Nutricosmetics: A new frontier in bioactive peptides' research toward skin aging.

Food derived bioactive peptides are small protein fragments (2-20 amino acids long) that can exhibit health benefits, beyond basic nutrition. For example, food bioactive peptides can act as physiological modulators with hormone or drug-like activities including anti-inflammatory, antimicrobial, antioxidant, and the ability to inhibit enzymes related to chronic disease metabolism. Recently, bioactive peptides have been studied for their potential role as nutricosmetics. For example, bioactive peptides can impart skin-aging protection toward extrinsic (i.e., environmental and sun UV-ray damage) and intrinsic (i.e., natural cell or chronological aging) factors. Specifically, bioactive peptides have demonstrated antioxidant and antimicrobial activates toward reactive oxygen species (ROS) and pathogenic bacteria associated with skin diseases, respectively. The anti-inflammatory properties of bioactive peptides using in vivo models has also been reported, where peptides have shown to decreased the expression of IL-6, TNF-α, IL-1ß, interferon-γ (INF-γ), and interleukin-17 (IL-17) in mice models. This chapter will discuss the main factors that trigger skin-aging processes, as well as provide examples of in vitro, in vivo, and in silico applications of bioactive peptides in relation to nutricosmetic applications.

Use of simplified models for theoretical prediction of the interactions between available antibodies and the receptor-binding domain of SARS-CoV-2 spike protein.

The negative impact of infectious diseases like COVID-19 on public health and the global economy is evident. This pandemic represents a significant challenge for the scientific community to develop new practical analytical methods for accurately diagnosing emerging cases. Due to their selectivity and sensitivity, new methodologies based on antigen/antibody interactions to detect COVID-19 biomarkers are necessary. In this context, the theoretical, computational modeling reduces experimental efforts and saves resources for rational biosensor design. This study proposes using molecular dynamics to predict the interactions between the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein simplified model and a set of highly characterized antibodies. The binding free energy of the antigen/antibody complexes was calculated for the simplified models and compared against the complete SARS-CoV-2 ectodomain to validate the methodology. The structural data derived from our molecular dynamics and end-point free energy calculations showed a positive correlation between both approximations, with a 0.82 Pearson correlation coefficient; t = 3.661, df = 3, p-value = 0.03522, with a 95% confident interval. Furthermore, we identified the interfacial residues that could generate covalent bonds with a specific chemical surface without perturbing the binding dynamics to develop highly sensitive and specific diagnostic devices. Communicated by Ramaswamy H. Sarma.

The carbon stable isotope compositions of glyphosate and aminomethylphosphonic acid (AMPA): Improved analytical sensitivity and first application to environmental water matrices.

RATIONALE: The presence of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in the environment has adverse effects on environmental quality, raising the need to better constrain their fates, in particular the processes that control their production and degradation. Our aim was to improve the sensitivity of their δ13 C analysis and demonstrate the feasibility of measuring them in natural surface water. METHODS: The δ13 C values of dissolved glyphosate and AMPA were determined using isotope ratio mass spectrometry (IRMS) (Delta V Plus instrument) coupled to a high-performance liquid chromatography (HPLC) unit, where glyphosate and AMPA were separated on a Hypercarb column. RESULTS: We demonstrated an improved sensitivity of the δ13 C analysis for glyphosate and AMPA by LC/IRMS compared with previous studies. For waters from the carbonate and silicate hydrofacies, while no pretreatment was required for the isotope analysis of glyphosate, removal by H3 PO4 acidification of dissolved inorganic carbon, that co-elutes with AMPA, was required prior to its analysis. We successfully tested a freeze-drying pre-concentration method showing no associated isotope fractionation up to concentration factors of 500 and 50 for glyphosate and AMPA, respectively. CONCLUSIONS: We demonstrated, for the first time, the feasibility of measuring the δ13 C values of glyphosate and AMPA in natural surface waters with contrasted hydrofacies (calcium carbonate and silicate types). This opens new fields in pesticide research, especially on the characterization of processes that control their degradation and the production of their secondary byproducts.

