Giovanni Carulli

Lithium and hematology: established and proposed uses

Lithium (as lithium carbonate) is an unexpensive drug, widely used in psychiatry for over 50 years in treatment of mood instability (bipolar disorder) and as an adjunct to antidepressants. Hematological effects of neutrophilia and increased circulating CD34+ cells of marrow origin have long been known. Lithium was at the center of hematological investigations in the 1980s, but no definitive use in hematology has yet emerged. We review evidence that lithium increases G‐CSF and augments G‐CSF effects. We suggest possible therapeutic uses of lithium in neutropenia. In bone marrow transplantation, preharvest lithium‐assisted hematopoietic stem cell mobilization may be useful as well.

Multiparameter immunophenotyping by flow cytometry in multiple myeloma: The diagnostic utility of defining ranges of normal antigenic expression in comparison to histology

Numerous studies have reported on the immunophenotype of plasma cells (PCs) in monoclonal gammopathy of undetermined significance (MGUS) and in plasma cell myeloma (PCM), but very few have examined the immunophenotype of normal PCs. In this study, an objective definition of normal range of expression for each antigen was found on normal control PCs. Using these new ranges of normal expression (new method) is different from using a static 20% of PCs cut‐off for all antigens as described in the literature (traditional method). These newly calculated normal ranges for each antigen were applied to our data, and compared to histologic and immunohistochemical findings.

Detection of Eosinophils in Whole Blood Samples by Flow Cytometry

A flow cytometric method to detect and study human eosinophils in whole blood was established. Normal subjects and patients with various types of eosinophilia (hypereosinophilic syndromes, allergic diseases, dermatitis, Hodgkins Disease, parasitosis) were studied. Whole blood samples were treated for 10 minutes at room temperature with a commercially available reagent (FACS Lysing Solution, Becton Dickinson) which acts both as a fixative and as a lysing agent. Eosinophils were identified as a granulocytic subpopulation with higher SSC and FSC properties. This cell population was characterized by evident autofluorescence and hypodiploid DNA features after propidium iodide staining. The purity of the eosinophil population sorted after electronic gating was close to 100%. A very significant correlation between eosinophil counting by our whole blood method and other two assays, namely routine automatic counting by the H*3 Bayer System and eosinophil detection by depolarized SSC, was obtained. The phagocytic properties of eosinophils were also studied by means of a commercially available diagnostic kit, thus demonstrating that our method is also suitable for the study of those granulocytic functions which can be evaluated by flow cytometry.

Aberrant expression of CD8 in B-cell non-Hodgkin lymphoma: a multicenter study of 951 bone marrow samples with lymphomatous infiltration.

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

Background Although cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation. Methodology/Principal Findings A variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca2+]i raise through the mobilization of intracellular Ca2+ stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients. Conclusions/Significance These results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca2+]i as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations.

In vivo Effectiveness of Lithium on Impaired Neutrophil Chemotaxis in Shwachman-Diamond Syndrome

The effect of lithium treatment on the impaired neutrophil chemotactic function of a patient affected by Shwachman-Diamond syndrome is reported. We found that (1) a cytoskeletal cellular defect seems to be involved in the impairment of neutrophil function (and perhaps of cellular secretion and chondrocyte function) in the syndrome; (2) intermittent neutropenia is always present in the syndrome, and (3) lithium seems capable, in addition to its capacity of inducing leukocytosis, of modulating leukocyte functions by modulating the microtubular system. The drug, at usual therapeutic dosage, was able to normalize neutrophil functions without side effects. As no therapy is available in this syndrome to date, our data suggest the therapeutic use of lithium in order to improve these cytoskeleton-mediated functions and the degree of neutropenia.

Motility of rhG‐CSF‐induced neutrophils in patients undergoing chemotherapy: evidence for inhibition detected by image analysis

The motility of circulating neutrophils from seven patients affected by intermediate and high‐grade non‐Hodgkins lymphoma was investigated before and after rhG‐CSF administration (5μg/kg/d for 5 d subcutaneously) in the course of chemotherapy. Random motility and bacterial lipopolysaccharide‐induced chemotaxis were studied by the micropore filter technique in a Boyden chamber. These functions were evaluated by a very sensitive technique, based on a computer‐assisted image processing system, capable of giving several parameters about the kinetics of cell migration. Along with a significant increase in neutrophil number, a significant decrease both in random and stimulated motility was found. The kinetics of cell migration showed that the cells maintained the typical gaussian pattern of random motility. On the contrary, neutrophils were found to have lost the typical stimulated migration peak. These findings are consistent with a rhG‐CSF‐induced impairment of the directional movement, rather than of the ability of moving at random. These effects were found in patients who, in the same experimental conditions, had displayed an enhanced phagocytosis and phagocytosis‐associated chemiluminescence along with an enhanced CD32 expression, not due to an aspecific cell manipulation. Two hypotheses may be taken into account: (i) an increased adhesiveness due to a direct or an indirect activity of the cytokine; (ii) an abnormality in the cytoskeleton maturation and/or rearrangement during the accelerated bone marrow transit of myeloid cells. These findings emphasize that rh‐GCSF administration can modulate several functions which play an important role in host defence, and suggest the utility of carrying out further studies to investigate the optimum dosage both to correct neutrophil number and preserve neutrophil functional activities.

FcRIII (CD16) expression on neutrophils from chronic myeloid leukemia. A flow cytometric study

FcRIII (CD16) expression on neutrophils from 17 patients with chronic myeloid leukemia (CML) was studied by flow cytometry using monoclonal antibodies. A variable proportion of CD16-negative neutrophils were found both in CML patients in chronic phase (3 out of 8 patients) and in CML patients in hematological remission (3 out of 9 patients). Neutrophils with reduced FcRIII expression showed more defective chemiluminescence and phagocytosis than neutrophils with normal FcRIII expression. Circulating myeloid cells from three patients in chronic phase, showing a normal percentage of CD16-positive neutrophils, were isolated and fractionated by discontinuous Percoll gradients. This study showed that CD16 appears at the stage of metamyelocyte, that band cells and segmented neutrophils display an identical pattern of membrane FcRIII, and that the fluorescence intensity shown by metamyelocytes is different from that displayed by more mature cells. The association between low FcRIII expression and function abnormality could be suggestive of a defect in CML neutrophil maturation.

The Role of CD19 and CD27 in the Diagnosis of Multiple Myeloma by Flow Cytometry: A New Statistical Model

We have developed a new statistical diagnostic model that examines the correlation between immunophenotype and clonality as detected by flow cytometry (FC) and histology, defining the diagnostic role of FC in multiple myeloma (MM). The 192 bone marrow samples from patients and control subjects were studied for routine diagnostic analysis of MM; a minimum of 100 plasma cells (PCs) were analyzed for each patient sample. A direct 7- or 8-color method was applied to study the immunophenotype of PCs, utilizing a FACSCanto II (BD Biosciences, San Jose, CA). Samples were labeled with fluorochrome-conjugated monoclonal antibodies (AmCyan, Pac Blue, fluorescein isothiocyanate, phycoerythrin [PE], PECy7, peridinin-chlorophyll protein, allophycocyanin [APC], and APC-Cy7) to the following antigens: CD138, CD81, CD200, CD221, CD45, CD38, CD28, CD19, CD27, CD117, CD38, CD33, CD20, CD56, CD10, and immunoglobulin κ and λ light chains. Among all antigens tested, CD19 and CD27, when applied to our model, resulted in optimal concordance with histology. This model defines the effective diagnostic role FC could have in MM and in the detection of minimal residual disease.

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis

A high expression of Wilms’ tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon.