Inflammatory cell burden and phenotype in endomyocardial biopsies with antibody-mediated rejection (AMR): a multicenter pilot study from the AECVP.

This multicenter case-controlled pilot study evaluated myocardial inflammatory burden (IB) and phenotype in endomyocardial biopsies (EMBs) with and without pathologic antibody-mediated rejection (pAMR). Sixty-five EMBs from five European heart transplant centers were centrally reviewed as positive (grade 2, n = 28), suspicious (grade 1, n = 7) or negative (n = 30) for pAMR. Absolute counts of total, intravascular (IV) and extravascular (EV) immunophenotyped mononuclear cells were correlated with pAMR grade, capillary C4d deposition, donor specific antibody (DSA) status and acute cellular rejection (ACR). In pAMR+ biopsies, equivalent number of IV CD3+ T lymphocytes (23 ± 4/0.225 mm(2) ) and CD68+ macrophages (21 ± 4/0.225 mm(2) ) were seen. IB and cell phenotype correlated with pAMR grade, C4d positivity and DSA positivity (p < 0.0001). High numbers of IV T lymphocytes were associated with low grade ACR (p = 0.002). In late-occurring AMR EV plasma cells occurring in 34% of pAMR+ EMBs were associated with higher IB. The IB in AMR correlated with pAMR+, C4d positivity and DSA positivity. In pAMR+ equivalent numbers of IV T lymphocytes and macrophages were found. The presence of plasma cells was associated with a higher IB and occurrence of pAMR late after transplantation.

A new HLA allele, HLA-B*08:122, described in an unrelated donor of Caucasian origin.

A new human leukocyte antigen-B allele was found in an unrelated Italian donor.

The distribution of KIR-HLA functional blocks is different from north to south of Italy.

The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells. Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival. We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification. We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A. We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy. By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0.012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 0.0003) and the Bw4 Ile80 epitope (P = 0.0005). The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -0.1211), possibly as a consequence of their similar function in inhibiting NK cells. The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 0.0067) and with HLA-B Bw4 Ile80 (P = 0.0027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 0.0044). Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections. Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate HLA-A and HLA-B ligands are selectively pressured in Italy according to geographical north-to-south distribution.

Identification of a novel HLA-C*08 variant allele, C*08:31. Sequence analysis from exons 1 through 8.

A novel allele, HLA- C*08:31 has been identified by sequence based typing in an Italian hematological patient undergoing bone marrow transplantation.

Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population.

BACKGROUND: Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 217 unrelated healthy Italian individuals from 22 immunogenetics laboratories, located in the northern, central and southern regions of Italy. METHODS: Two hundred and seventeen DNA samples were studied by a low resolution PCR-SSP kit designed to identify all KIR genes. RESULTS: All 17 KIR genes were observed in the population with different frequencies than other Caucasian and non-Caucasian populations; framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. Sixty-five different profiles were found in this Italian population study. Haplotype A remains the most prevalent and genotype 1, with a frequency of 28.5%, is the most commonly observed in the Italian population. CONCLUSION: The Italian Caucasian population shows polymorphism of the KIR gene family like other Caucasian and non-Caucasian populations. Although 64 genotypes have been observed, genotype 1 remains the most frequent as already observed in other populations. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation.

In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.

Enhancement of experimental metastatic ability by tumor necrosis factor-alpha alone or in combination with interferon-gamma.

The effects of recombinant tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on B16 mouse melanoma experimental metastatic ability and major histocompatibility complex (H-2b) antigens expression were studied. B16 cells exposed in vitro to TNF-alpha had an increased H-2 expression and were more metastatic than untreated cells. The simultaneous treatment with TNF-alpha and IFN-gamma amplified the enhancement of experimental metastasis and all other effects obtained with TNF-alpha alone. The B16 clone B78H1, selectively resistant to H-2 induction and to enhancement of metastatic ability by IFN-gamma, was not affected by treatment with TNF-alpha and with TNF-alpha + IFN-gamma. These findings contribute to a better understanding of the pleiotropic effects of TNF, some of which can have opposing actions in the complex tumor-host relationships.

A sensitive test for the detection of NK activity. Plasma clot clonogenic assay.

