Alfredo Minguela

Flow cytometric DNA analysis and p53 protein expression show a good correlation with histologic findings in patients with barrett's esophagus

There is a considerable degree of subjectivity and, therefore, substantial interobserver and intraobserver disagreement in the diagnosis and grading of dysplastic lesions in Barretts esophagus (BE). The aim of this study was to evaluate the usefulness of DNA flow cytometry and immunohistochemical staining for p53 protein as objective methods to complement the conventional histologic diagnosis of dysplasia in patients with this disease. The most common problems and the possible advantages of using these procedures are analyzed briefly in this article.

HLA polymorphism in the Murcia population (Spain): in the cradle of the archaeologic Iberians.

Human leukocyte antigen (HLA) study in Murcian individuals was performed in order to provide information of their historical origins and relationships with other Iberian and Mediterranean populations. HLA class I and class II alleles were determined in 173 unrelated Caucasoid donors from Murcia Region in the Southeast of Spain by serologic and DNA based polymerase chain reaction (PCR) typing. Class I antigen and class II allele frequencies of our series were not very different to those found in Spaniards. The analysis of extended haplotypes showed that the three haplotypes most frequent in our population were respectively, A29-B44-Cwb-DRB1*0701-DRB4*0101-DQA1*0201-DQB1*0202, A1-B8-Cw7-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201 and A30-B18-Cw5-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201. They were followed by A26-B38-Cwb-DRB1*1301-DRB3*0202-DQA1*0103-DQB1*0603, which could point to an ancestral relationship between Murcian and Portuguese Iberian populations, and by A2-B7-Cw7-DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 also present in all Iberian Peninsula populations. Allelic frequencies, populations distance dendrogram and correspondence analysis were used to study the relationships between Murcian and other populations. The closest relation was observed with Spaniards and Portuguese, followed in decreasing order by French, Italians, Algerians, Germans, Catalans, Basques, Cretans, Sardinians, and Greeks. Thus, Murcian population seems to belong to the European genetic pool, revealing a lesser genetic distance with the North Africans and the rest of populations from the Iberian Peninsula.

CD28 EXPRESSION ON PERIPHERAL BLOOD T LYMPHOCYTES AFTER ORTHOTOPIC LIVER TRANSPLANT : UPREGULATION IN ACUTE REJECTION

Despite immunosuppressive treatments, acute rejection remains a significant cause of graft loss. Efficient allorecognition implicates cognate T-cell interactions and requires costimulatory signals such as those delivered via CD28. Therefore, we have studied CD28 peripheral blood T-cell expression, analyzing its possible implications in liver allograft acute rejection. Fifty-five CsA-immunosuppressed orthotopic liver recipients, with or without acute rejection (AR and NAR) were immunocytometrically monitored after transplant and thirty healthy volunteers were studied as controls. In liver recipients the absolute number of CD28+ cells fell sharply immediately after transplant, but no significant differences were detected between the AR and NAR groups either in the absolute number or in the percentage of CD28+ lymphocytes. By contrast, both CD4+CD28+ and CD8+CD28+ T-cell subsets displayed a significant increase in CD28 intensity expression in AR recipients, whereas CD28 expression was significantly downregulated in the NAR recipients. This data suggests that CD28 molecule can be important in the immunologic events preceding acute rejection and that CD28 up- or downregulation could become a useful predictive marker for acute rejection or tolerance development in liver recipients.

Imaging cytometry for counting circulating tumor cells: comparative analysis of the CellSearch vs ImageStream systems

Circulating tumor cell (CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems.

CD28/CTLA-4 and CD80/CD86 costimulatory molecules are mainly involved in acceptance or rejection of human liver transplant

CD28/CTLA-4 interactions with their specific B7-ligands (CD80 and CD86) have decisive roles in antigenic and allogenic responses. Recently, experimental transplant studies demonstrated that donor-specific tolerance is achieved by blocking these interactions. The present study analyzes the expression of these co-stimulatory molecules in peripheral blood cells from 74 liver recipients and in 16 liver biopsies, which were classified into acute-rejection (AR, n = 27) and nonacute-rejection (NAR, n = 47) groups, as well as their influence on the in vitro response of in vivo allosensitized cells. The results clearly indicate that in human liver transplant too, B7 and CD28/CTLA-4 expression on B and CD4(+) peripheral lymphocytes respectively, contributes to graft acceptance or rejection, and appears to be of crucial importance in modulating the host alloresponse and specific-CTL generation. In the NAR-group, costimulatory molecule expression remained at basal levels after transplant, whereas in the AR-group these molecules were significantly upregulated on days of AR. CTLA-4 was observed in the infiltrating lymphocytes in most of the biopsies, but CD80 or CD86 were not. Moreover, specific cytotoxicity from the in vivo primed cells was clearly suppressed in the NAR-patients with low co-stimulatory molecule expression, whereas this activity was not modified but rather stimulated in the AR-group. Together, these findings indicate that intervention of CD28/CTLA-4/B7 signaling could be therapeutically useful in clinical transplantation.

Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture

The one‐way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable.

