Measurement of cell viability by 7-AAD staining 24 h after thawin

Measurement of cell viability by 7-AAD staining 24 h after thawing demonstrated that Teffs had a viability of 90%, whereas 70% of Tregs were viable (data not shown). We tested whether Gefitinib order the expression of any of the markers affected by GAD65 stimulation was related to clinical outcome of treatment. We found no significant correlation between expression of Treg markers used in this study and changes in stimulated C-peptide measured as ΔAUC or AUC 4 years after treatment. C-peptide secretion was not significantly different in patients where an FSChiSSChi population was induced by

GAD65 stimulation compared to those who did not respond in this way (data not shown). To test whether the function of Tregs in T1D children included in the Phase II trial was affected by GAD-alum or placebo administration, suppression assays

using sorted and expanded Tregs (CD4+CD25hiCD127lo) and Teffs (CD4+CD25–CD127+) were performed. Gates used to sort Tregs and Teffs are illustrated in Fig. 3a. Expanded Tregs from patients treated with GAD-alum suppressed proliferation of autologous Teffs to the same extent as Tregs from placebo patients Buparlisib (Fig. 3b). Tregs from both groups of patients displayed dose-dependent suppression of proliferation. As reported previously [25, 27, 28], we further found that suppression in autologous cultures of Tregs and Teffs was reduced in all patients (n = 7, placebo and GAD-alum

combined) compared to a healthy control (seven repeated measurements, Fig. 3c, P < 0·0001). To determine whether this attenuated suppression was intrinsic to Tregs or Teffs, we tested the suppression of Tregs from T1D patients (either GAD-alum- or placebo-treated, with similar results; Fig. 3b,d), and from a healthy control in autologous and cross-over culture suppression assays. As shown in Fig. 3c, T1D Tregs exerted the same level of suppression on Teffs coming from either T1D or healthy subjects. Selleckchem 5-FU In the reverse experiment, healthy Tregs were able to suppress Teffs from healthy or T1D subjects to a similar degree. Taken together, these results suggest that attenuated suppression from Tregs of T1D patients is due to reduced Treg efficacy rather than to increased Teff resistance to suppression. To determine whether there was a difference in reduced Treg-mediated suppression due to treatment, we tested if the suppression exerted in cross-over cultures of T1D Tregs versus healthy Teffs and healthy Tregs versus T1D Teffs was different between treatment arms. There was no difference in suppression exerted by Tregs from GAD-alum-treated patients compared to placebo Tregs in cross-over cultures with healthy Teffs, nor in the suppression exerted by healthy Tregs cultured with Teffs from GAD-alum-treated patients and Teffs from placebo subjects (Fig. 3d).

To verify Vα usage for DbNPCD8+ and DbPACD8+ T cells, PCR was per

To verify Vα usage for DbNPCD8+ and DbPACD8+ T cells, PCR was performed with a panel of Vα primers: Vα1, Vα2, Vα3, Vα4, Nutlin-3 solubility dmso Vα5, Vα6, Vα7, Vα8, Vα9, Vα10, Vα11, Vα13, Vα14, Vα16, Vα17, Vα18, Vα19, and Vα20 41. PCR products were cloned into a vector pCR2.1-TOPO, and colonies containing inserts were sequenced. The authors thank Dina Stockwell for technical assistance, Ken Field for FACS sorting and Serrin Rowarth for providing the A7 mice. This work was supported by Australian National Health and Medical Research Council (NHMRC) Project Grants to KK (AI454312) and PCD (AI454595), an NHMRC Program

Grant # 567122 (to PCD and SJT), and NIH grant AI170251. K. K. is an NHMRC RD Wright Fellow and S. J. T. is a Pfizer Senior Research Fellow. S. A. V. is a recipient of the Australian Postgraduate Award and E. B. D. of the NHMRC Postgraduate Biomedical Scholarship. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Caribbean hair sheep are more

resistant to gastrointestinal nematodes than conventional wool breeds, but mechanisms that confer resistance are not fully understood. This study compared immune effector cell populations and antibody BGJ398 datasheet concentrations in 12 hair and 12 wool lambs infected with the abomasal parasite Haemonchus contortus and sacrificed at 3 or 27 days Methocarbamol post-infection (p.i.) and 14 uninfected animals of each breed. Faecal egg counts were over 2·5-fold higher (P = 0·12) and packed cell volumes approximately 8% lower (P < 0·10) in infected wool lambs. Abomasal lymph nodes were heavier in infected animals (P < 0·05) and infected hair sheep had larger lymph nodes than infected wool sheep (P < 0·05). Tissue eosinophil concentrations were likewise larger (P = 0·07) in hair compared with wool sheep at 3 days p.i. Circulating levels of IgE and IgA in uninfected lambs were higher in hair sheep

