Rapid improvement in symptoms and physical function following ibrutinib initiation in chronic lymphocytic leukemia and the associated changes in plasma cytokines.

A prospective pilot study was carried out on 34 CLL patients treated with ibrutinib, evaluating the effects on symptoms and physical function with changes in plasma exosomes (EXs), ß2-microglobulin (ß2M) and 26 plasma cytokines. The revised Edmonton Symptom Assessment Scale (ESAS-R) demonstrated moderate fatigue, shortness of breath and a sense of unwellness before treatment, which significantly improved within 2 weeks of starting ibrutinib. These changes were associated with a rapid improvement in sit-to-stand and 4 m walking speeds. The plasma levels of CCL11, IL-7, -8 and -10 dropped initially while the levels of TNF-α/-ß, CCL3, CCL4, CCL17, and IL-16 continued to decline for 12 months. Despite the initial lymphocytosis, plasma ß2M levels fell but no consistent change in plasma EXs occurred. Thus, ibrutinib can produce a rapid and sustained improvement in symptoms and physical function in CLL, associated with a decline in multiple plasma cytokines.

Descriptive analysis of dosing and outcomes for patients with ibrutinib-treated relapsed or refractory chronic lymphocytic leukemia in a Canadian centre.

Background: Ibrutinib is an approved treatment for relapsed or refractory chronic lymphocytic leukemia (cll) and small lymphocytic lymphoma (sll). The effect of ibrutinib dose reduction compared with discontinuation in a population-based setting is unclear. Methods: To examine the patterns of ibrutinib use in a Canadian population-based setting, we analyzed a retrospective cohort of patients with relapsed or refractory cll or sll treated with ibrutinib. Results: The 64 patients diagnosed with cll or sll had a median age of 76.5 years. Most had unmutated ighv (immunoglobulin variable heavy chain). A hematologic response occurred in 39 patients regardless of the ibrutinib dose. The most common toxicities were infection, bruising or bleeding, and musculoskeletal problems, with a median time to first toxicity of 14 days. More than half the cohort experienced a dose reduction, with musculoskeletal problems, cytopenias, and infection being the leading causes; surgery was the most frequent indication for holding treatment. Only 26 of the 64 patients (40.6%) stayed on the recommended maximal dose of ibrutinib. No differences in reported toxicities or hematologic response rates were evident between the patients receiving maximal and submaximal therapy. At the end of the study period, 53 patients from the initial cohort remained on ibrutinib. Conclusions: More than half the study patients received ibrutinib therapy at a submaximal dose without evidence of increased frequency of toxicities or disease progression. The rate of ibrutinib discontinuation was lower in our cohort than has been reported in other settings. Submaximal ibrutinib dosing will have to be further systematically evaluated.

Lysosomotropic agents selectively target chronic lymphocytic leukemia cells due to altered sphingolipid metabolism.

Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL.

Relationship between progression-free survival and overall survival in chronic lymphocytic leukemia: a literature-based analysis.

BACKGROUND: The endpoints of progression-free survival (pfs) and time-to-progression (ttp) are frequently used to evaluate the clinical benefit of anticancer drugs. However, the surrogacy of those endpoints for overall survival (os) is not validated in all cancer settings. In the present study, we used a trial-based approach to assess the relationship between median pfs or ttp and median os in chronic lymphocytic leukemia (cll). METHODS: The pico (population, interventions, comparators, outcomes) method was used to conduct a systematic review of the literature. The population consisted of patients with cll; the interventions and comparators were standard therapies for cll; and the outcomes were median pfs, ttp, and os. Two independent reviewers screened titles, abstracts, and full papers for eligibility and then extracted data from selected studies. Correlation coefficients were calculated to assess the relationship between median pfs or ttp and median os. Subgroup correlation analyses were also conducted according to the characteristics of the selected studies (such as line of treatment and type of treatment under investigation). RESULTS: Of the 1263 potentially relevant articles identified during the literature search, twenty-three were included. On average, median pfs or ttp was 16.0 months (standard deviation: 12.4 months) and median os was 43.5 months (standard deviation: 31.2 months). Results of the correlation analysis indicated that median pfs or ttp is highly correlated with median os (Spearman correlation coefficient: 0.813; p ≤ 0.001). A significant correlation between median pfs or ttp and median os was observed in second- and subsequent-line therapies, but not in the first-line setting. CONCLUSIONS: Our study demonstrates a strong correlation between median pfs or ttp and median os in previously treated cll, which reinforce the hypothesis that pfs and ttp could be adequate surrogate endpoints for os in this cancer setting.

Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling.

Chronic lymphocytic leukemia (CLL) can be divided into groups based on biomarkers of poor prognosis. The expression of the tyrosine kinase ZAP-70 (member of the Syk tyrosine kinase family) in CLL cells is associated with shorter overall survival in CLL patients. Currently, there is a lack of targeted therapies for patients with ZAP-70 expression in CLL cells. The tyrosine kinase inhibitor gefitinib has been shown to be effective at induce apoptosis in acute myeloid leukemia through inhibition of Syk. In this study, we sought to test the efficacy of gefitinib in primary human ZAP-70+ CLL cells. We demonstrate that gefitinib preferentially induces cell death in ZAP-70-expressing CLL cells with a median IC50 of 4.5 µM. In addition, gefitinib decreases the viability of ZAP-70+ Jurkat T leukemia cells but fails to affect T cells from CLL patients. Western blot analysis shows gefitinib reduces both basal and B-cell receptor (BCR)-stimulated phosphorylation of Syk/ZAP-70, ERK, and Akt in ZAP-70+ CLL cells. Moreover, gefitinib inhibits the pro-survival response from BCR stimulation and decreases pro-survival proteins such as Mcl-1. Finally, ZAP-70 expression sensitizes Raji cells to gefitinib treatment. These results demonstrate that gefitinib specifically targets ZAP-70+ CLL cells and inhibits the BCR cell survival pathway leading to apoptosis. This represents the likelihood of tyrosine kinase inhibitors being effective targeted treatments for ZAP-70+ CLL cells.

Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B.

Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins.

Increased risk of second malignancies in chronic lymphocytic leukaemia patients as compared with follicular lymphoma patients: a Canadian population-based study.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) patients have an increased risk of other malignancies. This may be due to surveillance bias, treatment or immunosuppression. METHODS: Cohort study of 612 consecutively diagnosed CLL patients in a Canadian province, with comparisons to follicular lymphoma (FL) patients. RESULTS: Treated CLL patients had a 1.7-fold increased risk of second cancers compared with untreated CLL patients. As compared with untreated FL patients, untreated CLL patients had a two-fold increased incidence of second malignancies. CONCLUSION: Chronic lymphocytic leukaemia patients have an inherent predisposition to second cancers and the incidence is further increased by treatment.

High incidence of chronic lymphocytic leukemia (CLL) diagnosed by immunophenotyping: a population-based Canadian cohort.

Incidence and outcomes of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are not well established at the population level, especially since the widespread use of immunophenotyping. We studied the epidemiology of CLL in Manitoba (Canada) by combining data from a centralized flow cytometry facility and the provincial cancer registry for the period 1998-2003. Of 616 cases identified, 27% of patients identified by flow cytometry were not on the cancer registry. The age-adjusted incidence of 7.99/100,000 is substantially higher than the reported incidence in registry reports. We also noted differences in relative survival based on age and gender.

Myxoma virus M-T5 protects infected cells from the stress of cell cycle arrest through its interaction with host cell cullin-1.