La inexactitud de las ecuaciones predictivas de LaFarge para la estimación del consumo de oxígeno en niños menores de 3 años / The inaccuracy of LaFarge equations estimating the oxygen consumption in children under 3 years age

La medición precisa del consumo de oxígeno (VO2) es fundamental para una evaluación hemodinámica fiable, en especial en pacientes con cardiopatías congénitas intervenidos de cateterización cardiaca. Las ecuaciones predictivas de LaFarge pueden introducir estimaciones del VO2 no fiables, particularmente en población pediátrica. A nivel clínico, se podría traducir en fallos de cálculo en importantes parámetros hemodinámicos, con posibles consecuencias a nivel diagnóstico y terapéutico. Nuestro objetivo es validar las ecuaciones de LaFarge (las más usadas actualmente para la estimación del VO2), comparándola con la medición directa en niños durante la cateterización cardiaca en la sala de hemodinámica. Un estudio prospectivo observacional unicéntrico fue desarrollado con 21 pacientes (de 0-3 años de edad) con diferentes cardiopatías congénitas y programados para cateterización cardiaca diagnóstica. Bajo ventilación mecánica y con anestesia general, se monitorizó directamente el VO2 con un módulo metabólico en nuestra sala de hemodinámica. Por otra parte, se calculó el estimado con la ecuación de LaFarge. Para el análisis estadístico, se usaron el gráfico Bland-Altman, el coeficiente de Pearson y el porcentaje de error, entre otros. Las ecuaciones de LaFarge sobrestiman el valor de VO2 en todos los pacientes del estudio. Es por ello que, en la población pediátrica menor de 3 años, la utilización de métodos de medición directa del VO2 aporta más exactitud y aceptación, en lugar de su estimación con las ecuaciones de LaFarge

Accurate oxygen consumption measurement (VO2), is essential to obtain a reliable hemodynamic assessment, particularly in patients with congenital heart diseases undergoing cardiac catheterization. LaFarge equations can be unreliable in predicting VO2, particularly in the pediatric population. In a clinical setting, these inaccurate estimates can lead to important hemodynamic parameter miscalculations, with possible therapeutic or diagnostic consequences. Our aim is to validate LaFarge equations (the most widely used for estimating VO2) and compare them with direct measurement in children during cardiac catheterization in the cath lab. We performed a prospective observational study in 21 patients (0-3 years of age) with different congenital cardiac diseases, scheduled for diagnostic cardiac catheterization. Under general anesthesia and mechanical ventilation, VO2 was measured directly with a metabolic module in our cath lab, and also estimated using LaFarge equations. Statistical analysis included Bland-Altman plots, Pearson coefficient and percentage error, among others. LaFarge equations overestimated VO2 values in all study patients. Therefore, in pediatric patients under 3 years of age, the use of direct VO2 measurement methods are more accurate and acceptable than LaFarge equations

The inaccuracy of LaFarge equations estimating the oxygen consumption in children under 3 years age. / La inexactitud de las ecuaciones predictivas de LaFarge para la estimación del consumo de oxígeno en niños menores de 3 años.

Accurate oxygen consumption measurement (VO2), is essential to obtain a reliable hemodynamic assessment, particularly in patients with congenital heart diseases undergoing cardiac catheterization. LaFarge equations can be unreliable in predicting VO2, particularly in the pediatric population. In a clinical setting, these inaccurate estimates can lead to important hemodynamic parameter miscalculations, with possible therapeutic or diagnostic consequences. Our aim is to validate LaFarge equations (the most widely used for estimating VO2) and compare them with direct measurement in children during cardiac catheterization in the cath lab. We performed a prospective observational study in 21 patients (0-3 years of age) with different congenital cardiac diseases, scheduled for diagnostic cardiac catheterization. Under general anesthesia and mechanical ventilation, VO2 was measured directly with a metabolic module in our cath lab, and also estimated using LaFarge equations. Statistical analysis included Bland-Altman plots, Pearson coefficient and percentage error, among others. LaFarge equations overestimated VO2 values in all study patients. Therefore, in pediatric patients under 3 years of age, the use of direct VO2 measurement methods are more accurate and acceptable than LaFarge equations.