HLA-DRB1 and -DQB1 Polymorphism in Liver Recipients: Relationship Between HLA-DQB10302 Allele Frequency and Acute Rejection

Polymorphism of HLA-DRB1 and HLA-DQB1 loci was performed in fifty-three orthotopic liver graft recipients as well as in 108 unrelated healthy controls. Nonradioactive SSOPs were used to study PCR-amplified DNA from peripheral blood lymphocytes and biopsied material. The comparison frequency for DQB1 alleles did not reveal any significant differences between the total group of liver recipients and controls. However, when the liver recipients were subgrouped according to their rejection episode manifestations, increased and significant frequencies were observed for HLA-DQB1*0302 allele in patients showing acute rejection episodes compared to healthy controls or patients without acute rejection. This relationship did not appear influenced by the amino acid beta alanine residue in the 57th position. On the other hand, the study of the DRB1 allele frequencies did not show significant differences in any study. These results suggest that HLA-DQB1 genes could be important in the liver graft alloresponses, opening a way to a better understanding of the special tolerance state, normally observed in this type of transplant, leading us to consider the possible HLA-DQB1*0302 allele effect on tolerance rupture.

Large-volume-apheresis facilitates autologous transplantation of hematopoietic progenitors in poor mobilizer patients.

Given that pre‐apheresis CD34+ cell count (PA‐CD34) predicts the apheresis yield, a minimum of 5 to 20 PA‐CD34/μl is required in many institutions to initiate cell collection. The aim of this study was to clarify whether large‐volume‐apheresis (LVA) could facilitate progenitor cell transplantation in patients with low PA‐CD34. Apheresis was initiated in 226 patients, disregarding PA‐CD34, at days: +5 in G‐CSF, +10 in cyclophosphamide+G‐CSF, and +15 to +20 in other chemotherapy+G‐CSF mobilization, when leucocytes >2.5 × 109/L. Four times the blood volume was processed. Patients were grouped according to their PA‐CD34: ≥10/μl (group‐A, n = 143); <10/μl but ≥5/μl (group‐B, n = 40) and <5/μl (group‐C, n = 43). No differences were found in diagnoses, gender, age, previous treatments or mobilization regimen between groups. Enough CD34+ cells (>1.9 × 106/kg) were obtained in 31 patients (72%) from group‐C, although in this group two mobilizations were needed in 20 patients (46.5%), compared to 5 (3.5%) and 1 (2.5%) in groups A and B, respectively (P < 0.01). Evenly three apheresis or more were required in 28 patients (65.1%) from group‐C, compared to 8 (5.6%) and 6 (15.0%) in groups A and B, respectively (P < 0.01). In conclusion LVA can facilitate autologous transplantation in poor‐mobilizer‐patients, low PA‐CD34 should not be an inflexible exclusion factor. J. Clin. Apheresis, 2009.

Implication of soluble and membrane HLA class I and serum IL-10 in liver graft acceptance

Membrane HLA class-I expression (mHLA-I), soluble HLA class-I antigens (sHLA-I) and interleukin (IL)-10 are different factors implicated in the special acceptance of liver allograft. In this study, pre- and post-operative levels of mHLA-I in peripheral blood lymphocytes (PBL) and serum sHLA-I were analyzed in 86 liver transplants, immunosuppressed with Cyclosporine-A, methylprednisolone and azathioprine, and classified into acute-rejection (AR, n = 28) and non-acute-rejection (NAR, n = 58) groups. Serum IL-10 was studied in 47 recipients (AR-group, n = 16 and NAR-group, n = 31). Pre-transplant values of mHLA-I and sHLA-I showed a bimodal distribution (high/low) in NAR-recipients, but in AR-patients were mainly included in the low expression/secretion zone (mHLA-I, p < 0.02 and sHLA-I, p < 0.05). Consequently, average pre-transplant mHLA-I (868 +/- 109 versus 998 +/- 123, p < 0.05) and sHLA-I (1.3 +/- 0.4 versus 2.02 +/- 0.7 microg/ml, p < 0.01) was lower in the AR- than in the NAR-group. After transplant both parameters decreased in the NAR-group, but increased in AR-recipients previous to and on rejection diagnosis day. Additionally, serum IL-10 levels were significantly higher (p < 0.01) in the NAR than in the AR-group during the first 24 h post-transplant. In conclusion, low pre-transplant mHLA-I and sHLA-I levels pre-dispose liver recipients to acute rejection, whereas early post-transplant increases of serum IL-10 appear to be related to a good liver allograft acceptance.

Natural Killer Receptors on CD8 T Cells and Natural Killer Cells from Different HLA-C Phenotypes in Melanoma Patients

Purpose: Because immune mechanisms involved in cutaneous melanoma have not been fully elucidated, efforts have been made to achieve prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful thus far. Therefore, the goal of this study was to investigate the involvement of early changes in CD8 T cells and CD56 natural killer (NK) cells expressing NK receptors in different HLA-C dimorphism groups of melanoma patients. Experimental Design: CD8 T cells and CD56 NK cells were analyzed in 41 patients and 39 sex- and age-matched controls with different HLA-C genotypes by flow cytometry. HLA-C dimorphism at position 80 was tested by PCR sequence-specific primers and PCR sequence-specific oligonucleotide to examine whether it could mediate in the emergence of cells expressing killer cell immunoglobulin-like receptors. Results: Thirty-five of 41 patients had benign sentinel node, and showed an imbalance in the absolute number of CD8+DR+ or CD8+CD161+ peripheral blood T cells according to the CD28 coexpression compared with controls. CD8+CD28−CD158a+ T and CD56+CD158a+ NK cells were significantly increased in HLA-CLys80 homozygous nonmetastatic patients, whereas only CD56+CD158a+ NK cells increased in heterozygous ones. An up-regulation of the CD158a KIR receptor was also seen on NK cells but not in T cells of patients at advanced disease stages. Conclusions: This work provides, for the first time, evidence of immune activation in early stages of cutaneous melanoma, together with an increase of cells expressing CD158a in patients bearing the corresponding HLA-C ligand, which may be important to evaluate the disease progression and to use individualized immune therapeutic approaches.