(P < 0·05) and during infection, hair sheep had higher serum IgA than wool sheep at 3, 5, and 21 days p.i. (P < 0·05). Serum IgE in infected lambs did not differ between breeds, but concentrations of IgE in lymph nodes were higher (P < 0·01) at 27 days p.i. in infected hair sheep. Haemonchus contortus, a blood-feeding, abomasal parasite, is the most common and problematic of the gastrointestinal nematodes (GIN) of sheep in humid temperate and subtropical climates. The prevalence of GIN that are resistant to anthelmintic treatment is increasing, with almost all farms in the southeastern US having GIN that are resistant to one or more anthelmintics (1). In addition, consumers are driving the livestock industry to produce chemical-free products. Therefore, other methods of parasite control are needed. Caribbean hair sheep have greater resistance than conventional wool sheep to GIN parasites (2–4).

Larger SIEA diameters correlated with a

Larger SIEA diameters correlated with a GPCR & G Protein inhibitor decrease in diameter of ipsilateral DIEA perforators. Conclusion: The SIEA is present more frequently than previously demonstrated, but is typically too small for use in free tissue transfer. The variable degree of SIEA branching suggests that its territory of supply is also variable, and that preoperative imaging may be useful in planning SIEA flaps. © 2010 Wiley-Liss, Inc. Microsurgery 30:386–391, 2010. “
“Combined neurotization of both axillary and suprascapular nerves in shoulder reanimation

has been widely accepted in brachial plexus injuries, and the functional outcome is much superior to single nerve transfer. This study describes the surgical anatomy for axillary nerve relative to the available donor nerves and emphasize the salient technical aspects of anterior deltopectoral approach in brachial plexus injuries. Fifteen patients with brachial plexus injury who had axillary nerve neurotizations were evaluated. Five patients had complete avulsion, 9 patients had RAD001 C5, six patients had brachial plexus

injury pattern, and one patient had combined axillary and suprascapular nerve injury. The long head of triceps branch was the donor in C5,6 injuries; nerve to brachialis in combined nerve injury and intercostals for C5-T1 avulsion injuries. All these donors were identified through the anterior approach, and the nerve transfer was done. The recovery of deltoid was found excellent (M5) in C5,6 brachial plexus injuries with an average of 134.4° abduction at follow up

of average 34.6 months. The shoulder recovery was good with 130° abduction in a case of combined axillary and suprascapular nerve injury. The deltoid recovery was good (M3) in C5-T1 avulsion injuries patients with an average of 64° shoulder abduction at follow up of 35 months. We believe that anterior approach is simple and easy for all axillary nerve transfers in brachial plexus injuries. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“Peripheral neuropathy is the most common ADP ribosylation factor nerve disorder in human immunodeficiency virus (HIV) patients. Distal symmetrical sensory polyneuropathy (DSP) affects roughly one third of HIV patients. With the introduction of antiretrovirals, more patients are surviving longer, and chronic complications are surfacing. Three consecutive patients with at least a 5-year history of HIV presented during the period from 2007 to 2009. All three patients were on antiretrovirals and had no other comorbid conditions such as spinal pathology or diabetes. All patients had symptoms of pain, numbness, and weakness. Quantitative sensory testing and/or electromyography/nerve conduction testing (EMG/NCT) were performed preoperatively and correlated with the presence of Tinel signs. Targeted nerve releases were performed in four extremities, for a total of 18 nerves.

3a) There was no up-regulation of the gene expression

3a). There was no up-regulation of the gene expression Bafilomycin A1 in vivo of cytokines and chemokines in regions away from the inoculation site in either mouse strain (data not shown). These results suggest that

MPyV infection of the brain leads to CCL5 expression in both mouse strains, and that IFN-β and IL-6 are also induced in immunocompetent and immunocompromised mice, respectively. Finally, the experiments were performed to elucidate whether MPyV inoculation into the brain causes clinical manifestations in mice. The mice were mock-inoculated or inoculated with MPyV as described above, and body weights were recorded every 2 days for 14 days p.i. In both strains, the mean body weights of MPyV-inoculated mice were comparable to those of the controls at each time point, and

there were no significant differences between the two groups (Fig. 3b). In addition, BALB/c and KSN mice did not show any signs of disease, such as paralysis, paresis, or seizures, up to 30 days p.i. (data not shown). These observations indicate that MPyV asymptomatically infects mice after virus inoculation into the brain. In the current study, the modes of MPyV infection were quantitatively analyzed in adult mice after stereotaxic microinfusion of virus inoculum into the brain parenchyma. Intracranial inoculation tetracosactide by directly puncturing the skull with a needle connected to a syringe PD98059 concentration is frequently used as a way to introduce a virus into the cerebrum of mice (3); however, using this method, the accurate injection of a small amount of virus inoculum into an exact location within the brain tissue is difficult. Therefore, stereotaxic microinfusion can be regarded as a useful technique for quantifying virus spread within the brain. Since viral DNA levels peaked at 4 days p.i. in both BALB/c and KSN mice, it is thought that MPyV replicates in the adult mouse brain up to 4 days after stereotaxic inoculation.

In athymic KSN nude mice, the significant levels of MPyV genomes continued to be detected up to 30 days p.i., suggesting that MPyV establishes a long-term infection in the brains of immunocompromised mice. In BALB/c mice, the amount of virus was dramatically diminished from a peak at 4 days p.i., although low but detectable levels of viral DNA were seen at 30 days p.i.; thus, this observation suggests that the MPyV infection of the brain is controlled by T cell-mediated immunity in immunocompetent mice. Although the stereotaxic injection of MPyV led to a long-term infection in the brains of KSN mice, the viral DNA levels did not increase in a time-dependent manner between 4 and 30 days p.i.

Memory B cells might predict clinical prognosis more accurately t

Memory B cells might predict clinical prognosis more accurately than serum immunoglobulin concentrations [29]. In this regard it is interesting to note that memory B cells might be a predictive marker of outcome in hypogammaglobulinemia Transmembrane Transporters inhibitor during infancy [30].

Taking these observations into account, the establishment of age-dependent reference values for distinct B cell populations is of relevance. While this study was ongoing, age-dependent peripheral B cell frequencies have been published for children < 18 years by two other independent groups [19,20]. We present reference values of these B cell subsets for children, and additionally extended these data for adults up to the age of 50 years. While comparing our proposed reference values with those already published we could confirm the published data, highlighting the reproducibility of this flow cytometric approach [19,20]. Beyond the already published data we present age-dependent reference values for transitional B cells as well as CD21lowCD38low B cells in addition. Both B cell subsets as well as the proportion of CD27+IgD- memory B cells found implementation into the latest CVID classification approach (EUROclass) [14]. However, the proposed cut-off values

of this approach originated predominantly from data obtained by adult individuals. As we could show that transitional B cells and CD27+IgD- memory B cells underlie age-dependent developmental changes, the proposed cut-off values of the EUROclass approach might be misleading in childhood. According to our proposed reference values it seems obvious that a frequency of ≥ 2% switched memory B cells Wnt drug and < 9% transitional B cells (proposed as cut-off values in the EUROclass approach) can be applied only to individuals ≥ 18 years of age but not to younger individuals (Table 2). Recently, aminophylline low numbers of switched memory B cells (< 5/µl) have been suggested as the cut-off value in paediatric CVID, distinguishing a subgroup of patients with increased risk

of autoimmunity and severe infections [23]. Because numbers of switched memory B cells usually exceed this cut-off value in healthy individuals beyond the first year of life (Table 2), this cut-off value might be used to distinguish impaired from normal B cell differentiation. However, efforts should be undertaken to validate quantitative changes in peripheral B cell development as predictors for disease prognosis in childhood onset of autoimmune diseases and immunodeficiency. In summary, we have characterized the peripheral blood B cell compartment in detail during age. This study provides reference values of different B cell subpopulations from birth to 50 years of age. We would like to thank Gertraud Baier, Gaby Haase, Barbara Ottensmeier and Brigitte Wollny for excellent technical assistance. The study was supported by the German Research Foundation (Gi 295/3-1). We thank David Carr for statistical analysis. Nothing to disclose.

