Sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus.

UNLABELLED: To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10(5) PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE: Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

Does hypocortisolism predict a poor response to cognitive behavioural therapy in chronic fatigue syndrome?

BACKGROUND: There is evidence that patients with chronic fatigue syndrome (CFS) have mild hypocortisolism. The clinical significance of this is unclear. We aimed to determine whether hypocortisolism exerted any effect on the response of CFS to cognitive behavioural therapy (CBT). METHOD: We measured 24-h urinary free cortisol (UFC) in 84 patients with Centers for Disease Control and Prevention (CDC)-defined CFS (of whom 64 were free from psychotropic medication) who then received CBT in a specialist, tertiary out-patient clinic as part of their usual clinical care. We also measured salivary cortisol output from 0800 to 2000 h in a subsample of 56 psychotropic medication-free patients. RESULTS: Overall, 39% of patients responded to CBT after 6 months of treatment. Lower 24-h UFC output was associated with a poorer response to CBT but only in psychotropic medication-free patients. A flattened diurnal profile of salivary cortisol was also associated with a poor response to CBT. CONCLUSIONS: Low cortisol is of clinical relevance in CFS, as it is associated with a poorer response to CBT. Hypocortisolism could be one of several maintaining factors that interact in the persistence of CFS.

Chronic intermittent hypoxia and incremental cycling exercise independently depress muscle in vitro maximal Na+-K+-ATPase activity in well-trained athletes.

Athletes commonly attempt to enhance performance by training in normoxia but sleeping in hypoxia [live high and train low (LHTL)]. However, chronic hypoxia reduces muscle Na(+)-K(+)-ATPase content, whereas fatiguing contractions reduce Na(+)-K(+)-ATPase activity, which each may impair performance. We examined whether LHTL and intense exercise would decrease muscle Na(+)-K(+)-ATPase activity and whether these effects would be additive and sufficient to impair performance or plasma K(+) regulation. Thirteen subjects were randomly assigned to two fitness-matched groups, LHTL (n = 6) or control (Con, n = 7). LHTL slept at simulated moderate altitude (3,000 m, inspired O(2) fraction = 15.48%) for 23 nights and lived and trained by day under normoxic conditions in Canberra (altitude approximately 600 m). Con lived, trained, and slept in normoxia. A standardized incremental exercise test was conducted before and after LHTL. A vastus lateralis muscle biopsy was taken at rest and after exercise, before and after LHTL or Con, and analyzed for maximal Na(+)-K(+)-ATPase activity [K(+)-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase)] and Na(+)-K(+)-ATPase content ([(3)H]ouabain binding sites). 3-O-MFPase activity was decreased by -2.9 +/- 2.6% in LHTL (P < 0.05) and was depressed immediately after exercise (P < 0.05) similarly in Con and LHTL (-13.0 +/- 3.2 and -11.8 +/- 1.5%, respectively). Plasma K(+) concentration during exercise was unchanged by LHTL; [(3)H]ouabain binding was unchanged with LHTL or exercise. Peak oxygen consumption was reduced in LHTL (P < 0.05) but not in Con, whereas exercise work was unchanged in either group. Thus LHTL had a minor effect on, and incremental exercise reduced, Na(+)-K(+)-ATPase activity. However, the small LHTL-induced depression of 3-O-MFPase activity was insufficient to adversely affect either K(+) regulation or total work performed.

Aromatic L-amino acid decarboxylase (AAAD) inhibitors as carcinoid tumor-imaging agents: synthesis of 18F-labeled alpha-fluoromethyl-6-fluoro-m-tyrosine (FM-6-FmT).

The aromatic L-amino acid decarboxylase (AAAD) enzyme is significantly upregulated in neuroendocrine tumors and, thus, would be a good target for PET imaging agents. Alpha-fluoromethyl-DOPA (FMDOPA) is one of the most potent irreversible AAAD inhibitor and its non-catechol derivative, alpha-fluoromethyl-m-tyrosine (FMmT), is a promising AAAD imaging agent. We synthesized FMmT and its direct electrophilic fluorination provided a mixture of products identified by NMR analysis after HPLC purification as 6-fluoro-, 2-fluoro- and 2,6-difluoro-derivatives of FMmT. Using rat striatal homogenates, alpha-fluoromethyl-6-fluoro-m-tyrosine (FM-6-FmT) was found to have AAAD inhibitory activity comparable to that of FMDOPA. Electrophilic radiofluorination of FMmT using [18F]AcOF gave 18F labeled 6-fluoro-, 2-fluoro- and 2,6-difluoro-FMmT derivatives in 22.0%, 21.9% and 8.5% radiochemical yields, respectively. Based on its proposed mechanism of inhibition, FM-6-[18F]FmT is expected to irreversibly bind to AAAD and, hence, could be used as a PET agent to image tumors of endocrine origin containing high concentrations of AAAD. Since FM-6-FmT lacks the catechol moiety, it is expected to be better than FMDOPA since it is not a substrate for catechol-O-methyltransferase.

