P-selectin glycoprotein ligand-1 as a potential target for humoral immunotherapy of multiple myeloma.

Monoclonal antibodies (mAbs), successfully adopted in the treatment of several haematological malignancies, have proved almost ineffective in multiple myeloma (MM), because of the lack of an appropriate antigen for targeting and killing MM cells. Here, we demonstrate that PSGL1, the major ligand of P-Selectin, a marker of plasmacytic differentiation expressed at high levels on normal and neoplastic plasma cells, may represent a novel target for mAb-mediated MM immunotherapy. The primary effectors of mAb-induced cell-death, complement-mediated lysis (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), were investigated using U266B1 and LP1 cell-lines as models. Along with immunological mechanisms, the induction of apoptosis by PSGL1 cross-linking was assessed. The anti-PSGL1 murine mAb KPL1 induced death of MM cells in a dose- and time-dependent fashion and mediated a significant amount of ADCC. KPL1 alone mediated C1q deposition on target cells but proved unable to induce CDC due to inhibition of the lytic activity of complement by membrane complement regulators (mCRP) expressed on the cell surface. Consistently, CDC was induced by KPL1 upon mCRP blockage. Our results suggest a role for PSGL1 in MM humoral immunotherapy and support further in vivo studies assessing the effects of anti-PSGL1 mAbs on MM growth and interaction with the bone marrow microenvironment.

Different immunophenotypical apoptotic profiles characterise megakaryocytes of essential thrombocythaemia and primary myelofibrosis.

AIMS: Essential thrombocythaemia (ET) and primary myelofibrosis (PMF) share some clinical and pathological features, but show different biological behaviour and prognosis. The latest contributions to understanding the nature of these disorders have focused on bone marrow microenvironment remodelling and proliferative stress, recognising megakaryocytes (MKCs) as "key-cells". The aim of this study was to investigate the apoptotic profile of ET and PMF MKCs in order to further characterise the biology of these disorders. METHODS: Bone marrow biopsy samples from 30 patients with ET, and 30 patients with PMF, were immunophenotypically studied for the expression of pro-apoptotic (Fas, Fas-L, Bax, Bad) and anti-apoptotic (Bcl-2, Bcl-XL, hTERT (human telomerase reverse transcriptase)) molecules and the "executioner" molecule caspase-3. The fraction of MKCs undergoing apoptosis was assessed by deoxynucleotidyl transferase-mediated dUTP nick-end labelling. RESULTS: Only the mitochondrial pathway seemed to be involved in MKC apoptosis. The anti-apoptotic molecule Bcl-XL was predominantly found in ET MKCs (50.5% of ET MKCs versus 35% of PMF MKCs; p = 0.036), while pro-apoptotic molecules Bax and Bad showed a prevalent expression in PMF MKCs (30.5% of ET MKCs versus 55% of PMF MKCs; 41% of ET MKCs versus 52% of PMF MKCs; p = 0.001 and p = 0.068, respectively). A significant fraction of PMF MKCs were committed to apoptosis according to caspase-3 expression and TUNEL, while only few ET cells were committed to apoptosis. hTERT was significantly more expressed in PMF (32% of ET MKCs versus 46% of PMF MKCs; p = 0.022), in agreement with the proliferative nature of this disease. CONCLUSIONS: It was found that ET and PMF MKCs, which barely differ in terms of morphology and aggregation, are characterised by markedly different apoptotic profiles. The rather high apoptotic fraction of PMF was able to support the fibrotic nature of this process, while the anti-apoptotic profile of ET cells fits well with their "steady" maturative state.

Pleomorphic adenoma of the lateral nasal wall: case report.

Pleomorphic adenoma is the most common tumour of the salivary glands. However, it is extremely rare for these to originate in the nose and even when they do so, it is most commonly in the nasal septum. It is important to be aware of the paucity of presenting symptoms (nasal obstruction and epistaxis), as the lesion may not be recognized immediately. Growth is generally restricted locally and the tumour is not known to spread to the neighbouring structures. Surgical resection is the treatment of choice. Recurrences and evolution to malignancy are not frequent, but long-term follow-up is recommended. The case is presented of pleomorphic adenoma arising from the lateral wall of the right nasal cavity, in a 34-year-old male, which was resected completely, endoscopically. Histological and immunohistochemical evaluation revealed the presence of a pleomorphic adenoma.

Immunophenotypic profile and role of adhesion molecules in splenic marginal zone lymphoma with bone marrow involvement.

