Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.     

Sequestration of p27Kip1 protein by cyclin D1 in typical and blastic variants of mantle cell lymphoma (MCL): implications for pathogenesis

p27 is a cyclin-dependent kinase inhibitor that plays a critical role in regulating G(1)/S progression, and whose activity is, in part, regulated through interactions with D-type cyclins. Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation resulting in deregulated cyclin D1. We previously showed that p27 expression in MCL, as assessed by immunohistochemistry (IHC), does not show the usual inverse relationship to proliferate seen in most other lymphomas that do not overexpress cyclin D1. This suggested that the normal expression or control of p27 activity on cell growth might be altered through potential interactions with cyclin D1. Using Western blot and coimmunoprecipitation studies, we assessed the interrelationship between cyclin D1 and p27 in several cyclin D1(+) cell lines and primary MCL cases. Similar to our previous results by IHC, typical MCLs showed lower expression of p27 when compared to the more highly proliferative blastic cases or cell lines (mean arbitrary units: 58 versus 236 versus 120). Cyclin D1 was expressed at variable levels in both typical and blastic MCLs. p27 protein could be consistently coimmunoprecipitated with cyclin D1 from both cell lines and cases. Using techniques of exhaustive immunoprecipitation, we could demonstrate that most p27 protein was sequestered into complexes containing cyclin D1. We hypothesize that mantle cell lymphomagenesis results not only from direct consequences of inappropriate cyclin D1 expression, but also from the ability of overexpressed cyclin D1 to buffer physiologic changes in p27 levels, thereby rendering p27 ineffective as an inhibitor of cellular growth.     

Improvement in lipids after switch to boosted atazanavir or darunavir in children/adolescents with perinatally acquired HIV on older protease inhibitors: results from the Pediatric HIV/AIDS Cohort Study

Objectives

Dyslipidaemia is common in perinatally HIV‐infected (PHIV) youth receiving protease inhibitors (PIs). Few studies have evaluated longitudinal lipid changes in PHIV youth after switch to newer PIs.

Methods

We compared longitudinal changes in fasting lipids [total cholesterol (TC), triglycerides (TG), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), and TC:HDL‐C ratio] in PHIV youth enrolled in the Pediatric HIV/AIDS Cohort Study (PHACS) Adolescent Master Protocol (AMP) study who switched to atazanavir/ritonavir (ATV/r)‐ or darunavir/ritonavir (DRV/r)‐based antiretroviral therapy (ART) from an older PI‐based ART and those remaining on an older PI. Generalized estimating equation models were fitted to assess the association of a switch to ATV/r‐ or DRV/r‐based ART with the rate of change in lipids, adjusted for potential confounders.

Results

From 2007 to 2014, 47 PHIV children/adolescents switched to ATV/r or DRV/r, while 120 remained on an older PI [primarily lopinavir/r (72%) and nelfinavir (24%)]. Baseline age ranged from 7 to 21 years. After adjustment for age, Tanner stage, race/ethnicity, and HIV RNA level, a switch to ATV/r or DRV/r was associated with a more rapid annual rate of decline in the ratio of TC:HDL‐C. (β = ?0.12; P = 0.039) than remaining on an older PI. On average, TC declined by 4.57 mg/dL/year (P = 0.057) more in the switch group. A switch to ATV/r or DRV/r was not associated with the rate of HDL‐C, LDL‐C, or TG change.

Conclusions

A switch to ATV/r or DRV/r may result in more rapid reduction in TC and the TC:HDL‐C ratio in PHIV youth, potentially impacting long‐term cardiovascular disease risk.

     

Human umbilical vein endothelial cells secrete transcobalamin II

Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.     

Endoscopic ultrasound and computer tomography are inaccurate methods of classifying cystic pancreatic lesions

BACKGROUND: Despite advances in imaging modalities, preoperative diagnosis of pancreatic cystic lesions remains difficult. AIM: To assess the accuracy of endoscopic ultrasound and computer tomography to preoperatively distinguish benign from potentially malignant and malignant pancreatic cystic lesions. METHODS: Photograph series obtained from endoscopic ultrasound examinations of 66 patients with cystic pancreatic lesions were blindly reviewed by three endoscopic ultrasonographers. Forty-one of those 66 patients also underwent a computer tomography scan at our institution, which was blindly reviewed by a single radiologist. Computer tomography and endoscopic ultrasound classification into benign and malignant and potentially malignant pancreatic cystic lesions was correlated with the final diagnosis, which was established by surgical pathology (n = 43), diagnostic fine needle aspiration (n = 13) or follow-up imaging (n = 10). Interobserver agreement was measured using kappa statistics. RESULTS: Endoscopic ultrasound classification by the three examiners into benign versus malignant or potentially malignant cystic lesions was correct in 65-67%. Interobserver agreement was 50%. Kappa values for pairs of endoscopic ultrasound examiners were 0.16, 0.43 and 0.53. Computer tomography classification was correct in 71% and in agreement with the endoscopic ultrasound classification in 56-61% (kappa 0.12 to 0.27). CONCLUSIONS: Endoscopic ultrasound and computer tomography cannot accurately distinguish between benign pancreatic cystic lesions and malignant or potentially malignant ones. There is poor-to-modest interobserver agreement in classifying these lesions.     

Repeated measures study of human herpesvirus 8 (HHV-8) DNA and antibodies in men seropositive for both HHV-8 and HIV

OBJECTIVE: To study the natural history and pathogenesis of human herpesvirus 8 (HHV-8) infection in HHV-8-seropositive, immunosuppressed men. DESIGN: Longitudinal study of 87 HHV-8- and HIV-seropositive men [42 with Kaposi's sarcoma (KS)] during four visits over a 2 month period. METHODS:: Patients provided oral fluid and blood. HHV-8 antibody titers were measured with peptide-based enzyme-linked immunosorbent assays (ELISA) for ORF65 and K8.1; HHV-8 DNA was detected with polymerase chain reaction ELISA. RESULTS: HHV-8 DNA was present in oral fluid or peripheral blood mononuclear cells (PBMC) at one or more of the four visits in 71% of men with KS and 56% of men without KS. The strongest correlate of HHV-8 DNA in PBMC was the presence of KS [odds ratio (OR), 8.7; 95% confidence interval (CI), 3.4-22]. Detection of HHV-8 DNA in oral fluid or PBMC was often intermittent, but individuals who shed virus at one time point were more likely to shed at other times. Some men had incomplete epitope recognition in their anti-HHV-8 antibody response. High antibody titers were associated with the absence of circulating HHV-8, particularly for the ORF65 seroassay (OR, 0.16; 95% CI, 0.05-0.51). CONCLUSIONS: Among HHV-8 seropositive men, circulating virus is common even in the absence of disease. The link between KS and HHV-8 DNA in PBMC suggests that anti-herpes drugs may impede KS development or progression. Seroassays should target multiple epitopes to achieve maximal sensitivity. HHV-8 replication may be limited by high antibody titers or other immune function for which antibodies are a marker.     

Bone involvement in young patients with non-Hodgkin's lymphoma: efficacy of chemotherapy without local radiotherapy

Of 95 young non-Hodgkin's lymphoma patients entered consecutively on the National Cancer Institute (NCI) Protocol 7704, 26 (27.4%) had involvement of one or more bones. The mean age of these 26 patients was 16.6 years, and the male to female ratio was 3.3:1. Tumor histology included undifferentiated Burkitt's lymphoma in 12, undifferentiated non-Burkitt's lymphoma in two, undifferentiated, unspecified lymphoma in one, diffuse large cell lymphoma in three, and lymphoblastic lymphoma in eight patients. Most had extensive disease; two patients had isolated bone lesions, one had lesions of two bones without involvement of other tissues, and 23 had either multiple bone lesions or single bone lesions with involvement of other tissues. Eight of the 26 patients had bone marrow involvement. Of a subgroup of 12 patients with jaw disease, 11 had undifferentiated lymphoma and one had diffuse large cell lymphoma. Only one had primary a jaw tumor, with two quadrants of the jaw involved. All 26 patients were treated with chemotherapy; only two received radiotherapy initially for bone lesions. Predicted survival of the 26 patients at 5 years is 53.2%. The 12 patients who remain disease free have a mean survival of 62.1 months (range, 22 to 100 months). Our results call into question the role of radiotherapy in the treatment of bone lesions in non-Hodgkin's lymphoma.     

Multifarious characterization of leishmania tropica from a Judean desert focus, exposing intraspecific diversity and incriminating phlebotomus sergenti as its vector

The predominant sand fly species collected inside houses in Kfar Adumim, an Israeli village in the Judean Desert that is a focus of cutaneous leishmaniasis, was Phlebotomus papatasi, which was also caught attempting to bite humans. Phlebotomus sergenti, which is rarely seen inside houses, constituted the predominant sand fly species in caves near the village. Leishmania isolates from Ph. sergenti and humans typed as Leishmania tropica. Sand fly and human isolates produced similar small nodular cutaneous lesions in hamsters. Isolates produced excreted factor (EF) of subserotypes A(9) or A(9)B(2), characteristic of L. tropica and reacted with L. tropica-specific monoclonal antibodies. Isoenzyme analysis consigned the strains to the L. tropica zymodemes MON-137 and MON-275. Molecular genetic analyses confirmed the strains were L. tropica and intraspecific microheterogeneity was observed. Genomic fingerprinting using a mini-satellite probe separated the L. tropica strains into two clusters that were not entirely congruent with geographic distribution. These results support the heterogeneous nature of L. tropica and incriminate Ph. sergenti as its vector in this Judean Desert focus.     

Cardiac amyloidosis presenting with elevations of cardiac troponin I and angina pectoris.

We present the case of a 43-year-old male who was initially evaluated for angina pectoris and dyspnea. His CK, CK-MB, and cTnI were all elevated following a blood transfusion and he underwent coronary arteriography, which demonstrated no luminal obstructions. After several months, he was transferred to Mayo Clinic where diagnoses of fulminant cardiac amyloidosis and systemic multiple myeloma were established. The cTnI remained elevated despite normalization of the CK and CK-MB. Despite aggressive treatment, the patient died. Postmortem analysis demonstrated amyloid cardiac deposition including involvement of the coronary microvasculature. Electron microscopy revealed myocyte compression injury from amyloid infiltration. We believe this is the first report of elevated troponin I in a patient with cardiac amyloidosis. The electron microscopy in our case confirms cardiac damage as the mechanism for cTnI elevation. This observation strengthens our knowledge about the specificity of cTnI for the detection of cardiac injury.     

NIH conference. T-cell lymphoproliferative syndrome associated with human T-cell leukemia/lymphoma virus

Human T-cell leukemia/lymphoma virus is a unique family of T-cell tropic, human, type-C retroviruses. The discovery of this class of retroviruses provides the first proven link between retroviruses and cancer in humans. This virus is endemic in certain parts of the world, including the southeastern United States, and is associated with the development of adult T-cell leukemia/lymphoma, a fulminant lymphoproliferative disorder frequently accompanied by opportunistic infections and hypercalcemia. Over the last few years, major advances have been made in understanding the clinical, epidemiologic, molecular biologic, and immunologic features of this unique class of human RNA tumor viruses.