Giuseppe Claudio Viscomi

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients.

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.

Rifaximin: beyond the traditional antibiotic activity.

Rifaximin is a non-systemic oral antibiotic derived from rifampin and characterized by a broad spectrum of antibacterial activity against Gram-positive and -negative, aerobic and anaerobic bacteria. Rifaximin was first approved in Italy in 1987 and afterwards in many other worldwide countries for the treatment of several gastrointestinal diseases. This review updates the pharmacology and pharmacodynamics of rifaximin highlighting the different actions, beyond its antibacterial activity, such as alteration of virulence, prevention of gut mucosal adherence and bacterial translocation. Moreover, rifaximin exerts some anti-inflammatory effects with only a minimal effect on the overall composition of the gut microbiota. All these properties make rifaximin a good candidate to treat various gastrointestinal diseases.

Structure-activity of type I interferons

Type I IFNs constitute a family of proteins exhibiting high homology in primary, secondary, and tertiary structures. They interact with the same receptor and transmit signals to cellular nucleus through a similar mechanism, eliciting roughly homogeneous biological activity. Nevertheless, the members of that family, IFNα species, IFNβ and IFNω, due to local differences in the structure sometime show distinct properties. From the reported data it results that even minute changes or differences in the primary sequences could be responsible for a significant variety of biological actions, thus inducing to the hypothesis that Type I IFNs, rather than to be the result of a redundant replication during the evolution, play definite roles in the defense of living organisms to foreign agents.

Is generic rifaximin still a poorly absorbed antibiotic? A comparison of branded and generic formulations in healthy volunteers

Rifaximin is an antibiotic, locally acting in the gastrointestinal tract, which may exist in different crystal as well as amorphous forms. The branded rifaximin formulation contains the polymorph rifaximin-α, whose systemic bioavailability is very limited. This study was performed to compare the pharmacokinetics of this formulation with that of a generic product, whose composition in terms of solid state forms of the active pharmaceutical ingredient was found to be different. Two tablets (2×200mg) of branded and generic formulations were given to 24 healthy volunteers of either sex, according to a single-blind, randomized, two-treatment, single-dose, two-period, cross-over design. Plasma and urinary samples were collected at preset times (for 24h or 48h, respectively) after dosing, and assayed for rifaximin concentrations by high-performance liquid chromatography-mass spectrometry. Rifaximin plasma and urine concentration-time profiles showed relevant differences when generic and branded rifaximin were compared. Most pharmacokinetic parameters were significantly higher after administration of generic rifaximin than after rifaximin-α. In particular, the differences for Cmax, AUC and cumulative urinary excretion between the generic formulation and the branded product ranged from 165% to 345%. The few adverse events recorded were not serious and not related to study medications. The results of the present investigation demonstrate different systemic bioavailability of generic and branded formulations of rifaximin. As a consequence, the therapeutic results obtained with rifaximin-α should not be translated sic et simpliciter to the generic formulations of rifaximin, which do not claim containing only rifaximin-α and will display significantly higher systemic absorption in both health and disease.

The structure–property relationship of four crystal forms of rifaximin

The crystal structures of four crystal forms of rifaximin, named α, β, δ and e, have been elucidated by a combination of single crystal and powder diffraction experiments with synchrotron and X-ray sources. The crystal structures of the δ and e forms have been determined from powder diffraction data alone. Hot stage microscopy has also been of paramount importance in the understanding of the β → α conversion process.

Evaluation of the effects of human leukocyte IFN-α on the immune response to the HBV vaccine in healthy unvaccinated individuals

HBV vaccine needs 3 injections over 6 months to induce immunity. Thus, the use of adjuvants capable of inducing earlier immune protection would be highly desirable. Most adjuvants may act by inducing cytokines, and among them, type I interferons (IFNs), deserve a special attention in view of the potent immunomostimulatory activity observed in mouse models and on dendritic cell functions. The aim of the present trial was to evaluate the effects of IFN-alpha administered as an adjuvant of HBV vaccine in healthy unvaccinated individuals. No significant enhancing effect on the antibody response was observed, in spite of an early and transient upregulation of costimulatory molecule expression on peripheral blood mononuclear cells, which may be suggestive of an IFN-mediated activation of antigen presenting cells. We conclude that, under the conditions used in this trial, natural IFN-alpha does not act as an adjuvant of the HBV vaccine in healthy unvaccinated individuals.