Natural killer (NK) activity, which has been implicated in the immune response against viral infections and neoplasias, is currently evaluated by means of a chromium (51Cr) release assay. However, criticisms have been raised with regard to the reliability and reproducibility of the test. We have developed a different in vitro method for measuring NK activity, based on the inhibition of the target clone growth in plasma clot semisolid medium. This method overcomes the limitations inherent to the 51Cr release test and more closely mimics the in vivo situation. The inhibitory activity revealed by the cloning assay was always greater than the lytic activity in the 51Cr release assay. Moreover, effector/target ratios of 3:1 and 1.5:1 still produced clonal inhibition. B-CLL cells, used as effectors, showed no inhibitory activity and the Raji cell line employed as target was resistant in both techniques. Thus, the clonogenic assay appears to be more sensitive for the evaluation of low levels of NK activity, for basic studies on the effector/target interactions, for the evaluation of LAK cell activity, and in diseases in which an involvement of the NK compartment has been hypothesized.

Reduced incidence of GVHD without increase in relapse with low-dose rabbit ATG in the preparative regimen for unrelated bone marrow transplants in CML.

SUMMARY: Antithymocyte globulin (ATG) treatment prevents graft failure and results in a low incidence of GVHD, but an increased risk of relapse could be expected as a consequence of reduced GVHD. From September 1995 to June 2001, 28 consecutive chronic myeloid leukemia (CML) patients underwent unrelated bone marrow transplants: 21 were in chronic phase (CP) and seven in advanced phase (AP). Median age was 35.5 years (range 20-50). HLA typing was based on high-resolution molecular techniques; in eight cases there were one or more allele mismatches. The preparative regimen consisted of TBI, EDX 120 mg/kg and rabbit ATG 15 mg/kg. All patients engrafted and no rejection occurred. Acute GVHD grade III-IV occurred in six patients (21%). Chronic GVHD occurred in 10 (40%) and it was extensive in one. Four out of seven patients transplanted in AP had a hematological relapse. Of 21 in CP, there was one cytogenetic and one molecular relapse: these two patients are now in complete remission with imatinib mesylate. With a median follow-up of 45.7 months, the 5-year survival is 76.2% for those transplanted in CP. These data demonstrate that transplants performed in CP, with low-dose ATG, are associated with a good outcome, low incidence of GVHD and no increase of relapse.

Comparison of the DNA Content, Bromodeoxyuridine Incorporation and Ki-67 Antigen Expression in Human Acute Myeloid Leukemia.

The cell kinetics of twenty-two acute myeloid leukemias (AML) were investigated by means of flow cytometry evaluating the S-phase DNA content, bromodeoxyuridine labelling index (BrdUrd L.I.) and Ki-67 antigen expression. Eight patients showed a good correlation between the DNA content and BrdUrd L.I., while nine gave rise to divergent results. In the remaining five patients the S-phase DNA content could not be evaluated due to the presence of an additional aneuploid population. The Ki-67 antigen expression defined the extent of the growth fraction in all cases and allowed for better characterization of the cell cycle. These results suggest that the three methods explore only partly overlapping events; thus, it seems that a reliable picture of the cell kinetics in leukemic populations can only be achieved by combining all these methods.

Synergism between gamma interferon and doxorubicin in a human MDR colon adenocarcinoma cell line.

Recent interest in cancer therapy derives from the ability of interferons to synergistically increase the activity of chemotherapeutic agents. To understand the biological basis of this synergism we evaluated the effects of human recombinant IFN-gamma on the expression of the mdr1 gene and on the cellular growth of a human colon adenocarcinoma cell line (LoVo) and its MDR subline (LoVo/Dx) after coincubation with doxorubicin. Treatment with IFN-gamma showed unchanged levels of MDR1-glycoprotein, no perturbation on cell cycle distribution and a significant reduction of colony formation in both lines (P < 0.05) starting from 100 U/ml. A synergistic effect was observed in the LoVo/Dx cell line when doxorubicin was added after exposure to 0.1-10 U/ml of IFN-gamma. Our data indicate that the effects of IFN-gamma, independent from action on cell proliferation and from modulation of p-glycoprotein expression, are a cause of the synergistic activity between this lymphokine and conventional chemotherapeutic agents such as doxorubicin.

Intensification of GVHD prophylaxis with low-dose ATG-F before allogeneic PBSC transplantation from HLA-identical siblings in adult patients with hematological malignancies: results from a retrospective analysis.

