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Follicular lymphoma (FL) involving the spleen must be distinguished from reactive hyperplasia and from other lymphomas. A prior study reported that splenic FLs frequently lack BCL2 expression, further complicating diagnosis. We examined 16 cases of splenic FL, including 12 cases initially diagnosed at splenectomy. Two morphologic patterns were identified: one with architectural abnormalities (AA) and one with an extensive architectural preservation (AP) pattern. Newly diagnosed AP cases were associated with older age (P = .051) and grade 1 histologic features (P = .023). All cases displayed a CD10+/BCL2+ phenotype. Cytogenetics and FISH identified IGH/BCL2 or BCL6 translocations in all tested cases. Splenic FLs display phenotypic and cytogenetic findings similar to nodal FLs. However, splenic FLs frequently display an exclusively intrafollicular growth pattern resembling so-called in situ FL. Recognition of subtle FL with preserved architecture is important because patients may have overt FL at other sites or the FL may progress to overt nodal disease.

Context.—Chronic myelogenous leukemia (CML) and the assessment of the BCR-ABL transcript has become a new paradigm. Novel tyrosine kinase inhibitors as mainstream therapeutic options for the CML patient warrant routine quantification of the BCR-ABL transcript. The Xpert BCR-ABL Monitor assay is a nested reverse transcriptase polymerase chain reaction that greatly reduces technical time by using a single cartridge to isolate RNA and run a quantitative reverse transcriptase polymerase chain reaction. Objective.—To evaluate the Xpert BCR-ABL Monitor assay for quantitative assessment of the BCR-ABL transcript in CML patients. Design.—A standard curve of K-562 cells diluted in normal peripheral blood was used to test the sensitivity, linearity, and percent coefficient of variation of the assay. Specimen stability was tested by running standard curves immediately and after 24 hours or 96 hours of storage at 4°C. Specimens from normal controls, patients known to have CML, or patients suspected of having CML were also tested. Results.—The sensitivity of the assay was sufficient to detect 1 K-562 cell in 105 normal cells. The R2 of the standard curve was 0.98 and the percent coefficient of variation for each data point was 15% to 24%. Eleven of 14 patients with known CML on imatinib treatment tested positive for the BCR-ABL transcript, whereas 10 normal controls tested negative. Conclusions.—The Xpert BCR-ABL Monitor assay is a rapid, sensitive method for monitoring the presence of the BCR-ABL transcript in CML patients. The single-use cartridge minimizes hands-on technical time, minimizes the potential for contamination, and allows quantitative BCR-ABL testing to be performed in a random access fashion.

We used real-time polymerase chain reaction (PCR) technology to detect BK virus (BKV) in H and E-stained kidney biopsy sections, using laser capture microdissection. Renal allograft biopsy specimens from 4 patients with the histopathologic diagnosis of BKV-associated nephropathy (BKVAN; group 1) and 3 patients suspected to have BKVAN but without diagnostic histologic features (group 2) were retrieved. Diagnostic inclusion-bearing cells were microdissected by laser capture microscopy from group 1. Renal tubular epithelial cells were microdissected randomly in group 2. DNA was extracted and real-time amplification performed using primers targeting the large "T" and small "t" regions of the BKV and JC virus genomes. Tubular epithelial cells from a case without evidence of BKV infection were used as negative controls in a similar reaction. BKV presence was demonstrated only in epithelial cells containing typical viral inclusions. Group 2 and negative control samples were confirmed as negative for BKVAN. Real-time PCR technology can be used to detect BKV in H and E-stained, paraffin-embedded tissue sections. This technique detected BKV in tubular epithelial cells of renal allografts. To our knowledge, this is the first report of detecting BKV in laser capture microdissected renal biopsy specimens using real-time PCR.

The p90 ribosomal S6 kinase 2 (RSK2) is a serine/threonine kinase with high expression levels in adipose tissue. Numerous in vitro studies show that RSK2 is activated by a broad number of cellular stimuli and suggest that RSK2 is involved in the regulation of a variety of cellular processes. However, the physiological role of RSK2 still remains elusive. We therefore generated rsk2 knockout (KO) mice to better understand the function of RSK2 in vivo. Birth weights of RSK2 KO mice are normal, but the body weight is reduced with age, as compared with wild-type littermates. We found that the difference in body weight was largely caused by a specific loss of white adipose tissue that is accompanied by reduced serum levels of the adipocyte-derived peptide, leptin. KO mice also have impaired glucose tolerance and elevated fasting insulin and glucose levels that are restored following administration of low amounts of leptin, which do not affect food intake. We conclude that RSK2 plays a novel and an important role in regulation of adipose mass in mice and speculate that the reduction in fat tissue may negatively affect insulin sensitivity, as observed in human lipodystrophy, through reduced levels of adipocyte-derived factors, such as leptin.

