Ultrafast X-ray Auger probing of photoexcited molecular dynamics.

Molecules can efficiently and selectively convert light energy into other degrees of freedom. Disentangling the underlying ultrafast motion of electrons and nuclei of the photoexcited molecule presents a challenge to current spectroscopic approaches. Here we explore the photoexcited dynamics of molecules by an interaction with an ultrafast X-ray pulse creating a highly localized core hole that decays via Auger emission. We discover that the Auger spectrum as a function of photoexcitation--X-ray-probe delay contains valuable information about the nuclear and electronic degrees of freedom from an element-specific point of view. For the nucleobase thymine, the oxygen Auger spectrum shifts towards high kinetic energies, resulting from a particular C-O bond stretch in the ππ* photoexcited state. A subsequent shift of the Auger spectrum towards lower kinetic energies displays the electronic relaxation of the initial photoexcited state within 200 fs. Ab-initio simulations reinforce our interpretation and indicate an electronic decay to the nπ* state.

Strongly dispersive transient Bragg grating for high harmonics.

We create a transient Bragg grating in a high-harmonic generation medium using two counterpropagating pulses. The Bragg grating disperses the harmonics in angle and can diffract a large bandwidth with temporal resolution limited only by the source size.

Calibration of a high harmonic spectrometer by laser induced plasma emission.

We present a method that allows for a convenient switching between high harmonic generation (HHG) and accurate calibration of the vacuum ultraviolet (VUV) spectrometer used to analyze the harmonic spectrum. The accurate calibration of HHG spectra is becoming increasingly important for the determination of electronic structures. The wavelength of the laser harmonics themselves depend on the details of the harmonic geometry and phase matching, making them unsuitable for calibration purposes. In our calibration mode, the target resides directly at the focus of the laser, thereby enhancing plasma emission and suppressing harmonic generation. In HHG mode, the source medium resides in front or after the focus, showing enhanced HHG and no plasma emission lines. We analyze the plasma emission and use it for a direct calibration of our HHG spectra.

The development of a Th1-type response and resistance to Leishmania major infection in the absence of CD40-CD40L costimulation.

CD40-CD40L interactions have been shown to be essential for the production of IL-12 and IFN-gamma and control of L. major infection. In contrast, C57BL/6 mice deficient in CD28 develop a dominant Th1-type response and heal infection. In this study, we investigate the effects of a deficiency in both CD40L and CD28 molecules on the immune response and the course of L. major infection. We compared infection in mice genetically lacking CD40L (CD40L(-/-)), CD28 (CD28(-/-)), or both (CD40L(-/-)CD28(-/-)), and in C57BL/6 mice, all on a resistant background. Although CD40L(-/-) mice failed to control infection, CD28(-/-) and CD40L(-/-)CD28(-/-) mice, as well as C57BL/6 mice, spontaneously resolved their infections. Healing mice had reduced numbers of lesion parasites compared with nonhealing CD40L(-/-) mice. At wk 9 of infection, we detected similar levels of IL-4, IFN-gamma, IL-12p40, and IL-12Rbeta2 mRNA in draining lymph nodes of healing C57BL/6, CD28(-/-), and CD40L(-/-)CD28(-/-) mice, whereas CD40L(-/-) mice had increased mRNA levels for IL-4 but reduced levels for IFN-gamma, IL-12p40, and IL-12Rbeta2. In a separate experiment, blocking of the CD40-CD40L pathway using Ab to CD40L led to an exacerbation of infection in C57BL/6 mice, but had little or no effect on infection in CD28(-/-) mice. Together, these results demonstrate that in the absence of CD28 costimulation, CD40-CD40L interaction is not required for the development of a protective Th1-type response. The expression of IL-12p40, IL-12Rbeta2, and IFN-gamma in CD40L(-/-)CD28(-/-) mice further suggests the presence of an additional stimulus capable of regulating IL-12 and its receptors in absence of CD40-CD40L interactions.

