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 ß-chemokines.

Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembling nanocarrier formulation.

A unique formulation is described comprising liposomes containing glucosyl ceramide (GluCer) in the lipid bilayer to which bacteriophage T4 was attached. Binding of the phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous proposals that glucose is a major receptor moiety for T4 binding to Escherichia coli. By using the binding of T4 to liposomal GluCer, we further describe a formulation that can be used as a self-assembling combined antigen and adjuvant carrier. A peptide antigen derived from C-trimer (Ct) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of the Ct-Hoc construct on the T4 capsid, and specific binding of a human monoclonal antibody that recognizes a peptide sequence of Ct was demonstrated. Liposomes containing monophosphoryl lipid A (MPLA) have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and animals, and because of this, mice were immunized with the Ct-Hoc-T4 construct that was bound to liposomes containing both GluCer and MPLA, resulting in the induction of high titers of Ct-specific antibodies. We conclude that liposomes containing both GluCer and MPLA can spontaneously bind to a construct of T4 that displays antigens that spontaneously binds to the capsid of T4 bacteriophage. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.

Immune response to and histopathology of Campylobacter jejuni infection in ferrets (Mustela putorius furo).

Campylobacter jejuni is 1 of the most common enteric bacterial pathogens worldwide. The mechanisms of pathogenesis remain obscure, in part because of limitations of small animal models. Young ferrets develop diarrhea when fed C. jejuni, but their pathology and the immune response after infection have not been examined in detail. In the present study, we examined the pathogenesis of C. jejuni CG8421 and associated immune responses in ferrets. After oral infection with C. jejuni CG8421, 86.7% of the animals developed diarrhea and inflammatory responses that were similar to those seen in human infection. Pronounced histopathologic changes in the colonic mucosa of infected animals were observed during the acute phase (days 1 through 3) of infection. Electron micrographs of colonic epithelium revealed disruption of the villi and internalized bacteria that were not within membrane vacuoles. During the acute phase, C. jejuni was isolated from the livers of 7 of 9 (78%) animals, and bacteria were visualized immunohistochemically in the livers from 5 of the 7 animals (71%) from which C. jejuni was isolated. A vigorous systemic and mucosal immune response to Campylobacter antigens was elicited after infection of ferrets. The data presented contribute to the current knowledge of the pathogenicity of and immunologic response to C. jejuni CG8421 in ferrets and better understanding of this model.

Alpha-defensin-like product and asymmetric dimethylarginine increase in mesenteric lymph after hemorrhage in anesthetized rat.

Mesenteric lymph contains unidentified proinflammatory mediators that increase in concentration after hemorrhage. In the search for candidate mediators, we examined mesenteric lymph for the presence of proinflammatory substances that are known to be produced in the gut: (a) antimicrobial peptides and antimicrobial proteins produced in the Paneth cells of the intestine (alpha-defensin 4, secretory phospholipase A2 [sPLA2], and Reg 2 protein) and (b) asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. Anesthetized male rats were hemorrhaged to 40 mmHg and maintained at that pressure by intermittent blood withdrawal until the pressure fell to less than 40 mmHg (decompensation) at which point they were resuscitated with three times the shed blood volume of Ringer's lactate solution administered over 1 h. Mesenteric lymph samples were analyzed for ADMA by enzyme-linked immunosorbent assay and for alpha-defensin 4, sPLA2, and Reg2 by Western blotting. Protein concentration in lymph was unchanged by hemorrhage, but alpha-defensin 4 increased significantly (12-fold greater than control) as did ADMA (2-fold greater than control). The sPLA2 could not be detected in lymph, and Reg 2 was unchanged during hemorrhage. During resuscitation, lymph flow tended to increase, but the concentration of ADMA and alpha-defensin 4 by volume did not increase. Reg 2 decreased during resuscitation. The results indicate that ADMA and immunoreactive product to alpha-defensin 4 may contribute to the increase in inflammatory activity of mesenteric lymph during hemorrhage, but they are unlikely to be the mediators responsible for the increase in the concentration of inflammatory mediators in postresuscitation lymph.

Temporal analysis of Andes virus and Sin Nombre virus infections of Syrian hamsters.

