Preclinical investigation of the antitumour effects of anti-CD19-idarubicin immunoconjugates

Idarubicin (Ida), an analogue of daunomycin, was linked to a mouse monoclonal antibody against the B cell differentiation antigen CD19. Determination of the activity of both the antibody and drug moieties was carried out in vitro using a pre-B cell human acute lymphoblastic leukaemia cell line (NALM-6). A 23% loss in antibody binding and a 20-fold loss in drug activity were observed upon conjugation. Selective cytotoxicity for CD19⁺ cells, however, was obtained. Measurement of the cytotoxicity, antibody activity and release of the breakdown product, 14-OH-Ida, showed that the conjugates remained stable for more than 100 days after lyophilization and storage at –20° C. In vivo studies were performed in irradiatednu/nu mice bearing NALM-6 tumours. Initial dose response studies with free idarubicin demonstrated that three i.p. doses (every other day) of 10 g resulted in little antitumour activity, but the death of all the animals by day 23. The same treatment regime using 15 g Ida-anti-CD19 conjugate caused the disappearance of four out of five tumours with three complete cures and no evidence of toxicity as assessed by weight loss. Administration of a conjugate of idarubicin with an irrelevant antibody at this dose led to no significant antitumour response. The administration of free drug at a dose of 6 g resulted in a minor antitumour response but high toxicity, whereas injection of Ida-anti-CD19 conjugate at this dose caused no toxicity and a substantial antitumour effect with eradication of two out of five tumours. These results clearly demonstrate that the administration of Ida-anti-CD19 conjugates can result in complete tumour regression in an experimental model. Idarubicin-containing immunoconjugates should be useful for the treatment of patients with non-Hodgkin's lymphoma.     

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 g up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at _1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon , IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R⁺, CD4⁺ or CD8⁺ cells were found in the lymph nodes after therapy, but one patient showed activated CD8⁺ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8⁺ T cell activation and cytokine release, without CD4⁺ T cell activation.     

Molecular and Functional Studies of the Gamma Subunit of the Sodium Pump

This article reviews our studies of the subunit of the sodium pump. is a member of the FXYD family of small, single transmembrane proteins and is expressed predominantly in the kidney tubule. There are two major variants of which function similarly to bring about two distinct effects, one on K_ATP and the other, on K _K, the affinity of the pump for K ⁺ acting as a competitor of cytoplasmic Na⁺. In this way, is believed to provide a self-regulatory mechanism for maintaining the steady-state activity of the pump in the kidney. Our studies also suggest that K⁺ antagonism of cytoplasmic Na⁺ activation of the pump is relevant not only to the presence of in the kidney, but probably some hitherto undefined factor(s) in other tissues, most notably heart. The interesting possibility that not only but other members of the FXYD family regulate ion transport in a tissue-specific manner is discussed.     

Cord blood–derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19+ tumors

Background

Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19⁺ malignancies.

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells

Background

The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum.

Evidence that the low-affinity K+ transport system of Rhodobacter capsulatus is a uniporter. The effects of ammonium on the transporter

Comparison of the rate of accumulation of ⁸⁶Rb⁺ by intact cells of Rhodobacter capsulatus during short periods of illumination with the Rb⁺-dependent membrane ionic current measured by electrochromism supports the view that both activities reflect the operation of a low-affinity K⁺ transport system. In experiments performed under similar conditions the ratio of ⁸⁶Rb⁺ uptake to charge uptake was approx. 1.0, suggesting that the transport system operates as a uniporter. The addition of NH _inf4 ^sup+ to a cell suspension led to an increase in membrane ionic current but failed to inhibit the accumulation of ⁸⁶Rb⁺ during illumination. The presence of K⁺ and NH _inf4 ^sup+ inhibited the increase in cellular ATP levels at the onset of illumination. This effect was prevented by Cs⁺. The results are considered within the context of the hypothesis (Golby et al. Eur J Biochem 187: 589–597) that NH _inf4 ^sup+ can be transported by the K⁺ carrier and in the context of an alternative hypothesis that NH _inf4 ^sup+ increases the affinity of the K⁺ transport system for its natural substrate and for Rb⁺.Abbreviations pH pH difference across the cytoplasmic membrane - electrical potential difference across the cytoplasmic membrane     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3<Superscript>+</Superscript>, CD14<Superscript>+</Superscript> and CD19<Superscript>+</Superscript>

Background

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3⁺, CD14⁺ and CD19⁺. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.

Methods

PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3⁺, CD14⁺, CD19⁺ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.

Results

Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3⁺ cells then in 8/26 (30.8%) of CD14⁺ and CD19⁺ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3⁺ (2.4%), then in CD19⁺ (1.2%), and much lower in CD14⁺ (0.4%) cells.

Conclusions

Our results indicate that CD3⁺ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

     

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19⁺ tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19⁺ human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2^−/−γc^−/− immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.     

Pharmacokinetics of an HIV-1 gp120-specific chimeric antibody in patients with HIV-1 disease

The pharmacokinetics of mouse V/human C (1,) chimeric monoclonal antibody CGP47 439 specific for the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) was studied in patients with stage IV HIV-1 disease in an open-labeled phase I/IIA trial. Twelve male patients were enrolled and nine completed the study. Patients were divided into three groups according to the extent of CGP 47 439 to bind to gp120 from their viral isolates: undetectable for group 1, modestly reactive for group 2, and strongly reactive for group 3. A first dose of 1, 10, or 25 mg was administered by intravenous infusion to group 1, group 2 and group 3 patients, respectively. The patients then received seven doses of 50, 100, or 200 mg, respectively, every three weeks. CGP 47 439 serum concentrations were determined by an ELISA using monoclonal antibody AB19-4 specific for the idiotope of CGP 47 439. Half an hour after infusion only 25.5–36.1% of the administered antibody was found in the serum, reflecting its rapid distribution in the extravascular space and possibly binding to gp120 antigen in some of the patients. The terminal elimination half-life (T_1/2) was 16.2 days in group 1 patients, 9.7 days in group 2 and in group 3 patients 7.5 days and 9.1 days. An antibody response to CGP 47 439 was not a factor in determining elimination rates, because only very low and transient responses were found in three patients. These results suggest that the reactivity of CGP 47 439 with HIV-1 gp120 contributed to its elimination in HIV-1 infected patients.Abbreviations AIDS aquired immune deficiency syndrome - ARC AIDS-related complex - HIV-1 human immune deficiency virus type 1 - gp120 envelope glycoprotein with 120 KD molecular weight - V3 variable domain of gp120 - PND principle neutralizing determinant of gp120 - IgG immunoglobulin G - CD4⁺ lymphocytes: lymphocytes expressing the CD4 marker V_H and V_L variable heavy and variable light chain region of an antibody - C₁ and C_K constant heavy chain region of gamma l and constant K light chain region of an antibody - anti-id anti-idiotypic - AUC area under curve - T_1/2 terminal elimination half-life - ELISA enzyme-linked imuno sorbent assay - PBS phosphate buffered saline - NP-40 detergent - CDC center of disease control - GMP good manufacturing practice     

Rapid desensitization for brentuximab vedotin (Adceteris<Superscript>®</Superscript>) allergy: a case report

Background

Brentuximab vedotin (BV) is an antibody–drug conjugate formed by an anti-CD30 chimeric IgG₁ conjugated with monomethyl-auristatin-E. BV targets the CD30⁺ cells, which characterize Hodgkin lymphoma as well as anaplastic large cell lymphoma. Once bound to the CD30⁺ cells BV exerts its cytotoxic effect via the monomethyl-auristatin-E moiety. So far, accounts on immediate adverse reactions to BV remain anecdotal. Moreover, few reports exist on desensitization for BV.

Case presentation

A 20-year old male patient was diagnosed with Hodgkin lymphoma in July 2014. The first line treatment with adriblastine, bleomicine, vinblastine and dacarbazine lead to a partial remission. Thus, a treatment with BV was started. However, during the second BV infusion, he developed generalized urticaria and dyspnea. In order not to discontinue the treatment with BV, we performed a thorough allergological workup and designed a 12-step rapid desensitization protocol. Overall the desensitization procedure was well tolerated and no major adverse reactions occurred.

Conclusion

Rapid desensitization is a suitable and safe option in the case of BV allergy and prevents the BV treatment withdrawal.

     

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells

Summary Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2,7-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH₄ prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na⁺ removal or EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na⁺/H⁺ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na⁺/H⁺ exchange. Despite this heterogeneity, the Na⁺/H⁺ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4–7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na⁺/H⁺ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.     

Multiple pathways for amino acid transport inMytilus gill

Summary The epidermal tissues of marine mussels can accumulate amino acids from surrounding sea water. In the present study, gill tissue isolated from the California coastal mussel,Mytilus californianus, was used in conjunction with intact, actively pumping mussels to study epidermal transport processes. There appeared to be at least four pathways for this uptake: i) a -neutral pathway which transports taurine; ii) an -acidic pathway specific for substrates such as aspartate; iii) an -neutral pathway having a general specificity for this class of compound, but which also accepts the basic amino acid, lysine; and iv) a second -neutral pathway, also of broad specificity, capable of accepting the imino acid, proline, as a substrate. Replacement of Na in sea water with choline reduced uptake of leucine, taurine, aspartate, and proline by more than 95%, and reduced lysine uptake by 75%, suggesting that Na-independent pathways play no significant role in epidermal transport in the gill. Isolated gill tissue was used to estimate the maximum transport capacities (J _max's) of the pathways, which ranged from approximately 5 to 25 mol/(g·hr). Apparent Michaelis constants (K _t ^*'s) of the epidermal transporters were estimated using a convection-diffusion model introduced previously (Wright and Secomb, Am J Physiol 247:R346–R355, 1984). TheseK _t ^*'s ranged from 1 to 5 M. The characteristics of the epidermal transporters are such that they can play a significant role in both animal nutrition and in the reacumulation of endogenous amino acids lost from surface cells through passive diffusion.Abbreviation ASW artificial sea water     

Aluminum geochemistry in peatland waters

The chemical speciation of aluminum was examined in surface water samples from Sphagnum peatlands in north-central Minnesota, from peatlands along the Canadian east coast, and from bogs in the Pennine Mountain area of England. In highly organic ([DOC] 50 mg L^–1 ), low pH waters, 80–90% of total dissolved Al was complexed with organic matter (OM), while in waters with low DOC ([DOC] 5 mg L^–1) 54–86% of total dissolved Al existed as Al⁺³ or other inorganic Al species. Batch titrations of OM with Al revealed a high Al binding capacity, 1.4–2.8 mol (mg DOC)^–1, that generally was unsaturated with Al. Titrations of OM with Al in conjunction with a continuous distribution model were used to determine Al-OM conditional stability constants. Binding capacity (mol Al (mg DOC)^–1) and strength (formation constant) increased from pH 3 to 5 but decreased above pH 5 due to formation of AI-hydroxy species including A1(OH)₃ (s). The high binding capacity of OM in bog waters facilitates metal mobility, especially in low pH (< 5) wetlands where metal solubility is high and OM concentrations are highest. Results showed that the relative degree of organic matter saturation with metal ions was important in modeling AI speciation in bog waters.     

Bioprocess engineering characteristics of the horizontal stirred tank

Bioreactor performance studies of the recently developed horizontal stirred tank with a volume of 421 have been carried out for fermentation with Trichosporon cutaneum. Quantification on the basis of measured oxygen transfer capacity and power consumption is presented and compared with data for a conventional vertical tank bioreactor.During the experiments it has been observed that two different forms of morphology of Trichosporon, i.e. the normal yeast-form (Y) with single cells and a mycelium-form (M) with filamentous cells, are present in the horizontal stirred tank when working with the original strain (DSM 70698). After separation both forms were characterized and later on used for bioreactor performance studies in the horizontal and vertical stirred tank. Results of oxygen efficiency show the drastic effect of the morphology change on bioreactor performance. Finally different bioreactors are quantitatively compared on the basis of oxygen transfer, power consumption and productivity using the reference fermentation system Trichosporon cutaneum.List of Symbols F m³/h flow rate (volumetric) - k _La1/h volumetric transfer coefficient of OTR - M Nm torque - n 1/s rotational speed - P Nm/s power - V m³ volume - V _G1/min gas flow rate - x kg/m³ biomass concentration - ^* morphology index - ^* engineering (specific) viscosity - _app Ns/m² apparent viscosity - ₀ N/m² yield stress (Casson law) - t _1/e h measured time acc. to momentum method [17] - tEh characteristic time of electrode response - t _Gh mean residence time of gas phase Abbreviations CFR completely filled reactor - CRR cyclic ring reactor (torus) - JLR jet loop reactor - HSTR horizontal stirred tank reactor - M mycelium-form of Trichosporon cutaneum - O₂-eff O₂-efficiency - OUR O₂-uptake rate - OTR O₂-transfer rate - STR stirred tank reactor - ThLR thin layer reactor - VSTR vertical stirred tank reactor - Y yeast-form of Trichosporon cutaneum The work presented in this paper was supported by an Austrian Research Grant (FFWF, Project no. 4496)     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F₁ part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F₁ narrows and has three-fold symmetry at the end furthest from the F₀ part. The F₁ is linked to F₀ by a stalk approximately 45 Å long and 25–30 Å in diameter. The F₀ part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F₁ part. The and subunits are in a different conformation when ATP+Mg²⁺ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F₀ part of the complex.     

NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism

Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies.We, and others, have demonstrated that the outcome of CD40 receptor activation by its ligand CD40L/CD154 in carcinomas predominantly relies on the way the ligand is presented to the receptor at the cell surface, where membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) induces cell growth arrest and apoptosis. mCD40L-induced cell death occurs through a mechanism that involves the downregulation of pro-survival signals, mainly the PI3K/AKT pathway and the sustained activation of the pro-apoptotic JNK pathway, leading to caspase activation and subsequently apoptosis.^{1, 2} However, the role of CD40L in cell cycle regulation and growth arrest are still largely elusive; cell cycle regulation is a complex process that ensures correct cell division and is controlled by multiple mechanisms, governed by key regulatory proteins, including the cyclin-dependent kinases (CDKs) and their regulatory inhibitors. CDKs are a family of serine/threonine protein kinases that are activated at specific points of the cell cycle and are regulated by several different mechanisms.³CDK inhibitors function through inhibition of the G1 CDK cyclin complexes.⁴ Furthermore, the CDK inhibitor p21 has the ability to also inhibit DNA synthesis by binding to and inhibiting proliferating cell nuclear antigen.^{5, 6, 7} p21 protein expression is tightly regulated by the p53 tumour suppressor gene.⁸ The expression of p21 is also reported to be regulated by the recently identified Ras-associated factor 5 (NORE1A/RASSF5),⁹ a protein that is 41.8 kD and a member of the RASSF family of tumour suppressors.¹⁰ NORE1A expression is frequently downregulated by promoter methylation in human tumours.^{11, 12} Structurally, NORE1A contains a Ras-binding domain and was originally identified by a yeast two-hybrid screen.¹³ NORE1A can directly bind the Ras oncoprotein in a GTP-dependent manner.¹⁴ Exogenous expression of NORE1A has been reported to promote apoptosis by Ras-dependant and -independent mechanisms.¹² Furthermore, NORE1A protein expression results in a decrease in the number of cells in S phase of the cell cycle,¹⁵ indicating that NORE1A might function as a tumour suppressor; here, we show for the first time that mCD40L but not sCD40L induces the expression of NORE1A in an NFκB-dependant manner. NORE1A expression was found to contribute to mCD40L-induced cell death since inhibition of mCD40L-induced NORE1A expression resulted in reduced cell death by mCD40L. The role of CD40-induced NORE1A expression in cell cycle regulation was also examined and found to be mediated by p21 upregulation.     

Rituximab-induced direct inhibition of T-cell activation

Background

Rituximab, an anti-CD20 monoclonal antibody, is reported to increase the T-cell-dependent infection risk. The current study was designed to investigate whether rituximab interferes with T-cell activation.

Patients and methods

Patients with non-Hodgkin lymphoma receiving 4–6 courses of 375?mg/m² rituximab underwent detailed assessment of T-cell activation pre- and post-rituximab. A similar analysis assessed the in vitro effect of rituximab on T-cell activation in response to allogeneic dendritic cells (allo-DCs) and other stimuli.

Results

Patients receiving rituximab exhibited a significant decline in IL-2 and IFN-γ levels in peripheral blood, most prominent after repeated rituximab courses. Evaluation at 3?months after rituximab therapy showed restoration of inflammatory cytokine production. Similarly, in vitro stimulation of peripheral blood mononuclear cells in the presence of rituximab resulted in a significant decrease in T-cell activation markers, inflammatory cytokine production and proliferative capacity. These effects were also observed using B-cell-depleted T cells (CD3⁺CD25^?CD19^?) and were accompanied with disappearance of CD3⁺CD20^dim T-cell population.

Conclusion

Rituximab administration results in transient, dose-dependent T-cell inactivation. This effect is obtained even in B-cell absence and may increase the infection risk.     

Phase 1B study to improve immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D<Subscript>3</Subscript>

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34⁺ progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34⁺ cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D₃ could diminish CD34⁺ cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60 g) of 25-hydroxyvitamin D₃, with an emphasis on the six patients who received the maximum dosage of 60 g per day. Peripheral blood was collected at 0, 1, 2, 4, and 6 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D₃ reduces the number of immune suppressive CD34⁺ cells, increases HLA-DR expression, increases plasma IL-12 and IFN- levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D₃ treatment did not modulate plasma IL-1, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF- levels.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - high CD34⁺ patients patients with greater than 1% baseline CD34⁺ cell levels - HLA human leukocyte antigen - IFN interferon - IL interleukin - low CD34⁺ patients patients with less than 1% baseline CD34⁺ cell levels - OD optical density - TGF transforming growth factor