Electrophysiological evaluation of extracellular spermine and alkaline pH on synaptic human GABAA receptors.

Polyamines have fundamental roles in brain homeostasis as key modulators of cellular excitability. Several studies have suggested alterations in polyamine metabolism in stress related disorders, suicide, depression, and neurodegeneration, making the pharmacological modulation of polyamines a highly appealing therapeutic strategy. Polyamines are small aliphatic molecules that can modulate cationic channels involved in neuronal excitability. Previous indirect evidence has suggested that polyamines can modulate anionic GABAA receptors (GABAARs), which mediate inhibitory signaling and provide a direct route to reduce hyperexcitability. Here, we attempted to characterize the effect that spermine, the polyamine with the strongest reported effect on GABAARs, has on human postmortem native GABAARs. We microtransplanted human synaptic membranes from the dorsolateral prefrontal cortex of four cases with no history of mental or neurological disorders, and directly recorded spermine effects on ionic GABAARs responses on microtransplanted oocytes. We show that in human synapses, inhibition of GABAARs by spermine was better explained by alkalization of the extracellular solution. Additionally, spermine had no effect on the potentiation of GABA-currents by diazepam, indicating that even if diazepam binding is enhanced by spermine, it does not translate to changes in functional activity. Our results clearly demonstrate that while extracellular spermine does not have direct effects on human native synaptic GABAARs, spermine-mediated shifts of pH inhibit GABAARs. Potential spermine-mediated increase of pH in synapses in vivo may therefore participate in increased neuronal activity observed during physiological and pathological states, and during metabolic alterations that increase the release of spermine to the extracellular milieu.

Rotavirus VP6 protein as a bio-electrochemical scaffold: Molecular dynamics and experimental electrochemistry.

This paper reports a theoretical and experimental investigation on the recombinant protein rotavirus VP6 as a bioelectrochemical interface. Our motivation arises from the highly active zones of VP6 which can interact with biological structures and metals, as well as its useful features such as self-assembly, polymorphism, and active surface charge. A molecular simulation study was performed to analyze the charge transfer properties of theVP6 trimer under an applied electric field. The electrostatic properties were evaluated via the nonlinear second-order Poisson-Boltzmann equation, using finite element methods based on parameter discretization and calculation of solute/solvent interaction forces, which account for mean-field screening effects. The electrochemical study validated the theoretical predictions for VP6 in their different assemblies (trimers and nanotubes) when they are used as electrodes in 10 mM K3[Fe(CN)6], 1 M KCl. Applying a potential sweep promotes charge transfer, facilitates redox activity of the ferricyanide ion. Furthermore, protein assemblies decreased electrode electrical resistance and enabled gold particle electrodeposition on the protein VP6. These results suggest that VP6 is a promising conductive biomaterial that promotes charge transfer of redox probes and could be used as a new scaffold to create bio-electrochemical interfaces.

Identity, expression and functional role of the sodium-activated potassium current in vestibular ganglion afferent neurons.

Vestibular afferent neurons (VANs) transmit information from the vestibular end organs to the central nuclei. This information is encoded within the firing pattern of these cells and is heavily influenced by the K⁺ conductances expressed by vestibular neurons. In the present study, we describe the presence of a previously unidentified Na⁺-activated K⁺ conductance (KNa) in these cells. We observed that the blocking of Na⁺ channels by tetrodotoxin (TTX) or the substitution of choline for Na⁺ in the extracellular solution during voltage clamp pulses resulted in the reduction of a sustained outward current that was dependent on the Na⁺ current. Furthermore, increases in the intracellular concentration of Na⁺ that were made by blocking the Na⁺/K⁺ ATPase with ouabain increased the amplitude of the outward current, and reduction of the intracellular Cl⁻ concentration reduced the TTX-sensitive outward current. The substitution of Li⁺ for Na⁺ in the extracellular solution significantly reduced the amplitude of the outward current in voltage clamp pulses and decreased the afterhyperpolarization (AHP) of the action potentials in current clamp experiments. These electrophysiological results are consistent with the presence of mRNA transcripts for the KNa subunits Slick and Slack in the vestibular ganglia and in the sensory epithelium, which were detected using reverse-transcription polymerase chain reaction (RT-PCR). These results are also consistent with the immunolabeling of Slick and Slack protein in isolated vestibular neurons, in the vestibular ganglion and in the vestibular sensory epithelium. These results indicate that KNa channels are expressed in VANs and in their terminals. Furthermore, these data indicate that these channels may contribute to the firing pattern of vestibular neurons.

