Human herpesvirus 8 in primary effusion lymphoma in an HIV-seronegative male. A case report

BACKGROUND: AIDS-related body cavity-based lymphoma, or primary effusion lymphoma (PEL), is a distinct clinicopathologic entity that occurs predominantly in immunosuppressed patients infected with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. Although it rarely occurs in human immunodeficiency virus (HIV)-negative patients, we report such a case here. CASE: A 74-year-old male, who was HIV and Epstein-Barr virus (EBV) negative, was admitted to the hospital with dyspnea and chest pain. Chest radiography and computed tomography showed right pleural effusion. Cytologic analysis of the pleural effusion revealed a high grade lymphoma with round nuclei, prominent nucleoli and abundant cytoplasm. Polymerase chain reaction performed on the pleural effusion was positive for HHV-8 and negative for EBV. On molecular studies, the immunoglobulin heavy and kappa light chains were rearranged. Flow cytometry revealed a hyperploid fraction with DNA index of 1.29 expressing CD30. Immunostaining for HHV-8 from a cell block was positive. Electron microscopy revealed lymphomalike cells, many in various stages of apoptosis, with large nucleoli and clusters of viruslike particles in the nucleoplasm. CONCLUSION: A firm diagnosis of PEL can be established by the examination of cells from the lymphomatous effusion by a combination of cytology, molecular genetics, phenotypic features, immunostaining and electron microscopy. To our knowledge, this is the first case in which immunostaining for anti-HHV-8 monoclonal antibodies was used to support the diagnosis.     

Characterization, cloning, and expression of two diagnostic antigens for Taenia solium tapeworm infection

Adult and larval stages of Taenia solium cause 2 diseases in humans, i.e., taeniasis and cysticercosis, respectively. Diagnosis and treatment of taeniasis are the ultimate means to eliminate cysticercosis. A serological taeniasis diagnostic test has been developed for laboratory use. However, recombinant forms of the taeniasis diagnostic proteins are required to overcome the limited supply of native proteins and allow the development of a low-cost and field-applicable test with high sensitivity and specificity. Using 2-dimensional electrophoresis of T. solium excretory and secretory (TSES) products from hamster adult tape-worm in vitro cultures, we have identified 5 T. solium-specific protein spots, with molecular weights of 33 kDa (protein isoelectrofocusing point [pI]: 5.6, 5.3, 5.1) and 38 kDa (pI: 4.6, 4.5). Protein sequencing and molecular cloning of these proteins showed that although endowed with different pls, the proteins with the same molecular weights shared the same protein backbone, named TSES33 and TSES38. Their full-length complementary DNAs encode proteins with 267 and 278 amino acids, respectively. TSES33 and TSES38 were expressed in a baculovirus system. Both recombinant proteins were recognized by a panel of taeniasis, but not cysticercosis patient serum samples, indicating that they can potentially replace the native proteins in the development of a more efficacious taeniasis diagnostic test.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.     

The gamma subunit of Na+, K+-ATPase: role on ATPase activity and regulatory phosphorylation by PKA

In kidney, Na+, K+-ATPase is an oligomer (alphabeta gamma) with equimolar amounts of essential alpha and beta subunits and one small hydrophobic FXYD protein (gamma subunit). This report describes gamma subunit as an activator of pig kidney outer medulla Na+, K+-ATPase in aqueous medium. The effects of gamma subunit on Na+, K+-ATPase were dose-dependent and preincubation-dependent. Changes in alphabeta/gamma stoichiometry did not alter Km1 for ATP, and slightly increased Km2, but Vmax was increased at both catalytic and regulatory sites. Hydroxylamine treatment of enzyme phosphorylated by ATP (E-P), in the presence of additional gamma subunit, revealed that 52% of the E-P accumulation was not via acyl-phosphate formation. The gamma subunit was phosphorylated by endogenous kinases and by commercial catalytic subunit of protein kinase A (PKA). Additionally, we demonstrated that PKA phosphorylation of gamma subunit increased its capacity to stimulate ATP hydrolysis. These results suggest that gamma subunit can act as an intrinsic Na+, K+-ATPase regulator in kidney.