Ludmila V. Asher

Large Epidemic of Adenovirus Type 4 Infection among Military Trainees: Epidemiological, Clinical, and Laboratory Studies

Outbreaks of adenovirus type 4 (Ad4) acute respiratory disease (ARD) have reemerged among US military personnel during the past decade. A prospective epidemiological investigation of 678 military recruits was conducted at Fort Jackson, South Carolina, in the fall of 1998; 115 (17%) of the recruits were hospitalized for febrile ARD. Adenovirus types 4, 3, and 21 were recovered from the cultures of 70 (72%), 7 (7%), and 2 (2%) of 97 recruits, respectively. In addition, 69 (83%) of the 83 hospitalized and 82 (49%) of the 166 nonhospitalized unit contacts had seroconversion to Ad4, which indicates the very high susceptibility and communicability of Ad4 among military recruits. Young age (<20 years) and male sex increased the risk for anti-Ad4 seroconversion. Recruits from tropical areas had higher preexisting immunity than did recruits from temperate regions. Military recruits are highly susceptible to Ad4 infections. Prompt reinstitution of an adenovirus vaccination program in this high-risk population is urgently needed.

Opsonization by antigen‐specific antibodies as a mechanism of protective immunity induced by Plasmodium falciparum circumsporozoite protein‐based vaccine

Recently conducted trials involving the Plasmodium falciparum circumsporozoite (CS) protein‐based RTS,S malaria vaccine yielded unprecedented protection against a challenge with infectious sporozoites (spzs). The RTS,S vaccine induced high titres of CS protein‐specific antibodies (Abs) in many of the protected volunteers, but the contribution of these Abs to protection remains unknown. Because opsonization by Ab promotes the uptake and destruction of spzs by monocytes and macrophages in both rodent and primate malaria, we asked if the RTS,S‐induced Abs have antigen‐specific opsonizing activity. Screening plasma from a large number of subjects using spzs was impractical, therefore we developed an alternative assay based on cytofluorometry that allowed the detection of fluoresceinated‐Ag–Ab complexes endocytosed by the FcR+ THP‐1 human monocyte line. The results showed that plasma samples from RTS,S‐immunized subjects contained opsonizing CS protein‐specific Abs and the endocytic activity of these Abs in protected subjects was significantly higher than in subjects who were susceptible to infection with spzs. We also demonstrated by electron microscopy that live spzs exposed to RTS,S‐immune plasma could be internalized by the THP‐1 cells. These results suggest that opsonization by CS protein‐specific Abs might be one of the mechanisms that contributes to RTS,S‐induced protective immunity.

Cellular effects of leishmanial tubulin inhibitors on L. donovani.

To aid our investigation of tubulin as an antileishmanial drug target, the effects of the mammalian antimicrotubule agents ansamitocin P3, taxol, and hemiasterlin on Leishmania donovani promastigotes were described. These drugs affected the assembly of purified leishmanial tubulin and inhibited the growth of L. donovani promastigotes at micromolar concentrations. When promastigotes were treated with these agents, mitotic partitioning of nuclear DNA and cytokinesis were usually inhibited. The spatial orientation of kinetoplasts was often disturbed, suggesting a role for microtubules in the segregation of these organelles during mitosis. Aberrant cell types produced in drug-treated cultures included parasites with one nucleus and two geometrically distinct kinetoplasts, parasites with multiple kinetoplasts, and cytoplasts containing a kinetoplast but no nucleus. A subset of unique cell types, parasites containing two nuclei, a spindle fiber, and two geometrically distinct kinetoplasts, were observed in hemiasterlin-treated cultures. Flow cytometric analysis of L. donovani promastigotes treated with these three drugs indicated a dramatic shift toward the G2 + M phase of the cell cycle, with some cells containing four times the amount of DNA present in G1. These results were used to evaluate the cellular effects of WR85915, an aromatic thiocyanate with in vitro antileishmanial and anti-tubulin activity, on L. donovani. Treatment of parasites with WR85915 did not produce the unusual cell types described above and did not cause the accumulation of parasites in G2 + M, suggesting that WR85915 acts on target(s) in Leishmania in addition to tubulin. These studies validate tubulin as a suitable antileishmanial drug target and provide criteria to assess the cellular mechanism of action of new candidate antileishmanial agents.

Evaluation of a Rapid Quantitative Diagnostic Test for Adenovirus Type 4

Acute respiratory disease (ARD) due to adenoviruses is a reemerging disease in military recruits. It is a challenge for clinicians to accurately diagnose this disease and to appropriately treat affected individuals. This study investigated the utility of a quantitative, rapid-cycle, real-time fluorogenic polymerase chain reaction (PCR) technique for detecting adenovirus type 4 (Ad4) in a clinical setting. Throat swab specimens and clinical data were collected from US Army basic trainees hospitalized with ARD at Fort Jackson, South Carolina. A total of 140 throat swab specimens were collected from 83 subjects. Rapid PCR results (obtained in <2 h) had a sensitivity of 100% and a specificity of 100%, compared with viral culture. There was no difference, qualitative or quantitative, between frozen and fresh samples for PCR detection of Ad4. Individuals with test results positive for Ad4 were hospitalized longer than were individuals with negative test results. Higher virus loads at hospital admission corresponded to longer lengths of stay for Ad4-positive subjects.