Periodicity and patterns of Entamoeba histolytica and E. dispar infection in HIV+/AIDS patients in Mexico.

In a 12-month longitudinal study, a cohort of Mexican HIV+/AIDS patients was checked several times for Entamoeba infection, with the parasites identified, as E. histolytica or E. dispar, using PCR. The polymorphic region of the parasites' chitinase genes was investigated by PCR, with the variation in amplicon sizes being used as a measure of the genetic variation among the isolates. The patients found infected with Entamoeba at the start of the study displayed varied patterns of infection clearance and re-infection. The analysis of the polymorphisms in the chitinase gene revealed seven polymorphic patterns in the E. histolytica isolates investigated and three in the E. dispar isolates. Many of the patients were each re-infected with Entamoeba at least once during the 12 months of follow-up. As seen in a previous study in Mexico, none of the E. histolytica-infected patients developed any clinical symptoms of invasive amoebiasis during the follow-up period. The results highlight the complexity of the host-parasite relationship in human amoebiasis.

The muscarinic inhibition of the potassium M-current modulates the action-potential discharge in the vestibular primary-afferent neurons of the rat.

There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons. Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7-10) and held in primary culture (18-24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%+/-7% and 50%+/-3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%+/-12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

The phosphatidylinositol 3-kinase inhibitor LY294002 binds the estrogen receptor and inhibits 17beta-estradiol-induced transcriptional activity of an estrogen sensitive reporter gene.

Estrogen receptors (ERs) are members of the superfamily of ligand-activated transcription factors. In addition to the classical, hormone-mediated activation, ERs may alternatively be activated in a ligand-independent manner by a variety of agents including growth factors, neurotransmitters and cAMP. It has been demonstrated that the phosphatidylinositol 3 (PI3)-dependent kinase/Akt pathway may activate the ER alpha by increasing the activity of both estrogen independent activation function-1 and estrogen-dependent activation function-2 domains. The Akt phosphorylation site in the ER is Ser167. Phosphorylation of this residue is inhibited by LY294002, which blocks the PI3-kinase/Akt pathway. In the course of studies examining the effects of LY294002 on ligand-independent activation of ERs in L cells, we found that LY294002 exhibits antiestrogenic effects in a dose-dependent manner. By competition binding assays, we found that LY294002 specifically displaced radiolabelled estradiol from ERs with an IC(50) of 11+/-0.06 nM, being an estradiol competitor as effective as the antiestrogens ICI182,780 (IC(50), 21+/-0.13) and 4-OH-tamoxifen (IC(50), 15+/-0.09). Further, LY294002 irreversibly blocked estrogen-induced transactivation of an estradiol-sensitive reporter gene. These findings are of particular importance in the interpretation of studies demonstrating ERs inactivation by the PI3-kinase inhibitor. Our studies show that an apparent block of ER activation cannot be dissociated from inhibition of ligand-mediated events. Thus, this effect can be the result of the ability of LY294002 to bind the ERs and inhibit transactivation of estrogen-regulated genes.

Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.

