Loss of PKR Activity in Chronic Lymphocytic Leukemia

There are a number of observations that suggest the dsRNA-activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full-length PKR is present but not active in 21 of 28 patient samples from B-cell chronic lymphocytic leukemia (B-CLL). PKR from these patients was unable to auto-activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58(IPK). We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B-CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL. (C) 2004 Wiley-Liss, Inc.

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN > 44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Lymphoproliferative disease of the ocular adnexa: A clinical and pathologic study with statistical analysis of 69 patients

Purpose: To evaluate the clinical features, treatment, and outcomes of a cohort of patients with ocular adnexal lymphoproliferative disease classified according to the World Health Organization modification of the Revised European-American Classification of Lymphoid neoplasms and to perform a robust statistical analysis of these data. Methods: Sixty-nine cases of ocular adnexal lymphoproliferative disease, seen in a tertiary referral center from 1992 to 2003, were included in the study. Lesions were classified by using the World Health Organization modification of the Revised European-American Classification of Lymphoid neoplasms classification. Outcome variables included disease-specific Survival, relapse-free survival, local control, and distant control. Results: Stage IV disease at presentation, aggressive lymphoma histology, the presence of prior or concurrent systemic lymphoma at presentation, and bilateral adnexal disease were significant predictors for reduced disease-specific survival, local control, and distant control. Multivariate analysis found that aggressive histology and bilateral adnexal disease had significantly reduced disease-specific Survival. Conclusions: The typical presentation of adnexal lymphoproliferative disease is with a painless mass, swelling, or proptosis; however, pain and inflammation occurred in 20% and 30% of patients, respectively. Stage at presentation, tumor histology, primary or secondary status, and whether the process was unilateral or bilateral were significant variables for disease outcome. In this study, distant spread of lymphoma was lower in patients who received greater than 20 Gy of orbital radiotherapy.

Cutting edge: Species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified ohgonucleotides

Different DNA motifs are required for optimal stimulation Of mouse and human immune cells by CpG oligode-oxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CPG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 AWK than optimal motifs. When the CpG dinucleotide was inverted to GC In each ODN some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may he responsible for mediating many published CpG-independent responses to PS-ODN.

Modelling cell lifespan and proliferation: is likelihood to die or to divide independent of age?

In cell lifespan studies the exponential nature of cell survival curves is often interpreted as showing the rate of death is independent of the age of the cells within the population. Here we present an alternative model where cells that die are replaced and the age and lifespan of the population pool is monitored until a, steady state is reached. In our model newly generated individual cells are given a determined lifespan drawn from a number of known distributions including the lognormal, which is frequently found in nature. For lognormal lifespans the analytic steady-state survival curve obtained can be well-fit by a single or double exponential, depending on the mean and standard deviation. Thus, experimental evidence for exponential lifespans of one and/or two populations cannot be taken as definitive evidence for time and age independence of cell survival. A related model for a dividing population in steady state is also developed. We propose that the common adoption of age-independent, constant rates of change in biological modelling may be responsible for significant errors, both of interpretation and of mathematical deduction. We suggest that additional mathematical and experimental methods must be used to resolve the relationship between time and behavioural changes by cells that are predominantly unsynchronized.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

Unicellular pheromone glands of the pentatomid bug Nezara viridula (Heteroptera : Insecta): Ultrastructure, classification, and proposed function