The myxoma virus (MV) M-T5 gene encodes an ankyrin repeat protein that is important for virus replication in cells from several species. Insight was gained into the molecular mechanisms underlying the role of M-T5 as a host range determinant when the cell cycle regulatory protein cullin-1 (cul-1) was identified as a cellular binding partner of M-T5 and found to colocalize with the protein in both nuclear and cytosolic compartments. Consistent with this interaction, infection with wild-type MV (vMyxlac) or a deletion mutant lacking M-T5 (vMyxT5KO) differentially altered cell cycle progression in a panel of permissive and nonpermissive cells. Cells infected with vMyxlac transitioned rapidly out of the G0/G1 phase and preferentially accumulated at the G2/M checkpoint, whereas infection with vMyxT5KO impeded progression through the cell cycle, resulting in a greater percentage of cells retained at G0/G1. Levels of the cul-1 substrate, p27/Kip-1, were selectively increased in cells infected with vMyxT5KO compared to vMyxlac, concurrent with decreased phosphorylation of p27/Kip-1 at Thr187 and decreased ubiquitination. Compared to cells infected with vMyxlac, cell death was increased in vMyxT5KO-infected cells following treatment with diverse stimuli known to induce cell cycle arrest, including infection itself, serum deprivation, and exposure to proteasome inhibitors or double-stranded RNA. Moreover, infection with vMyxlac, but not vMyxT5KO, was sufficient to overcome the G0/G1 arrest induced by these stimuli. These findings suggest that M-T5 regulates cell cycle progression at the G0/G1 checkpoint, thereby protecting infected cells from diverse innate host antiviral responses normally triggered by G0/G1 cell cycle arrest.

Myxoma virus infection of primary human fibroblasts varies with cellular age and is regulated by host interferon responses.

Recent studies have indicated a critical role for interferon (IFN)-mediated antiviral responses in the host range of myxoma virus (MV), a pathogenic poxvirus of rabbits. To investigate the contribution of IFN to MV tropism in nonleporine cells, primary human dermal fibroblasts (HDFs) were tested for permissiveness to MV infection. Low-passage HDFs that underwent fewer than 25 population doublings (PD) were fully permissive for MV infection, supporting productive virus replication and cell-to-cell spread. In contrast, early and late viral gene expression was detectable in high-passage HDF (>75 PD), but MV failed to generate infectious progeny and could not form foci in these cells. Vesicular stomatitis virus (VSV) plaque reduction assays confirmed that constitutive IFN production progressively increased as HDFs were passaged, concurrent with an increase in the expression of transcripts for type I IFN and IFN-responsive genes involved in antiviral responses. These findings correlated with the enhanced sensitivity of higher-passage HDF to inducers of type I IFN responses, such as dsRNA. Furthermore, pretreatment of low-passage HDF with type I IFN abrogated MV spread and replication while treatment of mature HDF with neutralizing antibodies to IFN-beta, but not IFN-alpha, restored the capacity to form foci. These findings emphasize the importance of post-entry events in determining the permissiveness of human cells to MV infection and support a critical role for innate type I IFN responses as key determinants of poxvirus host range and species restriction.

Technical knockout: understanding poxvirus pathogenesis by selectively deleting viral immunomodulatory genes.

The study of viral pathogens with genomes as large and complex as poxviruses represents a constant experimental challenge. Advances in recombinant DNA technologies have provided sophisticated methods to produce mutants defective in one or more viral genes, termed knockout (KO) viruses, thereby facilitating research into the impact of specific gene products on viral pathogenesis. Such strategies have rapidly advanced the systematic mining of many poxvirus genomes and enabled researchers to identify and characterize poxvirus genes whose functions represent the culmination of host and pathogen coevolution. Of particular interest are the multiple classes of virus-encoded immunomodulatory proteins that have evolved specifically to allow poxviruses to evade, obstruct or subvert critical elements within the host innate and acquired immune responses. Functional characterization of these viral genes by generating KO viruses and investigating the phenotypic changes that result is an important tool for understanding the molecular mechanisms underlying poxvirus replication and pathogenesis. Moreover, the insights gained have led to new developments in basic and clinical virology, provided a basis for the design of new vaccines and antivirals, and increased the potential application of poxviruses as investigative tools and sources of biotherapeutics for the treatment of human diseases.

Role of the serine-threonine kinase PAK-1 in myxoma virus replication.