Novel in vitro functional assays for the determination of anthrax toxin components.

The characterisation and evaluation of the UK licensed human anthrax vaccine depends on several in vivo tests that determine its safety and potency. Assays for the determination of functionally active and/or immunoreactive toxin components and S-layer proteins have been developed and applied to the characterisation of anthrax vaccine. These technologies may support production of consistent and effective vaccines, and may ultimately reduce the requirements for in vivo testing.

Live high:train low increases muscle buffer capacity and submaximal cycling efficiency.

This study investigated whether hypoxic exposure increased muscle buffer capacity (beta(m)) and mechanical efficiency during exercise in male athletes. A control (CON, n=7) and a live high:train low group (LHTL, n=6) trained at near sea level (600 m), with the LHTL group sleeping for 23 nights in simulated moderate altitude (3000 m). Whole body oxygen consumption (VO2) was measured under normoxia before, during and after 23 nights of sleeping in hypoxia, during cycle ergometry comprising 4 x 4-min submaximal stages, 2-min at 5.6 +/- 0.4 W kg(-1), and 2-min 'all-out' to determine total work and VO(2peak). A vastus lateralis muscle biopsy was taken at rest and after a standardized 2-min 5.6 +/- 0.4 W kg(-1) bout, before and after LHTL, and analysed for beta(m) and metabolites. After LHTL, beta(m) was increased (18%, P < 0.05). Although work was maintained, VO(2peak) fell after LHTL (7%, P < 0.05). Submaximal VO2 was reduced (4.4%, P < 0.05) and efficiency improved (0.8%, P < 0.05) after LHTL probably because of a shift in fuel utilization. This is the first study to show that hypoxic exposure, per se, increases muscle buffer capacity. Further, reduced VO2 during normoxic exercise after LHTL suggests that improved exercise efficiency is a fundamental adaptation to LHTL.

Hyperopic laser in situ keratomileusis to treat overcorrected myopic LASIK.

PURPOSE: To evaluate the safety and efficacy of hyperopic laser in situ keratomileusis (LASIK) in treating hyperopia caused by overcorrected myopic LASIK and to evaluate a new technique to place the hyperopic treatment after lifting the initial myopic flap. SETTING: Open-access outpatient excimer laser surgical facility. METHODS: A retrospective analysis was performed of 54 eyes in 47 patients who had spherical hyperopic LASIK by 21 surgeons for the treatment of significant hyperopia after overcorrected LASIK for myopia. In 42 eyes, the initial LASIK flaps were lifted and in 12 eyes, new flaps were cut. The mean age of the 25 men (53%) and 22 women (47%) was 48.2 years +/- 8.4 (SD). Outcome measures included refractive error, uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), and complications. The mean follow-up was 2.97 months. RESULTS: In eyes in which postoperative emmetropia was attempted (n = 45), the mean spherical equivalent improved from +1.21 +/- 0.49 diopters (D) preoperatively to -0.38 +/- 0.50 D postoperatively (P <.001). The mean UCVA improved from 20/38.6 +/- 16.3 to 20/27.4 +/- 9.4 (P <.001). At the last follow-up, 69% of eyes were within +/-0.5 D and 96% were within +/-1.0 D of emmetropia; 42% had a UCVA of 20/20 and 96% had a UCVA of 20/40 or better. No eyes lost 2 or more lines of BSCVA. No vision-threatening complications occurred. Results in patients who had initial flaps lifted and those who had new flaps cut were statistically indistinguishable. On average, achieved hyperopic corrections were 18% greater than intended. CONCLUSION: Hyperopic LASIK was safe, predictable, and effective in the treatment of hyperopia caused by overcorrected myopic LASIK. Results were similar whether the original flap was lifted or a new one was cut.

CD95 expression in aged mice infected with tuberculosis.

The interaction between CD95 and its ligand is an important homeostatic mechanism that leads to the induction of apoptosis in activated T cells. In view of recent evidence that this pathway might be defective in aged mice, this study investigated CD95 expression on T cells in old mice activated by infection with Mycobacterium tuberculosis. The results of the study do not support the hypothesis that CD95 is poorly expressed on CD4 T cells from old mice; instead, it was found that similar numbers of T cells from young and old mice expressed CD95, with the intensity of expression if anything higher on the cells from the old mice. In addition, the study demonstrated that changes in CD44 and CD45RB expression previously observed in young infected mice proceeded in a similar fashion in old animals and, as would be predicted, that CD95(hi) expression was primarily associated with CD4 T cells expressing the activated CD44(hi) CD45RBhi phenotype.

Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis.