Demonstration of Hepatitis A Virus in Cell Culture By Electron Microscopy with Immunoperoxidase Staining

Virus-like particles were demonstrated by electron microscopy in BS-C-1 cells infected with hepatitis A virus (HAV). Particles were usually enclosed within vesicles and accompanied by myelin-like membranous structures. Less often they were seen free in the cytoplasm. They were never observed in the nucleus. By immunoperoxidase staining particles were found to contain HAV antigens. These antigens were also found in the membrane of the vesicles surrounding the masses of particles and adjacent parts of the mitochondrial membranes. Our results demonstrate the usefulness of an electron microscopic immunocytochemical technique to study replication of HAV.

An anti-phosphoinositide-specific monoclonal antibody that neutralizes HIV-1 infection of human monocyte-derived macrophages

HIV-1 entry into cells requires the interaction of both HIV-1 envelope proteins and membrane lipids. We investigated the mechanism of neutralization of HIV-1 infection of primary monocyte-derived macrophages (MDM) by a murine monoclonal antibody (mAb) WR321. WR321 specifically binds phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. These phosphoinositides are present not only on the inner surface of the plasma membranes of cells but also on the surface of virions. HIV-1 acquires these lipids during the budding process. Pre-incubation of WR321 with the virus but not with MDM neutralized HIV-1 infection of MDM. Our results demonstrate that WR321 was internalized only when it was bound to HIV-1. WR321 did not prevent the entry of HIV-1 into MDM. However, once WR321 was internalized along with HIV-1 the mAb acted intracellulary to prevent the release of virions from MDM and also triggered the release of β-chemokines.

Gene expressions in Jurkat cells poisoned by a sulphur mustard vesicant and the induction of apoptosis

The sulphur mustard vesicant 2‐chloroethylethyl sulphide (CEES) induced apoptosis in Jurkat cells. Akt (PKB), a pivotal protein kinase which can block apoptosis and promotes cell survival, was identified to be chiefly down‐regulated in a dose‐dependent manner following CEES treatment. Functional analysis showed that the attendant Akt activity was simultaneously reduced. PDK1, an upstream effector of Akt, was also down‐regulated following CEES exposure, but two other upstream effectors of Akt, PI3‐K and PDK2, remained unchanged. The phosphorylation of Akt at Ser473 and Thr308 was significantly decreased following CEES treatment, reflecting the suppressed kinase activity of both PDK1 and PDK2. Concurrently, the anti‐apoptotic genes, Bcl family, were down‐regulated, in sharp contrast to the striking up‐regulation of some death executioner genes, caspase 3, 6, and 8. Based on these findings, a model of CEES‐induced apoptosis was established. These results suggest that CEES attacked the Akt pathway, directly or indirectly, by inhibiting Akt transcription, translation, and post‐translation modification. Taken together, upon exposure to CEES, apoptosis was induced in Jurkat cells via the down‐regulation of the survival factors that normally prevent the activation of the death executioner genes, the caspases.

White particulate matter found in blood collection bags consist of platelets and leukocytes

BACKGROUND:  In late January 2003, some blood centers and hospitals throughout the US voluntarily sus‐pended the use of some RBC and plasma units for trans‐fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets.

Apoptosis of Crypt Cells and Inflammatory Reactions in the Small Intestine of Mice Challenged with Staphylococcal Enterotoxin B

Staphylococcal enterotoxin B (SEB), a superantigen that stimulates activation and proliferation of T cells bearing certain Vb elements of the T cell receptor, causes food poisoning and toxic shock in humans and in monkeys. The clinical symptoms of SEB toxicosis include nausea, vomiting, retching, abdominal cramping, diarrhea, headache, muscular cramping and marked prostration with a considerable drop in blood pressure in some cases. The mechanism by which SEB causes these symptoms is still obscure. Production of large amounts of cytokines following SEB challenge was assumed to lead to toxic shock. We have recently developed a SEB toxic shock model in mice by priming with actinomycin D (Act-D). Mice primed with Act-D become very sensitive to SEB-induced toxicosis and death. Enhancement of cytokine expression in the spleen and leukocyte sequestration in the lung as well as increased adhesive molecule expression were reported previously. This report describes the pathological reactions found in the small intestine and discusses the pathogenesis of those abnormalities.

Virus-like Particles in the Liver of an Owl Monkey Inoculated with Hepatitis C Virus

Hepatitis C virus (HCV) is infectious for chimpanzees; these animals, however, are in short supply. We attempted to infect New World monkeys that are more readily available and are susceptible to other hepatitis viruses. Six owl monkeys were inoculated intravenously with plasma obtained from two human cases of HCV infection and plasma from an infected chimpanzee. One monkey died 39 days after inoculation. Liver tissue and blood were collected within one hour after the animal’s death, fixed and examined by light and electron microscopy. On examination by light microscopy, the liver showed focal hemorrhagic necrosis with extensive erythrophagocytosis by Kupffer cells and hepatocytes. There was no portal inflammation. Examination by electron microscopy revealed groups of 50–60 nm enveloped virus-like particles, of a size consistent with HCV, in the cytoplasm of hepatocytes, the spaces of Disse, and in clotted blood. In addition, bundles of dense filaments were seen in large dilated spaces in the cytoplasm of hepatocytes.