Several studies have shown that chronic GVHD (cGVHD) is more frequent in patients receiving transplants from PBSC than in those receiving BM. In the setting of PBSC-unrelated transplants, the addition of anti-T-cell globulin (ATG) has shown a significant decrease in incidence/severity of cGVHD, without an increase in relapses or infections. However, no prospective data are yet available in the sibling setting. We retrospectively analyzed the effects of intensification of standard GVHD prophylaxis (CsA+MTX) by the addition of low-dose ATG in 245 patients receiving a transplant from HLA-identical sibling. From 1996 to 2001, patients received PBSC as the preferred source (group 2), and then ATG was added before transplant (group 3) because of a high cGVHD rate. Patients receiving BM in the same time period were analyzed as a control group (group 1). The incidence of grade III-IV acute GVHD and cGVHD was not significantly different in the three groups, but extensive cGVHD was highest in group 2 (38%) compared with group 3 (21%) or group 1 (28%; P=0.03). OS, TRM and time to relapse/progression were similar in the three groups. Our analysis shows that adding ATG to PBSC sibling allogeneic transplants can lower cGVHD, without an increase of relapse. Further prospective studies are needed to confirm these findings.

High incidence of allograft dysfunction in liver transplanted patients treated with pegylated-interferon alpha-2b and ribavirin for hepatitis C recurrence: possible de novo autoimmune hepatitis?

BACKGROUND: Interferon may trigger autoimmune disorders, including autoimmune hepatitis, in immunocompetent patients. To date, no such disorders have been described in liver transplanted patients. METHODS: 9 of 44 liver transplanted patients who had been receiving pegylated-interferon alpha-2b and ribavirin for at least 6 months for hepatitis C virus (HCV) recurrence, developed graft dysfunction despite on-treatment HCV-RNA clearance in all but one case. Laboratory, microbiological, imaging and histological evaluations were performed to identify the origin of graft dysfunction. The International Autoimmune Hepatitis scoring system was also applied. RESULTS: In all cases infections, anastomoses complications and rejection were excluded, whereas the autoimmune hepatitis score suggested a "probable autoimmune hepatitis" (score from 10 to 14). Three patients developed other definite autoimmune disorders (overlap anti-mitochondrial antibodies (AMA)-positive cholangitis, autoimmune thyroiditis and systemic lupus erythematosus, respectively). In all cases, pre-existing autoimmune hepatitis was excluded. Anti-lymphocyte antibodies in immunosuppressive induction treatment correlated with the development of the disorder, whereas the use of granulocyte colony-stimulating factor to treat interferon-induced neutropenia showed a protective role. Withdrawal of antiviral treatment and treatment with prednisone resulted in different outcomes (five remissions and four graft failures with two deaths). CONCLUSIONS: De novo autoimmune hepatitis should be considered in differential diagnosis along with rejection in liver transplanted patients developing graft dysfunction while on treatment with interferon.

Febrile nonhaemolytic transfusion reaction caused by antibodies against human platelet antigen 5a.

Anti-human platelet antigens (HPA) alloantibodies are seldom involved in febrile nonhaemolytic reactions (FNHTRs). We describe a case in which anti-HPA-5a alloantibodies are related to an FNHTR. We studied the specificity of the alloantibodies by flow cytometry, ELISA and MACE. Typing of donors and the patient was performed by sequence-specific polymerase chain reaction. The alloantibodies were found reactive with HPA-5a antigens. The patient was HPA-5b/b, whereas the donor of the platelet apheresis involved in the FNHTR was HPA-5a/a. Despite the low frequency of anti-HPA-5a antibodies, they might be responsible for FNHTR.

A new tool in white blood cell reduction for packed red cells: 5 log depletion.

The recent development of new filters used for leucocyte reduction aims at restricting the number of leucocytes to a threshold where their undesirable effects can be minimized or excluded. In this paper we describe the performance of a new filter named BIO R01 MAX and claimed by the manufacturer to perform 5 Log10 depletion. The results show that the efficiency of the filter reached 5 Log10 depletion and the absolute number of white blood cells in the post-filtration units is always less than 2 x 10(4) with considerable safety in the prevention of transfusion reactions.

Process control of filtered red blood cells: which counting method?

Various counting methods have been described and reported for process control of leuco-epleted blood components. The recent production of high-efficiency leucocyte removal filters intensifies the need for sensitivity in determining the ever lower residual concentration of white cells (WBCs) in filtered units. In order to assess which method was the most efficient and feasible in the laboratory for the control of WBC-reduced packed red blood cells, we compared the sensitivity of four counting methods: Nageotte chamber analysis, flow cytometry, the fluorochrome method by Borzini and Nageotte chamber analysis as modified by Prati. We observed a difference in the post-filtration WBC content depending on which method of counting was used and we feel it reasonable to ask what method should be employed in blood component process control. The answer must naturally consider that the method is for use by a large number of laboratories, while the sensitivity of the method needs to be appropriate to the goal desired.

Flow cytometry: a tool in immunohematology for D+W (Du) antigen evaluation?