Objectives: Recently described, aggressive B-cell lymphomas with genetic abnormalities involving MYC and BCL2 have been shown to have a poor prognosis when treated with regimens for diffuse large B-cell lymphomas. Similar data on cases with concurrent MYC and BCL6 translocation are still scant. Moreover, little is known regarding the morphologic and immunophenotypic characteristics of these cases, which further complicates their identification. This study describes six cases of aggressive B-cell lymphoma with translocations involving MYC and BCL6. Methods: Six cases of large B-cell lymphoma with translocation involving MYC and BCL6 confirmed by fluorescence in situ were identified. The morphologic, immunophenotypic, and clinical features of the cases were examined. Results: All the patients were older women, and in 50% of cases, the presentation was extranodal. In two cases, the liver was involved at presentation. A starry-sky pattern was a constant feature of the cases in which the morphology could be reliably assessed. Five of six cases had an immunophenotype corresponding to the germinal center B cells, and only one was positive for BCL2, an immunophenotype reminiscent of that of Burkitt lymphoma. Conclusions: B-cell lymphomas with translocations involving MYC and BCL6 have morphologic and immunophenotypic features suggestive of Burkitt lymphoma or gray zone lymphoma, and they tend to be diagnosed mainly in women, often in extranodal locations.

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R(GL) KO mice, despite the normal insulin activation of GS.

Studies in rodents have established that GLUT-4 translocation is the major mechanism by which insulin and exercise increase glucose uptake in skeletal muscle. In contrast, much less is known about the translocation phenomenon in human skeletal muscle. In the current study, nine healthy volunteers were studied on two different days. On one day, biopsies of vastus lateralis muscle were taken before and after a 2-h euglycemic-hyperinsulinemic clamp (0.8 mU ⋅ kg−1 ⋅ min−1). On another day, subjects exercised for 60 min at 70% of maximal oxygen consumption (V˙o 2 max), a biopsy was obtained, and the same clamp and biopsy procedure was performed as that during the previous experiment. Compared with insulin treatment alone, glucose infusion rates were significantly increased during the postexercise clamp for the periods 0–30 min, 30–60 min, and 60–90 min, but not during the last 30 min of the clamp. Plasma membrane GLUT-4 content was significantly increased in response to physiological hyperinsulinemia (32% above rest), exercise (35%), and the combination of exercise plus insulin (44%). Phosphorylation of Akt, a putative signaling intermediary for GLUT-4 translocation, was increased in response to insulin (640% above rest), exercise (280%), and exercise plus insulin (1,000%). These data demonstrate that two normal physiological conditions, moderate intensity exercise and physiological hyperinsulinemia ∼56 μU/ml, cause GLUT-4 translocation and Akt phosphorylation in human skeletal muscle.

To study the effects of contractile activity on mitogen-activated protein kinase (MAP kinase), p70 S6 kinase (p70S6K), and Akt kinase signaling in rat skeletal muscle, hindlimb muscles were contracted by electrical stimulation of the sciatic nerve for periods of 15 s to 60 min. Contraction resulted in a rapid and transient activation of Raf-1 and MAP kinase kinase 1, a rapid and more sustained activation of MAP kinase and the 90-kDa ribosomal S6 kinase 2, and a dramatic increase in c- fos mRNA expression. Contraction also resulted in an apparent increase in the association of Raf-1 with p21Ras, although stimulation of MAP kinase signaling occurred independent of Shc, IRS1, and IRS2 tyrosine phosphorylation or the formation of Shc/Grb2 or IRS1/Grb2 complexes. Insulin was considerably less effective than contraction in stimulating the MAP kinase pathway. However, insulin, but not contraction, increased p70S6Kand Akt activities in the muscle. These results demonstrate that contraction-induced activation of the MAP kinase pathway is independent of proximal steps in insulin and/or growth factor-mediated signaling, and that contraction and insulin have discordant effects with respect to the activation of the MAP kinase pathway vs. p70S6Kand Akt. Of the numerous stimulators of MAP kinase in skeletal muscle, contractile activity emerges as a potent and physiologically relevant activator of MAP kinase signaling, and thus activation of this pathway is likely to be an important molecular mechanism by which skeletal muscle cells transduce mechanical and/or biochemical signals into downstream biological responses.