Andes virus (ANDV) and Sin Nombre virus (SNV) are rodent-borne hantaviruses that cause a highly lethal hemorrhagic fever in humans known as hantavirus pulmonary syndrome (HPS). There are no vaccines or specific drugs to prevent or treat HPS, and the pathogenesis is not understood. Syrian hamsters infected with ANDV, but not SNV, develop a highly lethal disease that closely resembles HPS in humans. Here, we performed a temporal pathogenesis study comparing ANDV and SNV infections in hamsters. SNV was nonpathogenic and viremia was not detected despite the fact that all animals were infected. ANDV was uniformly lethal with a mean time to death of 11 days. The first pathology detected was lymphocyte apoptosis starting on day 4. Animals were viremic and viral antigen was first observed in multiple organs by days 6 and 8, respectively. Levels of infectious virus in the blood increased 4 to 5 logs between days 6 and 8. Pulmonary edema was first detected ultrastructurally on day 6. Ultrastructural analysis of lung tissues revealed the presence of large inclusion bodies and substantial numbers of vacuoles within infected endothelial cells. Paraendothelial gaps were not observed, suggesting that fluid leakage was transcellular and directly attributable to infecting virus. Taken together, these data imply that HPS treatment strategies aimed at preventing virus replication and dissemination will have the greatest probability of success if administered before the viremic phase; however, because vascular leakage is associated with infected endothelial cells, a therapeutic strategy targeting viral replication might be effective even at later times (e.g., after disease onset).

Characterization of monoclonal antibodies to hepatitis E virus (HEV) capsid protein and identification of binding activity.

Twenty-seven monoclonal antibodies (Mabs) recognizing the open reading frame 2 structural protein of the Pakistan strain of hepatitis E virus (HEV) were generated by conventional hybridoma technique. These Mabs were characterized by ELISA, affinity-capture reverse transcriptase-polymerase chain reaction (AC/RT-PCR), immune electron microscopy (IEM), and a RT-PCR based seroneutralization assay. Twenty-seven Mabs were positive by ELISA. By AC/RT-PCR, 24 Mabs bound to Pakistan and Namibia HEV strains. Thirteen Mabs were examined by IEM. Nine Mabs, positive by ELISA and AC/RT-PCR, bound and aggregated to Mexican HEV strain. We tested five Mabs that were positive by ELISA, AC/RT/PCR, and IEM by a RT-PCR based seroneutralization assay. Only one Mab (Mab 7) showed activity that inhibited the ability of HEV to attach to Alexander hepatoma cells (PLC-PRF-5). When Mab 7 was diluted to 1: 160, its inhibition activity persisted suggesting that Mab 7 might be a potential candidate for further evaluation in primates (passive protection experiments).

Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: a chemical analysis.

Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe(3+). Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide.

Bovine polymerized hemoglobin (hemoglobin-based oxygen carrier-201) resuscitation in three swine models of hemorrhagic shock with militarily relevant delayed evacuation--effects on histopathology and organ function.

OBJECTIVE: To test our hypothesis that hemoglobin-based oxygen carrier (HBOC)-201 resuscitation in hemorrhagic shock (HS) will not lead to increased organ injury and dysfunction. DESIGN: Three swine HS models simulating military-relevant delayed evacuation: a) moderate controlled HS, b) severe controlled HS, and c) severe uncontrolled HS. SETTING: Military research laboratory. SUBJECTS: Swine. INTERVENTIONS: Swine were anesthetized/intubated and instrumented. To induce HS, in two controlled hemorrhage experiments, 40% (moderate controlled HS) or 55% (severe controlled HS) of blood volume was withdrawn; in an uncontrolled HS experiment, the liver was crushed/lacerated. During a 4-hr "prehospital phase," pigs were resuscitated with HBOC-201 (HBOC) or Hextend (HEX) or were nonresuscitated (NON). Upon "hospital arrival," liver injury was repaired (severe uncontrolled HS), blood or saline was infused, hemodynamics were monitored, and blood was collected. Upon animal death and/or 72 hrs, necropsy was followed by histopathologic evaluation of organ injury (hematoxylin and eosin, electron microscopy) and immunohistochemistry of oxidative potential (3-nitrotyrosine). Significance (p < .05) was assessed by Kruskal-Wallis, analysis of variance/Bonferroni, and mixed procedure tests. MEASUREMENTS AND MAIN RESULTS: Survival was significantly higher with HBOC than HEX only with severe uncontrolled HS (p = .002). Myocardial necrosis/fibroplasia, fluid requirements, cardiac output, and cardiac enzymes were generally similar or lower in HBOC than HEX pigs, but creatine kinase-MB (but not creatine kinase-MB/creatine kinase ratio) was higher with HBOC in moderate controlled HS. Alveolar/interstitial pulmonary edema was similar with HBOC and HEX, but Po2 was higher with HBOC in severe uncontrolled HS. Jejunal villar epithelial and hepatocellular necrosis were similarly minimal to moderate in all groups. Minimal biliary changes occurred exclusively with HBOC. Aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase were generally higher with HBOC than HEX. Mild renal papillary injury occurred more frequently with HBOC, but consistent patterns for urine output, blood urea nitrogen, and creatinine, were not seen. The 3-nitrotyrosine staining intensity was not different. CONCLUSIONS: In comparison with hetastarch, HBOC-201 resuscitation of swine with HS increased survival (with severe HS), did not increase evidence of oxidative potential, and had histopathologic and/or functional effects on organs that were clinically equivocal (myocardium, lungs, hepatic parenchyma, jejunum, and renal cortex/medulla) and potentially adverse (hepatobiliary and renal papilla). The effects of HBOC-201-resuscitation in HS should be corroborated in controlled clinical trials.