Subversion or appropriation of cellular signal transduction pathways is a common strategy employed by viruses to promote an environment within infected cells that supports the viral replicative cycle. Using subsets of 3T3 murine fibroblasts previously shown to differ in their ability to support myxoma virus (MV) replication, we investigated the role of host serine-threonine kinases (STKs) as potential mediators of the permissive phenotype. Both permissive and nonpermissive 3T3 cells supported equivalent levels of virion binding, entry, and early virus gene expression, indicating that MV tropism in 3T3 cells was not determined by receptor-mediated entry. In contrast, late virus gene expression and viral DNA replication were selectively compromised in restrictive 3T3 cells. Addition of specific protein kinase inhibitors, many of which shared the ability to influence the activity of the STKs p21-activated kinase 1 (PAK-1) and Raf-1 attenuated MV replication in permissive 3T3 cells. Western blot detection of the phosphorylated forms of PAK-1 (Thr423) and Raf-1 (Ser338) confirmed activation of these kinases in permissive cells after MV infection or gamma interferon treatment, but the activated forms of both kinases were greatly reduced or absent in restrictive 3T3 cells. The biological significance of these activations was demonstrated by using the autoinhibitory domain of PAK-1 (amino acids 83 to 149), expression of which reduced the efficiency of MV infection in permissive 3T3 cells concurrent with a decrease in PAK-1 activation. In comparison, overexpression of a constitutively active PAK-1 (T423E) mutant increased MV replication in restrictive 3T3 cells. These observations suggest that induced signaling via cellular STKs may play important roles in determining the permissiveness of host cells to poxvirus infection.

Poxviruses and immune evasion.

Large DNA viruses defend against hostile assault executed by the host immune system by producing an array of gene products that systematically sabotage key components of the inflammatory response. Poxviruses target many of the primary mediators of innate immunity including interferons, tumor necrosis factors, interleukins, complement, and chemokines. Poxviruses also manipulate a variety of intracellular signal transduction pathways such as the apoptotic response. Many of the poxvirus genes that disrupt these pathways have been hijacked directly from the host immune system, while others have demonstrated no clear resemblance to any known host genes. Nonetheless, the immunological targets and the diversity of strategies used by poxviruses to disrupt these host pathways have provided important insights into diverse aspects of immunology, virology, and inflammation. Furthermore, because of their anti-inflammatory nature, many of these poxvirus proteins hold promise as potential therapeutic agents for acute or chronic inflammatory conditions.

Neurovirulence depends on virus input titer in brain in feline immunodeficiency virus infection: evidence for activation of innate immunity and neuronal injury.

Lentiruses cause neurological disease depending on the virus strain and its neurotropism, yet it remains uncertain to what the impact of infectious virus quantity in the brain early in infection is on the subsequent development of neurological disease or neurovirulence. We investigated the relationship between infectious virus input titer and the resulting neurovirulence, using ex vivo and in vivo assays of feline immunodeficiency virus (FIV)-induced neurovirulence. FIV infection of cell cultures and neonatal cats was performed using 10(2.5) (low-titer) or 10(4.5) (high-titer) 50% tissue culture infectious doses (TCID(50))/ml of the neurovirulent FIV strain, V1CSF. Ex vivo neurotoxicity assays revealed that conditioned medium (CM) from feline macrophages infected with high-titer (P <.001) or low-titer (P <.01) V1CSF induced greater neuronal death than CM from mock-infected cells. In vivo, animals infected intracranially with high-titer V1CSF showed neurodevelopmental delays compared to mock-infected animals (P <.001) and animals infected with low-titer V1CSF (P <.02), concurrent with reduced weight gains and greater depletion of CD4+ cells over a 12-week period. Neuropathological changes, including astrogliosis, macrophage activation, and neuronal damage, were evident in V1CSF-infected animals and were viral titer dependent. In vivo magnetic resonance (MR) spectroscopy and proton nuclear magnetic resonance ((1)H-NMR) spectroscopy of tissue extracts revealed evidence of neuronal injury, including reduced N-acetyl aspartate/creatine (P <.05) and increased trimethylamine/creatine (P <.05) ratios, in the frontal cortex of high-titer V1CSF-infected animals compared to the other groups. T2-weighted MR imaging detected increased signal intensities in the frontal cortex and white matter of V1CSF-infected animals relative to controls, which was more evident as viral titer increased (P <.01). The present findings indicate that lentivirus infectious titers in the brain during the early stages of infection determine the severity of neurovirulence, reflected by neurobehavioral deficits, together with neuroradiological and neuropathological findings of activation of innate immunity and neuronal injury.

Feline immunodeficiency virus xenoinfection: the role of chemokine receptors and envelope diversity.