Context.—Real-time polymerase chain reaction technologies have replaced many of the more labor-intense methods in the molecular diagnostics laboratory. Similarly, melt curve analysis can provide a rapid means of mutation screening. Objective.—To determine if real-time polymerase chain reaction and melt curve analysis using the SmartCycler II could be used as a screening tool for 3 common mutations in BRCA1 and BRCA2. Design.—Real-time polymerase chain reaction amplification with SYBR Green I detection was performed on DNA from cell lines known to carry the 185delAG or 5382insC mutation in BRCA1 or the 6174delT mutation in BRCA2. The melting temperatures and the melt curves were analyzed for differences between wild-type DNA and cell lines that were heterozygous for each mutation. Results.—Significant differences were present in the melt curves for each of the mutations compared with those of the wild-type sequences. The melt curve for the 185delAG mutation showed a separate peak at a lower temperature, which represented the melting temperature of the heteroduplex. For the 6174delT mutation, the melt curve had a shoulder at a lower temperature, while the melt curve for the 5382insC mutation was shifted to the left and was broader than that for the wild-type sequences. Conclusions.—High-resolution melt curve analysis is a quick, reliable method for identifying mutations due to small deletions or insertions. As a proof of principle, we used this assay to identify the 3 most common BRCA1 and BRCA2 mutations in the Ashkenazi Jewish population.

Classic splenic marginal zone lymphomas are CD5-, CD10-, CD23-, CD43-, and usually IgD+ with biphasic white pulp nodules. However, the 2008 World Health Organization classification accepts splenic marginal zone lymphomas with monophasic marginal zone-like white pulp nodules and recognizes a group of unclassifiable splenic small B-cell lymphomas. To explore the relationship of classic splenic marginal zone lymphomas to these other less well-defined splenic lymphomas, a multiparameter study of 47 splenic marginal zone lymphomas and unclassifiable splenic small B-cell lymphomas was performed. Seventeen of 31 splenic marginal zone lymphomas were biphasic, and 14 were monophasic (90%-100% marginal zone-like white pulp nodules). Sixteen cases were unclassifiable splenic small B-cell lymphomas, most lacking a marginal zone-type component. There were many clinical similarities between the 3 groups, including similar survivals. Monophasic and unclassifiable cases were less likely to have a typical splenic marginal zone lymphoma phenotype (28.6%, 23.1%) compared with biphasic cases (86.7%), usually because of IgD negativity (P < .003). Thirty-four of 42 (81%) cases had cytogenetic abnormalities by fluorescence in situ hybridization; and 17 of 20 (85%), by classical cytogenetics. The most frequent fluorescence in situ hybridization abnormalities among the splenic marginal zone lymphomas were del(7)(q31) (26%), +12 (25%), and +3q27 (27%); and among the unclassifiable cases, +12 (50%) and +3q27 (36%). Five of 6 unclassifiable cases with exclusively small non-marginal zone-like lymphocytes involving both white and red pulp had +12 compared with 9 of 34 other cases (P < .02). CDK6 (2 cases) and BCL3 (1 case) rearrangements were only seen in the unclassifiable group. These results support including both biphasic and monophasic cases as splenic marginal zone lymphomas, but suggest that the lack of a non-marginal zone-like population in the monophasic group is associated with some biologic differences. They also demonstrate a relatively large proportion of unclassifiable cases, including a group with frequent +12.

c-Jun NH2-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle.

Physical exercise is a potent stimulator of mitogen-activated protein (MAP) kinase signaling. To determine if this activation is secondary to systemic responses to exercise or due to muscle contractile activity per se, an isolated muscle preparation was developed. Contractile activity in vitro significantly increased p44MAPKand p42MAPKphosphorylation by 2.9- and 2.4-fold, respectively. Contraction-stimulated MAP kinase phosphorylation was not decreased in the presence ofd-tubocurarine or calphostin C, suggesting that neither neurotransmitter release nor diacylglycerol-sensitive protein kinase C mediates the contraction-induced activation of this signaling cascade. However, PD-98059, an inhibitor of MAP kinase kinase (MEK), inhibited the contraction-induced increases in MAP kinase phosphorylation. PD-98059 did not alter contraction-induced increases in glucose uptake or glycogen synthase activity, demonstrating that MAP kinase signaling is not necessary for these important metabolic effects of contractile activity in skeletal muscle. These data suggest that contractile activity of the skeletal muscle fibers per se, and not responses to neurotransmitter release, hormones, or other systemic factors, is responsible for the stimulation of MAP kinase signaling with physical exercise.

Exercise causes selective changes in gene expression leading to alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling pathways that enable exercise to modulate gene regulatory events. We determined the effects of exercise on c-Jun NH2-terminal kinase (JNK) activity, a signaling molecule involved in the regulation of transcription. Biopsies of vastus lateralis muscle were taken from eight subjects at rest and after 60 min of cycle ergometer exercise. Exercise increased JNK activity in all subjects (5.9 +/- 1.8 fold above basal). JNK activation was associated with an increased expression of its downstream nuclear target c-Jun mRNA. When two additional subjects were studied using a one-legged exercise protocol, JNK activity increased only in the exercising leg, indicating that exercise-induced JNK signaling represents an intrinsic response of the contracting muscle, rather than a systemic response to exercise. These studies demonstrate that the JNK pathway may serve as a link between contractile activity and transcriptional responses in human skeletal muscle.

Little is known about the regulation of the mitogen-activated protein (MAP) kinase signaling cascades by hormonal stimulation in vivo. The extracellular signal-regulated kinase (ERK) and the c-jun kinase (JNK) are two MAP kinase signaling pathways that could play a role in the cellular response to hormones such as insulin and epinephrine. We studied the effects of insulin (20 U/rat) and epinephrine (25 microg/100 g body wt) injected in vivo on ERK and JNK signaling in skeletal muscle from Sprague-Dawley rats. Insulin significantly increased ERK phosphorylation and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2 (RSK2), by 1.4-fold, but it had no effect on JNK activity. In contrast, epinephrine had no effect on ERK phosphorylation or RSK2 activity, but it increased JNK activity by twofold, an effect that was inhibited by the presence of combined alpha and beta blockade. Furthermore, the phosphorylation of both p46 and p55 isoforms of JNK, measured by phosphospecific antibody, was increased severalfold. The activity and phosphorylation of MAP kinase kinase (MKK)-4, an upstream regulator of JNK, was unchanged by epinephrine. Incubation of isolated soleus muscles in vitro with epinephrine (10(-5) mol/l) also increased JNK activity by twofold. These data are the first to demonstrate that epinephrine can increase JNK activity. Insulin and epinephrine have different effects on MAP kinase signaling pathways in skeletal muscle, which may be one of the underlying molecular mechanisms through which these hormones regulate opposing metabolic functions.

Contractile activity plays a critical role in the regulation of gene transcription in skeletal muscle, which in turn determines muscle functional capabilities. However, little is known about the molecular signaling mechanisms that convert contractile activity into gene regulatory responses in skeletal muscle. In the current study we determined the effects of contractile activity in vivo on the c-Jun NH2-terminal kinase (JNK) pathway, a signaling cascade that has been implicated in the regulation of transcription. Electrical stimulation of the sciatic nerve to produce contractions in anaesthetized rats increased JNK activity by up to 7-fold above basal. Maximal enzyme activity occurred at 15 min of contraction and remained elevated at 60 min of contraction. The upstream activators of JNK, the mitogen-activated protein kinase kinase 4 and the mitogen-activated protein kinase kinase kinase 1 followed a similar time course of activation in response to contractile activity. In contrast, contraction induced a rapid and transient activation of the extracellular-regulated kinase pathway, indicating that the regulation of JNK signaling is distinct from that of extracellular-regulated kinase. The activation of the JNK signaling cascade was temporally associated with an increased expression of c-jun mRNA. These results demonstrate that contractile activity regulates JNK activity in skeletal muscle and suggest that activation of JNK may regulate contraction-induced gene expression in skeletal muscle.

Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pathway. Needle biopsies of vastus lateralis muscle were taken from nine subjects at rest and after 60 min of cycle ergometer exercise. In all subjects, exercise increased MAPK phosphorylation, and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2. Furthermore, exercise increased the activities of the upstream regulators of MAPK, MAP kinase kinase, and Raf-1. When two additional subjects were studied using a one-legged exercise protocol, MAPK phosphorylation and p90 ribosomal S6 kinase 2, MAP kinase kinase 1, and Raf-1 activities were increased only in the exercising leg. These studies demonstrate that exercise activates the MAPK cascade in human skeletal muscle and that this stimulation is primarily a local, tissue-specific phenomenon, rather than a systemic response to exercise. These findings suggest that the MAPK pathway may modulate cellular processes that occur in skeletal muscle in response to exercise.

Studies in mammalian cells have established the existence of at least three distinct mitogen-activated protein kinase (MAP kinase) signaling pathways that are activated by a variety of growth factors and/or environmental stressors. We determined whether physical exercise, a physiological stressor, and insulin, a metabolic stimulator and growth factor, activate the c-jun NH2-terminus kinase (JNK), the p38 kinase, and/or the extracellular regulatory kinases (ERK; p42MAPK and p44MAPK) signaling pathways in rat skeletal muscle. Animals were studied immediately after running on a motorized treadmill for 10-60 min (20 m/min, 10% grade) or 5-30 min after an intraperitoneal injection of insulin (20 U/rat). Exercise increased skeletal muscle JNK activity by two- to threefold throughout the time course studied, whereas insulin did not significantly increase JNK activity. The p38 activity was slightly stimulated by exercise and not by insulin. The ERK kinase pathway, as assessed by ribosomal S6 kinase-2 activity assays and phosphospecific p42MAPK/p4NAPK immunoblotting, was stimulated by both exercise and insulin. These data are the first demonstration of exercise stimulating multiple intracellular signaling pathways in skeletal muscle. Activation of these MAP kinase signaling pathways may mediate changes in skeletal muscle growth and metabolism that occur in response to exercise.