This study aimed to evaluate clinical and evolutive features of I

This study aimed to evaluate clinical and evolutive features of IRIS associated cryptococcosis patients in Uberaba, Brazil. Patients: Eighty-one

AIDS individuals admitted at the teaching hospital with cryptococcal PCI-32765 supplier meningitis were evaluated and from these, 40 were prospectively followed. Of 40 patients with cryptococcosis, nine (22.5%) presented clinical and laboratory features of IRIS. Six (66.6%) were male, with a mean age of 37.2. Five (55.5%) presented cryptococcosis as first AIDS defining condition. In seven (77.9%) IRIS was characterised as a relapse of meningeal symptoms after 10 weeks, mean time of 72 days, of starting HAART whereas, two asymptomatic patients developed the syndrome as an unmasked cryptococcosis after 10 and 12 weeks on HAART. Lymphadenitis as isolated finding associated with IRIS was evidenced in three cases. Angiogenesis inhibitor All patients presented low CD4+ and high RNA viral load baseline values. Cultures of cerebrospinal fluid and lymph-node fragments tissues of these cases were negative. Six of nine individuals developed

high intracranial pressure requiring a daily relief lumbar puncture. No deaths occurred during the evolution of these patients. The incidence and clinical evolutive profile observed in this case series are in accordance with other reports elsewhere. “
“Pomegranate is a wonderful fruit from the paradise which contains a wide variety of precious phytochemical compounds applicable in the fields of therapeutics and health care. Candida albicans is the most common etiological agent for many Sinomenine clinical mycoses which could lead to human and animal death. Determination of the anticandidal activity of pomegranate peel extracts (PPE), and application of PPE aerosol

as sanitizer agent against C. albicans contamination were investigated. Agar diffusion assay and broth microdilution susceptibility test were applied for qualitative and quantitative determining the PPE anticandidal activity, respectively, versus commonly used fungicides. Aerosolization of PPE using an experimentally designed sanitizer room was applied for examining C. albicans sanitation potentiality of extract. PPE exhibited potent anticandidal activity against C. albicans strains comparing with standard fungicides in both used susceptibility techniques. Methanol, ethanol and water extracts were the most effective for inhibiting C. albicans growth. PPE aerosol was an efficient method for complete sanitizing of semi-closed places against C. albicans growth. Application of PPE aerosol is a proper sanitizing method for preventing C. albicans contamination and growth in suspected places. “
“The effects of the addition of different amino nitrogens on growth, morphology and secondary metabolism of Malassezia furfur were investigated. After primary culture on Dixon agar, M.

We discuss here important pro-inflammatory molecules and leucocyt

We discuss here important pro-inflammatory molecules and leucocyte populations that were identified as key players in the murine model of DENV-2 infection using the mouse-adapted strain P23085. The inflammatory response triggered by this model of DENV infection frequently leads to tissue damage and death. However, it is possible in this model to assess and distinguish mechanisms necessary for the host response

to deal with infection from those that cause unwanted, misplaced and uncontrolled inflammation and drive disease. RG7204 solubility dmso By understanding where/how host–pathogen interactions lead to disease, we may be able to suggest novel strategies to restrain severe systemic and local inflammatory responses. Chemokines are members of a structurally related family of cytokines involved in leucocyte Doxorubicin molecular weight traffic during infection and inflammation. They are classified according to the relative position of conserved N-terminal cysteine residues, in which CC

chemokines represent the most abundant family and have the first two cysteines placed adjacently.[72] Chemokine receptors are expressed on the surface of leucocytes and are G protein-coupled receptors containing seven transmembrane domains.[73] Experimental and epidemiological evidence suggests an important role for chemokines, especially those from the CC family, and their receptors in infectious diseases such as HIV and herpes simplex virus 1.[74, 75] The expression of CC chemokines dominates over the expression of CXC chemokines during

viral infections, although this observation does not represent a general rule.[75] Among the CC chemokines, CCL3/MIP-1α and CCL5/regulated upon activation, normal T cell-expressed and secreted (RANTES) are widely associated with viral infections [74, 76] During intranasal influenza virus infection in mice, CCL2/monocyte chemotactic protein-1 (MCP-1) is detected in the lungs at various time-points post-infection, whereas other chemokines, including CCL3 and CCL5, are not expressed.[77] On the other hand, respiratory syncytial virus-infected mice display high levels of expression of numerous Amoxicillin chemokines in the lungs, including CCL3 and CCL5.[78] Among flaviviruses, CC chemokine receptors play an important role in leucocyte recruitment to the central nervous system.[79] Besides a deleterious pro-inflammatory role that CC chemokines could play in central nervous system, a well-studied example involves acute infection by West Nile virus in mice, in which the lack of CCR2 and CCR5 leads to decreased leucocyte recruitment, increased viral load in the central nervous system and enhanced mortality. West Nile virus infection induces high and continuous levels of CCL2 and CCL5, which are required for the local accumulation of NK cells, macrophages and T lymphocytes to control infection.