Splenic Marginal Zone Lymphoma (SMZL), with or without villous lymphocytes (VL+/-), is a low-grade lymphoproliferative disorder with constant involvement of the bone marrow (BM). Different BM infiltration patterns, mainly intra-sinusoidal, interstitial and nodular, have been described. Adhesion molecules (AMs) constitute a heterogeneous group of antigenic receptors playing a major role in leukocyte recruitment, in lymphocyte homing and in cellular-mediated immune response. Evolution and pattern of the BM infiltrate could be influenced by a variable expression of AM on SMZL lymphocytes. The degree and pattern of BM infiltration and the immunohistochemical expression of AM (H-CAM, BL-CAM, L-selectin, PSGL-1, E-selectin, ICAM-1, VCAM-1 and Beta-1 integrin) among the different infiltration patterns were evaluated in BM biopsies of 38 patients with SMZL and graded according to a semi-quantitative score ranging from 0-4 and based on the percentage of positive cells. An intra-sinusoidal infiltration was constantly observed, alone or in conjunction with other patterns. H-CAM and BL-CAM showed a moderate-to-high degree of positivity in the intra-sinusoidal infiltrate (median expression grade-3) and were expressed in the neoplastic lymphocytes independently from the pattern. PSGL-1 was mostly expressed in the perisinusoidal region and in case of interstitial infiltration (grade-2). ICAM-1 and VCAM-1 were selectively expressed in the nodules as a reticular meshwork located in the core region (grade-2); VCAM-1 was also expressed in the perinodular endothelia. E-selectin, L-selectin and beta-1 integrin proved constantly negative. These data suggest that different expression of AM can influence the modality of BM infiltration in SMZL.

DNA ploidy and S-phase fraction, but not p53 or NM23-H1 expression, predict outcome in colorectal cancer patients. Result of a 5-year prospective study.

PURPOSE: The aim of this study was to determine TP53 and NM23-H1 immunoreactivity, DNA ploidy, and S-phase fraction (SPF) in a series of 160 patients undergoing resective surgery for primary operable colorectal cancer (CRC) and to establish whether these alterations have any clinical value in predicting CRC patients' prognosis. METHODS: TP53 and NM23-H1 expressions were evaluated on paraffin-embedded tissue by immunohistochemistry and DNA-ploidy and SPF on frozen tissue by flow-cytometric analysis. RESULTS: The median follow-up time in our study group was 71 months (range 34-115 months). P53 protein expression was associated with distal tumors (P<0.05) and DNA aneuploid tumors (P<0.05) tumors. DNA-aneuploidy was associated with distal tumors (P<0.01), histological grade (G3) (P<0.05), advanced Dukes' stage (C and D) (P<0.01), lymph node metastases (P<0.01) and high SPF (>18.3%) (P<0.01). The major significant predictors for both disease relapse and death were advanced Dukes' stage, DNA-aneuploidy, and high SPF, while lymphohematic invasion was the only independent factor for relapse and non-curative resection for death. CONCLUSIONS: Our results indicate that DNA aneuploidy and high SPF are associated in CRC with a poor clinical 5-year outcome, while in contrast the prognostic role of TP53 and NM23-H1 expression is still to be clarified.

Risk of HIV and other blood-borne infections in the cardiac setting: patient-to-provider and provider-to-patient transmission.

Health care workers (HCWs) face a well-recognized risk of acquiring blood-borne pathogens in their workplace, in particular hepatitis B and C viruses (HBV/HBC) and human immunodeficiency virus (HIV). Additionally, infected HCWs performing invasive exposure-prone procedures, including in the cardiac setting, represent a potential risk for patients. An increasing number of infected persons could need specific cardiac diagnostic procedures and surgical treatment in the future, regardless of their sex or age. The risk of acquiring HIV, HCV, HBV infection after a single at-risk exposure averages 0.5%, and 1-2%, and 4-30%, respectively. The frequency of percutaneous exposure ranges from 1 to 15 per 100 surgical interventions, with cardiothoracic surgery reporting the highest rates of exposures; mucocutaneous contamination by blood-splash occurs in 50% of cardiothoracic operations. In the Italian Surveillance (SIROH), a total of 987 percutaneous and 255 mucocutaneous exposures were reported in the cardiac setting; most occurred in cardiology units (46%), and in cardiovascular surgery (44%). Overall, 257 source patients were anti-HCV+, 54 HBsAg+, and 14 HIV+. No seroconversions were observed. In the literature, 14 outbreaks were reported documenting transmission of HBV from 12 infected HCWs to 107 patients, and 2 cases of HCV to 6 patients, during cardiothoracic surgery, especially related to sternotomy and its suturing. The transmission rate was estimated to be 5% to 13% for HBV, and 0.36% to 2.25% for HCV. Strategies in risk reduction include adequate surveillance, education, effective sharps disposal, personal protective equipment, safety devices, and innovative technology-based intraoperative procedures.

Isolation and characterization of two cDNA clones coding for isoforms of the Parietaria judaica major allergen Par j 1.0101.

Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a lambda ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodies from a pool of sera (n = 23) of patients allergic to Pj. Sequence analysis showed open reading frames of 176 and 138 amino acids. Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D. These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported. The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology. Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101. The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen.

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10-14 kDa native allergens and preincubation of sera from Pj-allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region.

Isolation of proteins and nucleic acids by electrophoresis on disposable gel columns.

A simple and cheap one-step method to isolate proteins or nucleic acids by electrophoresis in disposable gel columns is reported. A disposable syringe was modified to host a gel column and an elution chamber. Starting from a crude extract of E. coli, the laboratory-made devise allowed the isolation of the maltose binding protein (MBP) fused to a recombinant allergenic molecule with a molecular mass of 58 kDa, from a mixture of several proteins. Also, plasmid DNA could be isolated from a mixture containing chromosomal DNA and RNA, avoiding the use of organic solvents. Electrophoresis was performed at 150 V, 35 degrees C, pH 8.0 and 8.3 for protein and DNA, respectively. The protein or the DNA obtained showed a yield of 80% and a purity grade of 90%, as estimated by densitometry.