Edoardo Benedetti

Determination of the Relative Amount of Nucleic Acids and Proteins in Leukemic and Normal Lymphocytes by Means of Fourier Transform Infrared Microspectroscopy

Previous Fourier transform infrared (FT-IR) spectroscopic studies on neoplastic and normal cells have shown different band profiles and intensity associated with absorptions of proteins and nucleic acids. In the present study, an interpretation of such differences has been attempted by comparing the spectra of DNA/RNA/protein mixtures with the spectra, particularly, obtained for lymphocytes from B-chronic lymphatic leukemia (B-CLL) patients and normal donors. FT-IR microspectroscopy analysis showed a good agreement between the intensity and the band profile of the spectra of leukemic lymphocytes and those of the binary mixture made up of 75% human serum albumin and 25% DNA. The addition of small amounts of RNA (1–5%) modified the band shape, making it more similar to the spectrum of normal lymphocytes. An attempt was also made to estimate the relative amounts of DNA and RNA. The results demonstrated an increase in the DNA/RNA ratio value in neoplastic lymphocytes with respect to that reported in literature for normal ones.

Progressive multifocal leukoencephalopathy: report of three cases in HIV-negative hematological patients and review of literature

Progressive multifocal leukoencephalopathy (PML) is a central nervous system (CNS) disease usually observed in immunodeficient patients, especially human immunodeficiency virus (HIV)-positive, caused by John Cunningham virus. This infectious complication has been described in many HIV-negative hematological patients, especially affected by lymphoproliferative diseases. PML has been observed after both chemotherapy and bone marrow transplantation and, recently, in association with rituximab. Diagnosis can be complicated, and often a CNS biopsy is required. Current treatment approaches are not effective in both HIV-positive and HIV-negative patients, and the outcome remain very poor in the majority of cases, even after combination therapies. We report three cases of PML in hematological patients, treated respectively with conventional chemotherapy and autologous and haploidentical transplantation, and review the literature on PML. All of them received rituximab, which has recently been in the focus of a Food and Drug Administration warning.

Peripheral blood stem cell contamination evaluated by a highly sensitive molecular method fails to predict outcome of autotransplanted multiple myeloma patients

Summary. To evaluate the clinical impact of minimal residual disease in multiple myeloma, apheretic products from 51 autotransplanted patients were tested by fluorescent (GeneScan) polymerase chain reaction (PCR). Sixty‐nine per cent of harvests were contaminated when evaluated for IgH rearrangement. Forty‐six patients responded to transplant, with 52·9% achieving complete response (CR). The clinical response of patients was significantly influenced by the number of re‐infused CD34+ cells. Positive PCR results of re‐infused harvests were not significantly related to patient outcome. Median overall survival (OS) was 33 months, and a significant advantage for patients transplanted by 12 months from diagnosis was observed. Moreover, OS was longer for patients receiving PCR‐negative stem cells, with 72% of patients surviving to 70 months in the group receiving PCR‐negative harvests vs 48% in the group transplanted with contaminated precursors (not statistically significant). Ex vivo purging caused a reduction of contamination of up to 3 logs; nevertheless, 80% of purged harvests remained PCR‐positive and the purging procedure did not alter response or survival rates. Thus, the failure of a predictive role for this highly sensitive molecular method could be explained by the assumption that in vivo persisting malignant cells are the true source of relapse in MM.

Hyperbaric oxygen therapy in BKV-associated hemorrhagic cystitis refractory to intravenous and intravesical cidofovir: Case report and review of literature

Hemorrhagic cystitis is a common complication in hematopoietic stem cell transplant recipients. We report here a case of severe BKV-associated hemorrhagic cystitis who did not respond to intravenous cidofovir. Overt hematuria successfully resolved after a few days on hyperbaric oxygen and intravesical instillations of cidofovir, while BK viruria dropped after a few weeks and remained low. We review the literature for therapeutic options in hemorrhagic cystitis and try to explain how hyperbaric oxygen stimulates mucosal repair in the urinary bladder.

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% α-helix (amide I and III components at 1653 cm−1, and 1312 and 1284 cm−1, respectively), 40% β-sheets (amide I and III components at 1625 and 1231 cm−1, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm−1, and 1249 cm−1, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin β-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (α-helix, β-sheets, and turns) are accurate.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF THE SECONDARY STRUCTURE OF CYTOCHROME C LANGMUIR-BLODGETT FILMS

A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100 degrees C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the alpha-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O-H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility.