Effects of bovine polymerized hemoglobin on coagulation in controlled hemorrhagic shock in swine.

HBOC-201, a bovine polymerized hemoglobin, has been proposed as a novel oxygen-carrying resuscitative fluid for patients with hemorrhagic shock (HS). Herein, we evaluated the hemostatic effects of HBOC-201 in an animal model of HS. A 40% blood loss-controlled hemorrhage and soft tissue injury were performed in 24 invasively monitored Yucatan mini-pigs. Pigs were resuscitated with HBOC-201 (HBOC) or hydroxyethyl starch (HEX), or were not resuscitated (NON) based on cardiac parameters during a 4-h prehospital phase. Afterward, animals received simulated hospital care for 3 days with blood or saline transfusions. Hemostasis measurements included in vivo bleeding time (BT), thromboelastography (TEG), in vitro bleeding time (platelet function; PFA-CT), prothrombin time (PT), and partial thromboplastin time (PTT). Serum lactate was measured and lung sections were evaluated for microthrombi by electron microscopy. During the prehospital phase, BT remained unchanged in the HBOC group. TEG reaction time increased in HBOC pigs during the late prehospital phase and was greater than in NON or HEX pigs at 24 h (P = 0.03). TEG maximum amplitude was similar for the two fluid-resuscitated groups. PFA-CT increased in both resuscitated groups but less with HBOC (P = 0.02) in the prehospital phase; this effect was reversed by 24 h (P = 0.02). In the hospital phase, PT decreased (P < 0.02), whereas PTT increased above baseline (P < 0.01). Lactic acidosis in HBOC and HEX groups was similar. Aspartate aminotransferase was relatively elevated in the HBOC group at 24 h. Electron microscopy showed no evidence of platelet/fibrin clots or microthrombi in any of the animals. Twenty-four-hour group differences mainly reflected the fact that all HEX animals (8/8) received blood transfusions compared with only one HBOC animal (1/8). In swine with HS, HBOC resuscitation induced less thrombopathy than HEX during the prehospital phase. Mild delayed effects on platelet and clot formation during the hospital phase are transient and likely related to fewer blood transfusions. In swine with HS, HBOC resuscitation induced less thrombopathy than HEX during the prehospital phase but more thrombopathy in the hospital phase. The delayed effects on platelet and clot formation during the hospital phase are transient and may be related to the need for fewer blood transfusions.

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.

Spontaneous acute tumor lysis syndrome in a DBA/1J mouse: a case report and review.

Spontaneous acute tumor lysis syndrome (ATLS) was diagnosed in a 10-month-old female DBA/1J sentinel mouse with leukemic lymphoma. The mouse was unable to maintain balance and died shortly after being observed rolling around in its cage. Disseminated neoplastic disease, including a large cranial mediastinal mass, enlarged lymph nodes and splenomegaly, was present at necropsy. Histopathologic examination revealed widespread massive necrosis of lymphoblastic tumor cells, and widely disseminated microemboli composed of nuclear and cytoplasmic cell debris. Although ATLS is widely recognized as an oncologic emergency in humans, acute lesions of ATLS have not been described. The mechanical obstruction of capillary beds by microemboli originating from disintegrating necrotic tumor cells was the likely cause of clinical signs and death in this mouse. We propose that similar microemboli may contribute to the pathogenesis of the acute renal failure and other clinical signs associated with ATLS in humans. Recognition of spontaneous ATLS in laboratory animals is especially important in studies that assess the efficacy and/or toxicity of anticancer treatments, where early deaths due to ATLS might mistakenly be attributed to a direct test article effect.

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.

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

1. The sulphur mustard vesicant 2-chloroethylethyl sulphide (CEES) induced apoptosis in Jurkat cells. 2. 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. 3. 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. 4. The phosphorylation of Akt at Ser(473) and Thr(308) was significantly decreased following CEES treatment, reflecting the suppressed kinase activity of both PDK1 and PDK2. 5. 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. 6. 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. 7. 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.

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.