IL-10 mediates susceptibility to Leishmania donovani infection.

Human visceral leishmaniasis (VL) results in a severe and potentially fatal systemic disease, accompanied by cellular immune depression. The production of IL-10 correlates with ongoing disease and it has been suggested that the cellular immune depression that accompanies active disease may be due to a predominance of IL-10 production rather than a lack of IFN-gamma production, which is essential for optimal macrophage activation and parasite elimination. To examine the role of IL-10 in resistance during L. donovani infection (a causative agent of VL), the course of infection was examined in mice lacking the gene for IL-10. BALB/c IL-10-/-, as well as C57BL/6 IL-10-/- mice, were highly resistant to L. donovani infection, as evidenced by liver parasite burdens which were tenfold lower than those in control mice after 14 days of infection. Enhanced resistance was accompanied by increased production of IFN-gamma and nitric oxide in BALB/c IL-10-/- mice. Susceptibility to infection in BALB/c IL-10-/- mice was enhanced following in vivo treatment with a neutralizing antibody to IFN-gamma or IL-12. Together these studies demonstrate for the first time that IL-10 is a critical component of the immune response that inhibits resistance to L. donovani.

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice.

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.

Anti-TGF-beta treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production.

CB6F1 mice display intermediate susceptibility to Leishmania major infection compared with the highly susceptible BALB/c and resistant C57BL/6 parental strains. During early weeks of infection, these mice develop dominant Th2 type responses to L. major, although they eventually exhibit a Th2 to Th1 switch and spontaneously resolve their infections. In this study, we have examined the effects of either IL-12 or anti-TGF-beta therapy on the immune response and course of disease in chronically infected CB6F1 mice. Local treatment with IL-12 inoculated into the parasitized lesion at 4 wk of infection induced a marked increase in IFN-gamma production but did not result in a significant reduction in numbers of parasite or promote more rapid healing. However, local treatment with an Ab to TGF-beta led to both a decrease in parasite numbers and more rapid healing, despite the fact that such treatment did not significantly alter the pattern of IL-4 and IFN-gamma production. Immunohistochemical studies showed that anti-TGF-beta treatment resulted in increased nitric oxide production within parasitized lesions. Our results suggest that TGF-beta may play an important regulatory role during chronic stages of a L. major infection by suppressing macrophage production of nitric oxide and that, in the absence of TGF-beta, even the relatively low levels of IFN-gamma observed in mice with dominant Th2-type responses are sufficient to activate macrophages to destroy amastigotes within parasitized lesions.

Leishmania major: a clone with low virulence for BALB/c mice elicits a Th1 type response and protects against infection with a highly virulent clone.

BALB/c mice are highly susceptible to infection with Leishmania major and generally develop a severe, nonhealing form of disease following parasite inoculation. As opposed to protective Th1 type immune responses which develop in resistant strains of mice, BALB/c mice develop predominant Th2 type responses characterized by the production of high levels of IL-4, but only low levels of IFN-gamma. However, BALB/c mice will develop resistance and Th1 type responses following the inoculation of very low numbers of L. major promastigotes. In this study, we have examined the effects of parasite virulence on the immune response and disease phenotype in susceptible BALB/c mice. Two clones of L. major were isolated which differed with respect to their in vitro growth rates as promastigotes and their virulence for mice. One rapidly growing clone, L.m.F1, was highly virulent in BALB/c mice and produced nonhealing infections characterized by predominant Th2 type responses. In contrast, a slow-growing clone, L.m.S2, was less virulent in BALB/c mice and produced self-healing infections at parasite doses equivalent to those which produced progressive disease with the more virulent clone. Mice which healed infections with the L.m.S2 clone developed responses characterized by elevated production of IFN-gamma and were resistant to a challenge infection with the virulent L.m.F1 clone. These results suggest that the virulence of individual parasite clones may influence both the course of disease and the phenotype of the immune response which develops during infection.