The results of this study provide the first evidence that two completely separate vaccine approaches, one based on a subunit vaccine consisting of a mild adjuvant admixed with purified culture filtrate proteins and enhanced by the cytokine interleukin-2 and the second based on immunization with DNA encoding the Ag85A protein secreted by Mycobacterium tuberculosis, could both prevent the onset of caseating disease, which is the hallmark of the guinea pig aerogenic infection model. In both cases, however, the survival of vaccinated guinea pigs was shorter than that conferred by Mycobacterium bovis BCG, with observed mortality of these animals probably due to consolidation of lung tissues by lymphocytic granulomas. An additional characteristic of these approaches was that neither induced skin test reactivity to commercial tuberculin. These data thus provide optimism that development of nonliving vaccines which can generate long-lived immunity approaching that conferred by the BCG vaccine is a feasible goal.

Disruption of the cellular inflammatory response to Listeria monocytogenes infection in mice with disruptions in targeted genes.

The results of this study to dissect the nature of the acquired immune response to infection with Listeria monocytogenes in mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of alphabeta T cells and the capacity to elaborate gamma interferon. In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days. The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; alphabeta-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in gammadelta-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower. In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the gammadelta-T-cell-knockout animals. These data further support the hypothesis that gammadelta T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.

Comfort and frictional properties of dental gloves.

OBJECTIVES: A survey of general dental practitioners and dental surgery assistants was carried out to ascertain their preferences and opinions on powder-free hydrogel-coated gloves compared with starch-powdered gloves. The aim was to relate the survey findings to laboratory measurements of the frictional characteristics of glove inner surfaces and their water absorptive capability. METHODS: The survey was carried out using a questionnaire given to local dental practitioners. Glove friction and water absorption measurements were made using specially designed equipment. RESULTS: The survey showed that a selected group of dentist and dental surgery assistants preferred hydrogel-coated gloves, particularly for damp donning, durability and long-term wear comfort. Laboratory measurements showed that the hydrogel coating gave a low friction coefficient against damp skin. The coating was durable, and absorbed water more readily than other treatments. CONCLUSION: A survey of dental practitioners and dental surgery assistants and laboratory measurements indicates that hydrogel-coated gloves have superior properties, and are preferred to other non-sterile glove types.

The role of interleukin-12 in acquired immunity to Mycobacterium tuberculosis infection.

The early phase of acquired cellular immunity to Mycobacterium tuberculosis infection is mediated by the emergence of protective CD4 T lymphocytes that secrete cytokines including interferon-gamma (IFN-gamma), a molecule which is pivotal in the expression of resistance to tuberculosis. Recent evidence demonstrates that infection with M. tuberculosis induces peripheral blood mononuclear cells to release the cytokine interleukin-12 (IL-12), a molecule that promotes the emergence of T-helper type-1 (Th1), IFN-gamma-producing T cells. We demonstrate here that IL-12 mRNA expression was induced by M. tuberculosis infection both in vivo and in vitro and that exogenous administration of IL-12 to mice transiently resulted in increased resistance to the infection. IL-12 also increased the production of IFN-gamma by both splenocytes derived from infected animals treated in vivo and by antigen-stimulated CD4 cells from untreated infected animals, with maximal effects at times associated with the expansion of antigen-specific CD4 T cells in vivo. In the absence of a T-cell response, as seen in SCID mice or nude mice, IL-12 only slightly augmented the moderate bacteriostatic capacity of these immunocompromised mice. Neutralization of IL-12 by specific monoclonal antibodies resulted in a reduction in granuloma integrity and slowing of the capacity of the animal to control bacterial growth.

Cytokine secretion by CD4 T lymphocytes acquired in response to Mycobacterium tuberculosis infection.

The results of this study, in which CD4 T cells were harvested from mice at various times during the course of a virulent Mycobacterium tuberculosis infection and examined for their secretion of cytokines during culture in vitro with bone marrow-derived macrophages presenting mycobacterial Ag, provide evidence for the existence of two separate waves of cytokine-producing CD4 cells. The first, which peaks at the time at which protective immunity was maximally expressed, was characterized as a IFN-gamma-secreting cell population that preferentially recognized macrophages presenting mycobacterial culture filtrate proteins, or that were infected with the live organism. A second population, which emerged 20 to 40 days later at a time when the infection had been contained, secreted IL-4 in response to the filtrate proteins, but also reacted particularly strongly to the 65-kDa (hsp60) heat shock protein molecule of M. tuberculosis. These data indicate that acquired immunity to tuberculosis infection involves the production of both Th1- and Th2-like cell populations that differ in terms of their kinetics of emergence and loss, and in terms of their Ag specificity.

Inhibition of growth of Mycobacterium avium in murine and human mononuclear phagocytes by migration inhibitory factor.