Impact of crystal polymorphism on the systemic bioavailability of rifaximin, an antibiotic acting locally in the gastrointestinal tract, in healthy volunteers

Background Rifaximin is an antibiotic, acting locally in the gastrointestinal tract, which may exist in different crystal as well as amorphous forms. The current marketed rifaximin formulation contains polymorph alpha, the systemic bioavailability of which is very limited. This study compared the pharmacokinetics of this formulation with those of the amorphous form. Methods Amorphous rifaximin was specifically prepared for the study and formulated as the marketed product. Two doses (200 mg and 400 mg) of both formulations were given to two groups of 12 healthy volunteers of either sex according to a single-blind, randomized, two-treatment, single-dose, two-period, cross-over design. Plasma and urine samples were collected at preset times (for 24 hours or 48 hours, respectively) after dosing, and assayed for rifaximin concentrations by high-performance liquid chromatography-mass spectrometry. Results For both dose levels, peak plasma concentration, area under the concentration-time curve, and cumulative urinary excretion were significantly higher after administration of amorphous rifaximin than rifaximin-α. Ninety percent confidence intervals for peak plasma concentration, area under the concentration-time curve, and urinary excretion ratios were largely outside the upper limit of the accepted (0.80–1.25) range, indicating higher systemic bioavailability of the amorphous rifaximin. The few adverse events recorded were not serious and not related to the study medications. Conclusion Rifaximin-α, a crystal polymorph, does differ from the amorphous form, the latter being systemically more bioavailable. In this regard, care must be taken when using – as a medicinal product – a formulation containing even small amounts of amorphous form, which may alter the peculiar pharmacologic properties of this poorly absorbed antibiotic.

Antigenic characterization of recombinant, lymphoblastoid, and leukocyte IFN-alpha by monoclonal antibodies.

To gain more insight into similarities of different interferon-alpha (IFN-alpha) species, we evaluated neutralization and immunoactivity of a variety of IFN preparations with various monoclonal antibodies (IFN-alpha mAb). Nine IFN-alpha mAb obtained through immunization with recombinant IFN-alpha (rmAb), lymphoblastoid IFN-alpha (LY mAb), and leukocyte IFN-alpha (LE mAb) were tested. The IFN-alpha mAb were evaluated for their ability to neutralize the antiviral activity of 11 recombinant IFN-alpha subtypes, two recombinant IFN-alpha hybrids, and lymphoblastoid and leukocyte IFN-alpha preparations. The same IFN-alpha mAb were also used in immunoblotting, and some of them were used in immunoaffinity chromatography. The results of the neutralization assay reveal that the IFN-alpha mAb significantly differ in their ability to neutralize the individual IFN-alpha species. Interestingly, none of the IFN-alpha mAb was able to neutralize all the IFN-alpha species. In particular, rmAb were unable to neutralize LE-IFN-alpha or LY-IFN-alpha, whereas LE mAb and LY mAb efficiently neutralized rIFN-alpha2. In some cases, the epitopes to which IFN-alpha mAb are directed were identified through the use of synthetic fragments of IFN-alpha2 or by evaluating the selectivity in binding to IFN-alpha subtypes.

Human Serum in Leukocyte Cultures Producing Human Interferon Alpha

Interferon Alpha (IFNα) is an antiviral, antiproliferative and immunostimulant cytokine world-wide applied in the treatment of several viral and oncologic diseases. Among the differentpreparations of IFNα, namely leukocyte IFNα (LE-IFα), lymphoblastoid IFNα and recombinant IFNα LE-IFNα is particularly relevant, since it is better tolerated and it does not induce any antigenic reaction to the administred drug. LE- IFNα is produced through the induction by viruses, such as Sendai virus, of human purified leukocyte cultures, whose media contain human agamma serum (HAS). Any attempt to omit the use of HAS caused poor yield of LE-IFNα Therefore, the role of HAS was investigated testing different preparations, performing dose (HAS concentration)/response (IFNα yield); curves, and evaluating the importance of the presence of HAS during the time of the culture. No differences in LE-IFnα production were detected based on the different HAS preparations. It was evident the dependence of LE-IFNα yield on the HAS concentration, even if some differences could be detected between different preparations. Optimal amount of HAS was depended on the cell concentrations. Reduction of HAS concentration was possible only after having started the culture at the optimized conditions. It could be concluded that an optimal concentration of HAS per cell can be defined and the presence of HAS is critical in particular at the beginning of leukocyte cultures, even before the addition of Sendai virus, suggesting that the main role of HSA could be to restore the capacity of purified leukocytes to produce IFNα rather than to support the IFNα expression during the culture.