekitu nitrotetrazolowego przez granulocyty u pracowników obslugujacych aparature rentgenowska.

In 44 persons (25 women and 15 men), workers handling X-ray equipment in X-ray laboratories the ability was studied of nitroblue tetrazolium (NBT) reduction by neutrophils. The obtained results were compared with a control group. Besides that subgroups were isolated taking into account the sex of the studied subjects and duration of their work with X-ray equipment (over five years and below five years). In the workers handling X-ray equipment and in the isolated subgroups of women and men, the index of spontaneous NBT reduction turned out to be statistically significantly higher than that in the control group. In the group of studied workers the index of latex-stimulated NBT reduction was statistically significantly lower than that in the control group. Taking into account the duration of work with X-ray equipment in the subgroups of women and men working for over five years, the lowest index of phagocytes was noted. On the basis of the obtained results the following conclusions were formulated: 1. In workers handling X-ray equipment disturbances occur of peripheral blood neutrophil metabolism assessed in the NBT test. 2. Neutrophil phagocytic activity is weakened in persons working over five years with X-ray equipment.