Infections caused by Mycobacterium avium, the most common form of diseminated bacterial disease in AIDS patients, are difficult to treat because of their resistance to many antimycobacterial drugs. The results of the present study show that recombinant migration inhibitory factor, a 12-kDa molecule recently isolated by COS-1 cell expression screening of cDNA from a human T-cell hybridoma, has potent inhibitory activity on the growth of a panel of clinical isolates of M. avium within both bone-marrow-derived murine macrophages and cultured human blood monocytes. These cells cultured in recombinant migration inhibitory factor exhibit various signs of activation, including cell division, morphological changes such as evidence of substantial phagolysosomal fusion, and enhanced secretion of tumor necrosis factor.

Dissemination of enteric Mycobacterium avium infections in mice rendered immunodeficient by thymectomy and CD4 depletion or by prior infection with murine AIDS retroviruses.

This study shows that infection of mice with the murine AIDS virus LP-BM5 or Du5H profoundly depressed the capacity of splenic T cells from these animals to respond to the T-cell mitogen phytohemagglutinin or concanavalin A or to alloantigens. Similar effects were also observed if mice were thymectomized and then infused with monoclonal anti-CD4 antibody (TxCD4- mice). When such mice were infected intravenously with Mycobacterium avium, growth of the infection was markedly exacerbated in the TxCD4- mice or in mice given murine AIDS virus 2 months earlier. In view of these data, we then investigated whether such treatments might cause dissemination of M. avium following enteric implantation of bacteria into the mouse cecum; this route was chosen in an attempt to model events in AIDS patients, in which the gut appears to be one of the major portals of M. avium infection. In this model, the entry and hematogenous dissemination of four clinical isolates of M. avium were monitored against time and found to be accelerated and enhanced in T-cell-deficient mice. In view of this finding, these novel approaches for enteric infection that use immunodeficient mice are presented as potential new models for the evaluation of immunotherapy and chemotherapy in a setting that bears some similarity to events believed to occur in AIDS patients.

Capacity of Mycobacterium avium isolates to grow well or poorly in murine macrophages resides in their ability to induce secretion of tumor necrosis factor.

The results of this study show that clinical isolates of Mycobacterium avium fall into two categories in terms of their capacity to grow within murine bone marrow-derived macrophage cultures: those that grow progressively and those that are incapable of growing within such cells. Members of the first category were invariably of the smooth-transparent colonial type, while most of the second were of the smooth-doomed type. In addition, this paper shows that although all isolates induced tumor necrosis factor (TNF) secretion by host cells to some extent, this production was always delayed in isolates that subsequently grew well in the host cells. This observation, coupled with the demonstration that the growth of the latter isolates was inhibited by the exogenous addition of TNF, leads us to hypothesize that the ability of a given isolate to somehow avoid host macrophage TNF production early during the course of the infection is a key factor in the pathogenesis of the disease.

Structural basis of capacity of lipoarabinomannan to induce secretion of tumor necrosis factor.

The results of this study show that lipoarabinomannans (LAM) isolated from a virulent strain and from an avirulent strain of Mycobacterium tuberculosis, which have recently been shown to differ markedly in terms of the structures of their nonreducing termini, also differ markedly in the capacity to induce the secretion of tumor necrosis factor from murine macrophages. It was found that LAM from the avirulent H37Ra strain was 100-fold more potent at inducing tumor necrosis factor secretion than LAM from the virulent Erdman strain, thus leading us to hypothesize that the structure of LAM from a given mycobacterial isolate may directly influence its ability to elicit, or avoid, cytokine-mediated mechanisms of host resistance.

T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. Evidence for different kinetics and recognition of a wide spectrum of protein antigens.

Recent evidence suggests the existence of at least two pathways of acquired specific resistance to Mycobacterium tuberculosis infection; the first consisting of cytokine-mediated activation of parasitized host cells by protective T cells, and the second involving the lysis of these cells by cytolytic T cells. Evidence presented in this report shows that both of the above mechanisms are operative in experimentally infected mice, but that they differ markedly in terms of their kinetics of emergence and loss. It was found that protective T cell activity was acquired very early during the course of the infection, and was temporally associated with the onset of bacterial elimination; however, cytolytic activity did not peak until 10 to 20 days later. This report shows further that the target Ag of these effector T cell populations were apparently numerous with no evidence for preferential recognition of a few immunodominant Ag. In view of the preponderance of target proteins in the bacterial filtrate, we present the hypothesis that such proteins secreted or otherwise leaked from the dividing mycobacterium are pinocytosed from the phagosome and used by the infected macrophage as the key protective Ag leading to T cell sensitization. This hypothesis thus explains the preferential requirement for the viable bacterium in the generation of specific resistance, and further explains why protective immunity is generated even while the organism is still multiplying in an apparently unrestrained manner.