The use of chemokine receptors as cell recognition signals is a property common to several lentiviruses, including feline, human, and simian immunodeficiency viruses. Previously, two feline immunodeficiency virus (FIV) isolates, V1CSF and Petaluma, were shown to use chemokine receptors in a strain-dependent manner to infect human peripheral blood mononuclear cells (PBMC) (J. Johnston and C. Power, J. Virol. 73:2491-2498, 1999). Since the sequences of these viruses differed primarily in regions of the FIV envelope gene implicated in receptor use and cell tropism, envelope chimeras of V1CSF and Petaluma were constructed to investigate the role of envelope diversity in the profiles of chemokine receptors used by FIV to infect primate cells. By use of a receptor-blocking assay, all viruses were found to infect human and macaque PBMC through a mechanism involving the CXCR4 receptor. However, infection by viruses encoding the V3-to-V5 region of the V1CSF surface unit was also inhibited by blockade of the CCR3 or CCR5 receptor. Similar results were obtained with GHOST cells, human osteosarcoma cells expressing specific combinations of chemokine receptors. CXCR4 was required for infection by all FIV strains, but viruses expressing the V3-to-V5 region of V1CSF required the concurrent presence of either CCR3 or CCR5. In contrast, CXCR4 alone was sufficient to allow infection of GHOST cells by FIV strains possessing the V3-to-V5 region of Petaluma. To assess the role of primate chemokine receptors in productive infection, Crandell feline kidney (CrFK) cells that expressed human CXCR4, CCR3, or CCR5 in addition to feline CXCR4 were generated. Sustained infection by viruses encoding the V3-to-V5 region of V1CSF was detected in CrFK cells expressing human CCR3 or CCR5 but not in cells expressing CXCR4 alone, while all CrFK cell lines were permissive to viruses encoding the V3-to-V5 region of Petaluma. These results indicate that FIV uses chemokine receptors to infect both human and nonhuman primate cells and that the profiles of these receptors are dependent on envelope sequence, and they provide insights into the mechanism by which xenoinfections may occur.

Envelope gene-mediated neurovirulence in feline immunodeficiency virus infection: induction of matrix metalloproteinases and neuronal injury.

The release of neurotoxins by activated brain macrophages or microglia is one mechanism proposed to contribute to the development of neurological disease following infection by lentiviruses, including feline immunodeficiency virus (FIV). Since molecular diversity in the lentiviral envelope gene influences the expression of host molecules implicated in neuronal injury, the role of the envelope sequence in FIV neuropathogenesis was investigated by using the neurovirulent FIV strain V1CSF, the nonneurovirulent strain Petaluma, and a chimera (FIVCh) containing the V1CSF envelope gene in a Petaluma background. All three viruses replicated in primary feline macrophages with equal efficiency, but conditioned medium from V1CSF- or FIVCh-infected cells was significantly more neurotoxic than medium from Petaluma-infected cultures (P < 0.001) and could be attenuated in a dose-dependent manner by treatment with either the matrix metalloproteinase (MMP) inhibitor prinomastat (PMT) or function-blocking antibodies to MMP-2. Although FIV sequences were detectable by PCR in brain tissue from neonatal cats infected with each of the viral strains, immunohistochemistry revealed increased astrogliosis and macrophage activation in the brains of V1CSF- and FIVCh-infected cats relative to the other groups, together with elevated markers of neuronal stress that included morphological changes and increased c-fos immunoreactivity. Similarly, MMP-2, but not MMP-9, mRNA and protein expression was increased in brain tissues of V1CSF- and FIVCh-infected cats relative to Petaluma-infected animals (P < 0.01). Infection with V1CSF or FIVCh was also associated with greater CD4(+) cell depletion (P < 0.001) and neurodevelopmental delays (P < 0.005), than in Petaluma-infected animals; these deficits improved following PMT therapy. These findings indicated that diversity in the envelope gene sequence influenced the neurovirulence exhibited by FIV both in vitro and in vivo, possibly through a mechanism involving the differential induction of MMP-2.

Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases.