Male Nezara viridula produce sex pheromones from many independent single cells, each with a duct that opens onto the ventral abdominal surface. Despite the presence of along duct and an associated end complex (in the form of a cupule and microvillus saccule), the structural organization of the cells that comprise the gland conform to Class 1 epidermal gland cell classification : a single cell surrounds the entire secretory complex. Each cuticular cupule contains a central bed of filaments and opens into a narrow tubular ductule that leads from the base of the cupule through the epidermis to the cuticle to open externally as a pore. The cuticle of the cupule is continuous with that of the ductule and has the appearance of three layers, although the inner (middle) layer may be a gap formed during construction of the complex. In young adult males, just molted, the ultrastructure of the cells and their inclusions indicate that they are not active. The region of the cell that is distal to the abdominal cuticle is reduced and the proximal region, surrounding the duct, is enlarged when compared with sexually mature (3-4 weeks old) adult males. At maturity the pheromone cells are enlarged distally around the cupule, but are reduced to a narrow sleeve proximally, around the ductule. Two characteristic cell profiles are evident, based on the shape of the cupule and the organelle content. Type A shows a broad opening to the cupule, an abundance of mitochondria, and few vesicular bodies. Type B has an elongated, narrow, vase-like opening to the cupule, few mitochondria, and numerous vesicular bodies. Type B cells are smaller and more abundant than Type A. Distribution within the epidermal layer also differs. It is likely that the different types represent cells producing different secretion profiles. However, the secretions retained by the standard fixation protocol within mature cells of both types look similar and appear to collect as crystalline bodies within the lumen. This may represent a common storage mechanism.

Periocular and Orbital Amyloidosis. Clinical Characteristics, Management, and Outcome

Objective: To present the clinical features and management outcome in a large series of patients with periocular and orbital amyloidosis. Design: Retrospective, noncomparative, interventional case series. Patients: All patients diagnosed with periocular and orbital amyloidosis in 6 oculoplastic and orbital units. Methods: Clinical records of all patients were reviewed. Main Outcome Measures: Clinical presentation, radiological and histological findings, treatment modalities, and outcome. Results. The study included 24 patients (15 female, 9 male) with a mean age of 57 17 years. Nineteen cases were unilateral, and 5 were bilateral. Clinical signs and symptoms included a visible or palpable periocular mass or tissue infiltration (95.8%), ptosis (54.2%), periocular discomfort or pain (25%), proptosis or globe displacement (21%), limitations in ocular motility (16.7%), recurrent periocular subcutaneous hemorrhages (12.5%), and diplopia (8.3%). Seven cases had orbital involvement, and 17 were periocular. Immunohistochemistry in 7 patients showed B cells or plasma cells producing monoclonal immunoglobulin chains that were deposited as amyloid light chains. Only 1 patient was diagnosed with systemic amyloid light chain amyloidosis. Treatment modalities were mainly observation and surgical debulking. During a mean follow-up period of 39 months, 21% showed significant progression after treatment, whereas 79% were stable or showed no recurrence after treatment. Conclusion: Periocular and orbital amyloidosis may present with a wide spectrum of clinical findings and result in significant ocular morbidity. Complete surgical excision is not feasible in many cases, and the goal of treatment is to preserve function and to prevent sight-threatening complications.

Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.

Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins

Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr etoposide treatment (80 muM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p < 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.

Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

T cells signaled by NF-kappa B- dendritic cells are sensitized not anergic to subsequent activation

Paradoxically, while peripheral self-tolerance exists for constitutively presented somatic self Ag, self-peptide recognized in the context of MHC class II has been shown to sensitize T cells for subsequent activation. We have shown that MHC class II(+)CD86(+)CD40(-) DC, which can be generated from bone marrow in the presence of an NF-kappaB inhibitor, and which constitutively populate peripheral tissues and lymphoid organs in naive animals, can induce Ag-specific tolerance. In this study, we show that CD40(-) human monocyte-derived dendritic cells (DC), generated in the presence of an NF-kappaB inhibitor, signal phosphorylation of TCRzeta, but little proliferation or IFN-gamma in vitro. Proliferation is arrested in the G(1)/G(0) phase of the cell cycle. Surprisingly, responding T cells are neither anergic nor regulatory, but are sensitized for subsequent IFN-gamma production. The data indicate that signaling through NF-kappaB determines the capacity of DC to stimulate T cell proliferation. Functionally, NF-kappaB(-)CD40(-)class II+ DC may either tolerize or sensitize T cells. Thus, while CD40(-) DC appear to prime or prepare T cells, the data imply that signals derived from other cells drive the generation either of Ag-specific regulatory or effector cells in vivo.

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.