This co-aggregation mechanism allows tyrosine phosphorylation of

This co-aggregation mechanism allows tyrosine phosphorylation of the ITIM by the

kinases associated with the activating receptor. This leads to the recruitment of phosphatases, such as Src homology 2 (SH2) domain-containing phosphatase-1 (SHP-1) or SH2 domain-containing inositol phosphatase-1 (SHIP-1), to the phosphorylated ITIM. These phosphatases are then ideally localized to allow them to find their respective substrates and be recruited to the activating receptor or plasma membrane to impede ITAM-initiated signalling, including activation of kinases, adapter proteins or specific membrane effector GSK126 supplier recruitment. Human CD89 (FcαRI), which is not expressed in rodents, is found on the surface of myeloid cells, including monocytes/macrophages, neutrophils and eosinophils, and binds to both IgA1 and IgA2. FcαRI is expressed simultaneously with or without physical association with the FcRγ-chain homodimer [4,5]. FcαRI plays a role in a variety of inflammatory diseases via its powerful proinflammatory function. Recently, we reported that FcαRI and its associated FcRγ subunit exhibit a novel anti-inflammatory function

for homologous immunoreceptors [6]. Inhibitory cross-talk was dependent on the FcRγ inhibitory ITAM (iITAM); it APO866 chemical structure occurred without co-aggregation and was triggered after monomeric targeting of FcαRI with anti-FcαRI (A77) fragment antigen-binding (Fab) or immunoglobulin (Ig)A ligand binding. Similar to ITIM-mediated signals, down-regulation of the response involved the association of receptors with the tyrosine phosphatase SHP-1. Such dual receptor functions have since been observed for other ITAM-bearing receptors, including several innate immune receptors [7,8], suggesting that they might represent a widespread mechanism of immune regulation. Recent discovery of the family of Toll-like receptors (TLRs) has focused attention on

the disease processes, as TLRs mediate pathogen recognition and immune activation [9,10]. Bacterial DNA has been shown to be a pathogen-derived structure that Nintedanib molecular weight activates the innate immune system through TLR-9 [11]. This activity depends on unmethylated cytosine-guanine dinucleotides (CpG), in particular base contexts [CpG oligodeoxynucleotides (CpG-ODNs)][12]. Recently, it has been shown that CpG-ODNs induce nuclear factor (NF)-κB activation, p38 phosphorylation, extracellular signal-regulated kinase (ERK) and the synthesis and release of tumour necrosis factor (TNF)-α in macrophages [13]. TLR-mediated immune activation may play a role in immune complex diseases of the kidney triggered by infections. Horse apoferritin-induced glomerulonephritis (HAF-GN) is a model of immune complex GN that is characterized by circulating HAF-specific antibodies, mesangioproliferative GN, glomerular macrophage accumulation and proteinuria [14].

89 Resistance is much less common than with lamivudine: 0% at one

89 Resistance is much less common than with lamivudine: 0% at one year and 29% at 5 years.90 This makes adefovir an option as add-on therapy in patients who have developed lamivudine resistance.91 Adefovir has not been well examined in patients with renal failure. A French study used adefovir in a composite series of 12 patients with CKD,92 all of whom had lamivudine-resistant HBV. There was a significant fall in HBV DNA levels after a median of 15 months of therapy. Only one of these patients was actually receiving dialysis during the study. A case report described successful treatment of HBV infection in

a dialysis-dependent liver transplant recipient who had lamivudine-resistant infection and cirrhosis of the allograft.93 Entecavir is a promising drug in the management GSK458 of HBV infection. In patients with normal renal function, entecavir has been shown to be superior to lamivudine94 and adefovir95 in reducing HBV DNA levels. Although there are not the long-term data that exist for lamivudine, resistance

rates appear to be low. Entecavir has not been studied in dialysis patients, although the dose should be reduced in renal failure.79 Tenofovir, a nucleotide reverse transcriptase inhibitor, is recommended as a buy MLN0128 first-line oral antiviral in HBV patients with normal renal function.96 Although larger series have not found tenofovir to be culpable in HIV patients with Erastin mouse renal failure,97 there have been a number of case reports of tubular toxicity and acute kidney injury98–100 with tenofovir use. This raises concern regarding the potential for nephrotoxicity in dialysis patients with residual renal function. A case report showed that tenofovir was effective in a single HBV-infected HD patient. This paper also assessed tenofovir pharmacokinetics,101 and recommended