Human endogenous retroviruses (HERVs) have been implicated as causative agents in diseases characterized by inflammation and macrophage activation, such as multiple sclerosis. Because monocyte activation and differentiation influence retroviral transcription and replication, we investigated the contribution of these processes to the expression of four HERV families (HERV-W, HERV-K, HERV-E, and HERV-H) in human monocytes, and autopsied brain tissue from patients with brain diseases associated with increased macrophage activity. Reverse transcriptase-polymerase chain reaction analysis of primary macrophages and U937 monocytoid cells stimulated with phorbol-12-myristate-13-acetate or lipopolysaccharide revealed three- to ninefold increases in HERV-W, HERV-K, and HERV-H RNA levels. In addition, elevated reverse transcriptase activity and HERV RNA were detectable in supernatants from PMA-stimulated U937 cultures, properties that could be attenuated with the inhibitor of monocyte differentiation threonine-lysine-proline. In contrast, stimulation of monocytes decreased or had no effect on HERV-E expression. Compared with controls, HERV-W and HERV-K expression was increased in brain tissue from patients with multiple sclerosis or human immunodeficiency virus infection or AIDS, with concomitant elevated tumor necrosis factor-alpha levels. Similarly, elevated HERV-W levels were detected in patients with Alzheimer's dementia only when tumor necrosis factor-alpha expression was also evident (2 of 6 cases). The detection of several HERVs in inflammatory brain diseases and the capacity to augment HERV expression in monocytes with compounds that influence cellular activity suggest that increased expression of these viruses is a consequence of increased immune activity rather than causative of distinct diseases.

Xenoinfection of nonhuman primates by feline immunodeficiency virus.

New viral infections in humans usually result from viruses that have been transmitted from other species as zoonoses. For example, it is accepted widely that human immunodeficiency virus (HIV) is the result of the propagation and adaptation of a simian immunodeficiency virus (SIV) from nonhuman primates to man [1]. Previously, we reported productive infection of primary human cells in vitro by feline immunodeficiency virus (FIV) [2], a lentivirus that causes an immunodeficiency syndrome in cats similar to HIV in humans [3]. The present study extends these findings by demonstrating that cynomolgus macaques (Macaca fasicularis) infected with FIV exhibited clinical signs, including depletion of CD4+ cells and weight loss, that are consistent with FIV infection. The development of an antibody response to FIV gag-encoded proteins and detection of virus-specific sequences in sera, blood-derived cells, and necropsied tissue accompanied these changes. Moreover, the reactivation of FIV replication from latently infected cells was observed after stimulation in vitro with phorbol esters and in vivo with tetanus toxoid. The proposed use of lentiviruses in human gene therapy [4, 5] and of nonhuman cells and organs in xenotransplantation [6] has raised concerns about zoonoses as potential sources of new human pathogens. Therefore, the study of FIV infection of primate cells may provide insight into the principles underlying retroviral xenoinfections.

Diminished adenosine A1 receptor expression on macrophages in brain and blood of patients with multiple sclerosis.

The nucleoside adenosine has been shown to control the production of proinflammatory molecules through its actions on cell surface purine receptors. Previously, we have reported that the adenosine A1 receptor (A1AR) regulates tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) expression and exhibits diminished function in patients with multiple sclerosis (MS; Mayne et al., Ann Neurol 1999;45:633-639). In the present study, A1AR expression in both brain and peripheral blood mononuclear cells (PBMC) from MS and control groups was characterized by fluorescence-activated cell sorting (FACS), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical analyses. FACS analyses of PBMC revealed that A1AR expression was chiefly detectable on CD14-positive cells and was reduced by 53.1% (p < 0.01) in MS patients compared to controls. A1AR mRNA levels were reduced by 43.1% (p < 0.001) in the brains of MS patients compared to patients with other neurological diseases and controls. A1AR protein expression in brain was detected primarily in CD45-positive glial cells and was markedly diminished in MS patients. The analysis of A1AR transcripts in the brain revealed that the A1AR-beta transcript was diminished (49.2%) in MS patients compared to controls (p < 0.002). These results indicate that the A1AR, expressed principally on cells of monocyte/macrophage lineage in both brain and blood, is selectively diminished in MS patients. Reduction of the A1AR-beta transcript in MS patients suggests that dysregulated splicing may influence A1AR protein levels, potentially leading to increased macrophage activation and central nervous system inflammation.