To investigate to what extent myeloablation, graft size, and ex vivo manipulation influence the engraftment and long-term survival of transduced murine hematopoietic cells, groups of C57BL/6J (CD45.2) mice receiving total body irradiation (TBI) (1-9 Gy) or no irradiation were transplanted with either transduced bone marrow (BM) cells, at two cell doses, or with fresh BM cells from B6/SJL (CD45.1) congenic mice. Short (40 days) and long-term (5 months) engraftment and transgene expression were measured by FACS analysis. No donor cells were detected in the hematopoietic tissues of non-myeloablated mice, whereas in the irradiated animals, levels of engraftment correlated well with the dose of TBI administered. Similar percentages of transgene-expressing cells were found in the grafted hematopoietic cells of all groups of mice, regardless of the dose of TBI administered or the level of engraftment achieved. This suggests that the engrafted animals could become tolerant to the transgene product (enhanced green fluorescent protein, EGFP). Our results indicate that TBI facilitates the engraftment of manipulated hematopoietic cells in a dose-dependent manner, that mice engrafted with EGFP(+) hematopoietic cells probably acquire tolerance to EGFP, and that increasing the graft size and reducing the ex vivo manipulation required for retroviral gene transfer of hematopoietic cells also enhances their engrafting potential.

E1A modulates phosphorylation of p130 and p107 by differentially regulating the activity of G1/S cyclin/CDK complexes.

We have previously shown that the adenoviral 12S E1A protein modulates the phosphorylation status of p130 and p107 without apparent changes in the cell cycle dependent phosphorylation of the retinoblastoma protein. Here we report on the mechanisms by which E1A modifies differentially the phosphorylation status of pocket proteins. In human U-2 OS osteosarcoma cells transiently expressing E1A, ectopic expression of D-type cyclins alone or combined, but not cyclins E and/or A, fully rescues E1A-mediated block in hyperphosphorylation of p130 to form 3. However, cyclins E and A, individually or together, induce hyperphosphorylation of p130 to species with intermediate mobility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of sites phosphorylatable by CDKs. One of these sites is Ser-1044. The effects of blocking the activities of endogenous and exogenous cyclins with p16 and dominant negative CDK2 in E1A expressing cells further indicate that p130 is phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that E1A modulates the activity of these G1/S CDKs by independent mechanisms. Stable expression of E1A in MC3T3-E1 cells leads to downregulation of D-type cyclins, and upregulation of cyclins E and A. This is accompanied by increased CDK2 kinase activity. Downregulation of D-type cyclins in these cells correlates with a block on both p130 hyperphosphorylation to form 3 and hyperphosphorylation of p107. This is rescued by D-type cyclins but not by cyclin E. In addition, we show that the upregulation of cyclins E and A is at least partially dependent on an intact pocket protein/E2F pathway, but downregulation of D-type cyclins is not. Moreover, we provide evidence that while the lack of a functional pRB pathway also results in a block on hyperphosphorylation of p130 to form 3, this is not sufficient to induce constitutive expression of p130 form 2b.

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice.

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34(+) cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45(+) cells expressed the transgene 90 to 120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45(+) cells expressing the EGFP transgene at this time. The transplantation of purified EGFP(+) cells increased the proportion of CD45(+) cells positive for EGFP expression to 57. 7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, respectively. Interestingly, the transplantation of EGFP(-) cells purified at 24 hours after infection also generated a significant engraftment of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.

E1A blocks hyperphosphorylation of p130 and p107 without affecting the phosphorylation status of the retinoblastoma protein.

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.

Occurrence of Hodgkin's disease and cutaneous B-cell lymphoma in the same patient: a report of two cases.

The occurrence of Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) in the same patient is well known. The most frequent observation has been the development of large B cell lymphoma in patients affected with the nodular form of lymphocytic predominant HD. A less common situation is the development of NHL among patients successfully treated for HD. In such patients the second lymphoma has been thought to be related to the previous therapy or the immunodeficiency state that can accompany HD. Histologically, these NHL lymphomas often are intermediate to high grade and frequently extranodal. We report two patients successfully treated for HD who also developed NHL of the skin. Both patients presented with strikingly similar findings regarding to sex, age and subtype of HD. Clinical, histopathological and immunophenotypical findings were consistent with cutaneous low-grade B cell lymphoma of the marginal zone type. Both cases remain in complete remission of HD after standard therapy. In both patients the cutaneous lymphoma followed an indolent clinical course after a long follow-up period. This observation expands the spectrum of alterations possibly related to HD.