Evaluation of the MDR1, ABCG2, Topoisomerases IIα and GSTπ gene expression in patients affected by aggressive mantle cell lymphoma treated by the R-Hyper-CVAD regimen

The genomic profile of mantle cell lymphoma (MCL) has been reported to be significantly different from that of other indolent lymphoproliferative disorders, Topoisomerase IIα, glutathione-s-transferaseπ (GSTπ) and ABCG2 (BCRP) chemoresistance genes being over-expressed in MCL. In our study, expression levels of the above mentioned genes plus MDR1 were tested on bone marrow samples from 20 patients treated with Rituximab plus hyper-CVAD regimen, in order to evaluate a possible impact of the chemoresistance phenomenon on this promising treatment regimen. All patients expressed ABCG2 and MDR1 genes; 85% of cases expressed GSTπ and topoisomerase IIα. Only ABCG2 were over-expressed in comparison both with marrow from healthy donors and tonsilar CD5+/CD20+ lymphocytes (adopted as normal counterpart of the neoplastic population). The overall response rate of the entire series was 87.5%, with 44% of complete responses. Fifty-seven percent of patients achieved the clearance of minimal residual disease. Levels of tested genes did not condition either quality of clinical response or PFS (76% at 24 months). Nevertheless, an ABCG2 higher expression appeared associated with a worse PFS and levels of this gene paralleled the status of minimal residual disease. A further evaluation of ABCG2 expression in larger series of MCL patients would be suitable.

Panobinostat for the treatment of acute myelogenous leukemia

ABSTRACT Introduction: Therapeutic strategies in patients with acute myeloid leukemia (AML) have not changed significantly over the last decades. Appropriate strategies are ultimately driven by the assessment of patients’ fitness to define suitability for intensive induction chemotherapy, which produces high initial remission rates but, increased likelihood of relapse. Old/unfit AML patients still represent an urgent and unmet therapeutic need. Epigenetic deregulation represents a strategic characteristic of AML pathophysiology whereby aberrant gene transcription provides an advantage to leukemic cell survival. Efforts to re-establish impaired epigenetic regulation include hypomethylating agents and histone deacetylase inhibitors (HDACi). Areas covered: The review discusses the underlying mechanisms leading to disruption of lysine acetyltransferases (KAT or HAT)/deacetylase (KDAC or HDAC) balance and the rationale for using the HDACi panobinostat (LBH-589) in AML. Expert opinion: Although panobinostat has produced significant results in myeloma, its efficacy remains limited in AML. Panobinostat exerts pleiotropic activity and lack of specificity, which likely contributes to its inadequate safety in elderly AML patients. Phase I-II trials, utilizing panobinostat associated with well-known chemotherapeutic agents are ongoing and combinations with other druggable targets may likely be evaluated in future trials. The clinical use of this HDACi in AML the near future does not appearing promising.

Hypercytokinemia-induced metabolic encephalopathy in a multiple myeloma patient on hemodialysis undergoing autologous stem cell transplantation: Clinical response after plasma exchange

We report here a 50-years old female with multiple myeloma-associated chronic renal failure who underwent high-dose chemotherapy supported by autologous hematopoietic stem cell transplantation. She developed progressive encephalopathy on day 5 progressing to coma despite hemodialysis and no obvious organ failure. She finally recovered after a single 1-liter plasma exchange. The final diagnosis was metabolic encephalopathy due to hypercytokinemia, particularly high serum TNF levels. We discuss here the pathogenesis and raise an alert for monitoring cytokine levels in patients with renal failure undergoing high-dose chemotherapy.

Enhancement of hematopoietic stem cell engraftment by inhibition of CXCL12 proteolysis with sitagliptin, an oral dipeptidyl-peptidase IV inhibitor: A report in a case of delayed graft failure

Hematopoietic stem cell (HSC) transplantation fails if grafted HSCs fail to find and settle in an appropriate bone marrow niche where they can receive required trophic factors from stromal cells. Since intravenously injected HSCs are known to migrate into the bone marrow mainly in response to gradients of the CXCL12 chemokine, we reasoned that drugs that inhibit catabolism of CXCL12 could eventually increase the CXCL12 gradient and enhance engraftment. dipeptidyl peptidase IV (DPP-IV) catalyzes the first step in CXCL12 degradation, and oral DPP-IV inhibitors, such as sitagliptin, have been recently approved for type II diabetes mellitus. We report here a case of delayed graft failure in a T-cell-depleted (TCD) HSC transplant recipient, which was refractory to both retransplantation and high-dose granulocyte-colony stimulating factor (G-CSF). After beginning sitagliptin therapy our patient showed a sustained neutrophil rise and a reduction in transfusion dependence, which worsened again after drug discontinuation. Since these changes were paralleled by fluctuations in CXCL12 levels in serial peripheral blood samples, we discuss here the potential benefit of this class of drugs.