We performed a flow-cytometric analysis of the expression of D antigen in D+W samples (previously termed Du). We also analysed a series of D-positive and D-negative (cde phenotype) samples to obtain positive and negative controls, respectively. The evaluation was carried out by immunofluorescence and the intensity of positivity was expressed as mean channel value (MCV) of fluorescence. Results demonstrated that D+W samples have lower expression (less than 1 log) than D-positive cases (p > 0.001, Student t test), while cde samples show the same MCVs as negative controls. Moreover, it was also possible to set a grading of D antigen expression and to analyze cases difficult to assess by agglutination only.

Comparative analysis of six different white cell-reduction filters for packed red cells.

BACKGROUND: The reduction of white cells in blood components before transfusion by filters with at least 3 log10 depletion may prevent adverse transfusion reactions such as HLA alloimmunization, febrile reactions, transmitted infections, and immunomodulation. A new generation of filters with 4 log10 depletion is now available. STUDY DESIGN AND METHODS: The aim of this study is to compare the efficiency of white cell reduction by six commercial filters for packed red cells with 3 and 4 log10 depletion (claimed by manufacturers). The analysis of white cell concentration in the white cell-reduced units was performed by flow cytometry and with a Nageotte chamber. RESULTS: The last generation of filters (BPF4, RC400, R01 Plus) show mean residual white cell numbers of 0.18 +/- 0.14, 0.26 +/- 0.21, and 0.25 +/- 0.15 x 10(6), respectively, by flow cytometric analysis and 0.05 +/- 0.04, 0.18 +/- 0.15, and 0.38 +/- 0.23 x 10(6), respectively, by Nageotte chamber evaluation. The 3 log10 depletion filters (R01, Leucostop-4LT-mono, R200) have mean residual white cell numbers of 1.41 +/- 0.92, 2.4 +/- 1.99, and 1.05 +/- 0.64 x 10(6), respectively, by flow cytometric analysis and 3.56 +/- 1.7, 1.67 +/- 1.3, and 3.21 +/- 4.1 x 10(6), respectively, by Nageotte chamber evaluation. The data show that the BPF4, RC400, and R01 Plus filters are likely to be more efficient by 1 log10 reduction than the R01, Leucostop-4LT-mono, and R200 filters. CONCLUSION: The most recent generation of filters is able to deplete white cells from packed red cells by 4 log10; in particular, with one of the filters, the residual WBC content was less than 0.5 x 10(6) per unit in all experiments, while two other filters reached that level in 9 of 10 experiments.

In situ immunocytochemical staining of cell colonies growing in plasma clot.

The procedures involving the growth of cell colonies in semi-solid media, such as methyl cellulose or agar, provide a score of colony-forming-units (CFUs) by means of morphology, and allow the application of cytochemistry. However, a better characterization of the growing cells by employing monoclonal antibodies is impaired by the medium itself. Plasma clot is a possible alternative, allowing immunofluorescence as well as immunoenzymatic techniques. We have developed a staining procedure which can be performed using both peroxidase- or alkaline phosphatase-conjugated reagents; the colonies, growing in plasma clot, can be stained in situ, without transferring the cells. In this paper we report on the study of six different cell lines stained by immunocytochemical techniques with appropriate monoclonal antibodies.

Flow cytometry immunophenotyping and polymerase chain reaction-site-specific primers genotyping for HPA-1 alloantigens in an Italian blood donor population.

BACKGROUND AND OBJECTIVES: There is increasing interest in the development of rapid and reliable techniques for human platelet alloantigen (HPA) typing. This study investigates the reliability of flow cytometry for large-scale immunophenotyping of platelet alloantigens. MATERIALS AND METHODS: We used flow cytometry and polymerase chain reaction-site-specific primer (PCR-SSP) for the characterization of the human platelet antigen 1 (HPA-1) mosaic in blood donors. RESULTS: By using specific alloantisera and immunofluorescence labelling 9 (2.6%) out of 351 samples were HPA-1a-negative. To confirm this antigenic phenotype, all of the latter samples were submitted to PCR-SSP analysis, showing an HPA1-b/b genomic pattern. In HPA-1a-positive donors, flow cytometry was unable to distinguish HPA-1a/b heterozygous from HPA-1a/a homozygous subjects who were clearly identified by genotyping. CONCLUSIONS: Flow cytometry is a valuable tool for large-scale screening to identify HPA-1a-negative persons, whereas genotyping is the assay of choice for zygosity testing, antenatal diagnosis, and for thrombocytopenic alloimmunized patients.