Cardiac involvement and molecular staging in a fatal case of mycosis fungoides.

Polymerase chain reaction (PCR) amplification of T-cell receptor-gamma gene rearrangement was used for molecular staging in a case of primary cutaneous T-cell lymphoma (CTCL) with fatal evolution. Although initial evaluation was negative for systemic involvement, the patient died due to heart failure. Autopsy findings revealed lymphomatous myocardial infiltration, but other tissues and organs examined, including lymph nodes, liver, spleen, lung and bone marrow, appeared to be free of disease. Molecular analysis from frozen samples obtained during the initial evaluation, as well as paraffin-embedded material obtained during autopsy, revealed the presence of clonal rearranged bands in all tissues examined except the bone marrow. Subsequent hybridization of PCR products with a tumour-specific oligoprobe confirmed the PCR results, suggesting widespread dissemination of the lymphomatous process. The use of molecular analysis can add significant information about the extent of disease in patients with CTCL and may be helpful in the establishment of therapeutic options.

Retroviral gene transfer into human hematopoietic cells: an in vitro kinetic study.

BACKGROUND AND OBJECTIVE: Successful gene therapy applications require optimized strategies to increase gene transfer efficiency into hematopietic progenitor cells (HPCs) with long-term repopulating ability. One of the issues that needs to be clarified is how hematopoietic cells proliferate, differentiate and express the transgene after each cycle of transduction. We investigated the kinetics of cell expansion, CD34 antigen expression and transduction efficiency of human hematopoietic cells in culture conditions commonly used in retroviral gene transfer protocols. DESIGN AND METHODS: Purified CD34+ cells from cord blood (n=5) or leukapheresis products (n=9) and a retroviral vector encoding an enhanced version of the green fluorescent protein (EGFP) were used. Target cells were exposed daily to vector-containing supernatants and a combination of interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF) and Flt3-ligand (FL). Cell samples were harvested from the cultures and analyzed at 24 hour intervals for seven consecutive days. RESULTS: We found that CD34+ cells proliferated and differentiated under our culture conditions. The number of genetically modified cells increased after each cycle of transduction. Median numbers of cells positive for both CD34 and EGFP increased steadily over the culture period, but after day four most of the EGFP+ cells had a low CD34 expression. INTERPRETATION AND CONCLUSIONS: Culturing and transducing CD34+ cells for longer periods of time under these conditions might be detrimental for ex vivo gene transfer applications since the transduced cells are likely to have a decreased potential for long-term engraftment and repopulation in vivo.

Immunomagnetic bone marrow (BM) and peripheral blood progenitor cell (PBPC) purging in follicular lymphoma (FL).

Twenty-nine B cell follicular lymphoma (FL) patients had their BM (n = 12) or PBPC (n = 17) purged using a panel of monoclonal antibodies and immunomagnetic beads (IMB). The median recovery of nucleated cells (NC) and CD34+ cells was 59.3% (40.5-74) and 56.1% (30.8-82.9) in BM and 77.2% (64.7-88.3) and 73.5% (61.5-98.6) in PBPC (P<0.0005). A median of >1.62 and >1.02 log of target cell depletion was achieved as judged by flow cytometry analysis in BM and PBPC, respectively. Of 29% of initial harvests that had a bcl2 PCR-amplified signal, 37.5% became PCR negative in the final purged products. Absorbed cells containing IMB-target cell complexes gave bcl2 rearrangement signal in 20% of samples in which the start and final purged components were negative. Twenty-three of 26 patients receiving an autologous purged product are evaluable for engraftment. Median time to reach an ANC >0.5x10(9)/l and platelet count >20x10(9)/l was 21 (11-43) and 41 days (13-70) for BM (n = 9) and 14 (10-31) and 14 (8-37) for PBPC (n = 14) autografted patients (P = 0.01 and 0.001). One patient did not engraft and was rescued with a back-up BM. These data demonstrate that this indirect immunomagnetic technique is able to achieve a high grade of lymphoma cell depletion in BM and PBPC and that these purged products are capable of rapid engraftment after autologous transplantation.