a dose of 300 mg once a week to prevent accumulation. This was endorsed by the manufacturers in a study of nine HD patients.102 In summary, lamivudine has the most solid body of experience to support its use. Tenofovir and entecavir are likely to be more effective, and tenofovir has been shown to be safe in HD patients, but neither drug has any significant evidence base from this patient group. Determining which dialysis patients with chronic HBV infection to treat is a matter of controversy. In the case of patients with normal renal function, treatment is recommended for those with active HBV replication (HBeAg positive and/or HBV DNA positive) and raised alanine transaminase (ALT) levels.103 It is clear that patients with ESRD exhibit a different clinical and biochemical picture in chronic HBV infection.104 HD patients with HBV infection are less likely to have a symptomatic acute illness, and are more likely to develop chronic carrier status.

Recombinant Tax1 and Tax2A (subtype A) proteins were purified as

Recombinant Tax1 and Tax2A (subtype A) proteins were purified as described recently in detail [24, 25]. Truncated Tax2A/NH2term-His tagged sequence aa 1–198 (Tax2A/1–198) (MRGSHHHHHHGS AHFPGFGQSL LYGYPVYVFG DCVQADWCPV SGGLCSTRLH RHALLATCPE HQL TWDPIDG RVVSSPLQYL IPRLPSFPTQ RTSRTLKVLT PPTTPVSPKV PPAFFQSMRK HTPYRNGCLE

PTLGDQLPSL AFPEPGLRPQ NIYTTWGKTV VCLYLYQLSP PMTWPLIPHV IFCHPRQLGA FLTKVPLKRL EELLYKM LDLQPSLIS) and truncated Tax2A/COOHterm-His tagged sequence aa 135–331 (Tax2A/135–331) (MRGSHHHHHHGS EPGLRPQNIY TTWGKTVVCL YLYQLSPPMT WPLIPHVIFC HPRQLGAFLT KVPLKRLEEL LYKMFLHTGT VIVLPEDDLP TTMFQPVRAP CIQTAWCTGL LPYHSILTTP GLIWTFNDGS PMISGPCPKA GQPSLVVQSS LLIFEKFQTK AFHPSYLLSH QLIQYSSFHN LHLLFDEYTN IPVSILFNKE EADDNGD LDLQPSLIS) fragments of Tax2A protein containing NF-κB domains [28, 29] were subcloned from PET29a-Tax2-H6 [30] to pQE-30 (Amp-resistant) vector and transformed into the Esherichia coli BL21(DE3) strain (subcloning generation and protein production serviced by Promab Biotechnologies, Inc., Richmond, RXDX-106 ic50 CA, USA). An extract was prepared in an identical manner from E. coli

cells containing the empty vector for use as a mock control. Determination of protein concentrations was performed using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Endotoxin concentration for all protein recombinants at the concentration (100 pM) used in the in-vitro experiments were found to be endotoxin-free, as determined Dichloromethane dehalogenase by the limulus amoebocyte lysate test (E-TOXATE; Sigma) [24]. Recombinant replication-deficient adenoviruses expressing Tax2B (subtype B) or green fluorescent protein (GFP), used to control the efficiency of transduction (Ad-Tax2B or Ad-GFP, respectively) [31], were propagated and titrated as described recently [25]. The recombinant adenovirus containing the dominant negative mutant of IκBα with serine to alanine substitutions at amino acids 32 and 36, and therefore resistant to phosphorylation-induced degradation (a NF-κB super-repressor

designated NF-κB/SR), was obtained commercially (Vector Biolabs, Philadelphia, PA, USA). In this study the two major subtypes of HTLV-2 Tax, Tax2A (expressed as recombinant protein) and Tax2B (recombinant adenovirus) were assessed to determine whether both Tax2 subtypes were able to induce the production of CC-chemokines in peripheral blood mononuclear cells. PBMCs (1 × 106/ml) in complete RPMI medium were treated with extracellular Tax (recombinant Tax1 and Tax2A) proteins at 100 pM for 1, 2, 3, 6, 12 and 24 h to determine CC-chemokine production, and for 1 and 2 h for the determination of canonical NF-κB pathway activation. Mock-treated and untreated controls were used in all experiments.