Successful therapy of chronic, nonhealing murine cutaneous leishmaniasis with sodium stibogluconate and gamma interferon depends on continued interleukin-12 production.

Treatment of nonhealing forms of human leishmaniasis with antimonial drugs in combination with gamma interferon (IFN-gamma) may promote healing more effectively than conventional drug therapy. Although the natures of immune responses in patients prior to treatment are often unclear, it is generally assumed that such therapy also promotes a switch from a Th2-type response to a dominant Th1-type response. We have examined the efficacy of IFN-gamma therapy, in combination with drug therapy, to promote healing and a Th2-to-Th1 switch in highly susceptible BALB/c mice infected with Leishmania major. Short-term treatment with the antileishmanial drug sodium stibogluconate failed to significantly alter the course of disease or the immune response when it was given during the third and fourth weeks of infection. IFN-gamma therapy, administered over the same time period, also failed to induce cure or a Th1 dominant response. In contrast, mice treated with a combination of drug and IFN-gamma therapy resolved their infections and developed Th1-type responses. However, administration of an antibody to interleukin 12 (IL-12) reversed the therapeutic effects of therapy with drug plus IFN-gamma, suggesting that IFN-gamma promotes cure through an IL-12-dependent mechanism. Analysis of mRNA levels within parasitized lesions suggests that drug treatment plus IFN-gamma treatment, in addition to reducing parasite numbers, results in reduced levels of IL-4, IL-10, and transforming growth factor beta transcripts but increased levels of transcripts of the p40 chain of IL-12 and inducible nitric oxide synthase, which catalyzes the production of nitric oxide. Together, these results suggest that such immunotherapy may promote the development of a protective Th1-type response in susceptible mice by a mechanism which involves both suppression of regulatory cytokines and enhancement of IL-12 and nitric oxide production.

In vivo alterations in cytokine production following interleukin-12 (IL-12) and anti-IL-4 antibody treatment of CB6F1 mice with chronic cutaneous leishmaniasis.

CB6F1 mice exhibit intermediate resistance to infection with Leishmania major compared to their highly susceptible (BALB/c) and resistant (C57BL/6) parental strains. Unlike the C57BL/6 and BALB/c strains, which rapidly develop dominant Th1- or Th2-type responses, respectively, after infection, CB6F1 mice develop responses in which both Th1- and Th2-type cytokines are elevated through at least the first month of infection before Th1 responses become dominant as cutaneous lesions gradually heal. We have examined the effects of interleukin-12 (IL-12) and/or anti-IL-4 antibody treatment on cytokine production and the course of disease in CB6F1 mice with chronic L. major infections. When administered at 1 month of infection, IL-12 treatment led to a rapid decrease in mRNA levels for IL-4 within parasitized lesions and a moderate increase in gamma interferon (IFN-gamma) transcript levels in lymph nodes draining the site of infection. When IL-12 and anti-IL-4 antibody were administered together, they induced a marked decrease in IL-4 and transforming growth factor beta mRNA expression within lesions and a more dramatic increase in lymph node IFN-gamma transcript levels within 4 days after treatment. In comparison, similar treatment of infected BALB/c mice led to only a moderate increase in IFN-gamma transcripts but no decrease in mRNA levels for Th2-type cytokines. Treatment of CB6F1 mice with either IL-12 or anti-IL-4 antibody had no significant effect on the subsequent course of infection, whereas combined IL-12 plus anti-IL-4 treatment resulted in a decrease in lesion size and parasite numbers and a shift towards a Th1-dominant response. These results suggest that the immediate effects of cytokine or anti-cytokine therapy may be predictive of the long-term effects on the course of infection and that down-regulation of Th-2 type cytokines may be critical to the development of a Th1-dominant response.

In search of clinical excellence.

This article was presented as a lecture at the 1995 Combined Sections Meeting by Joseph P. Farrell, MS, PT, who received the Orthopaedic Section's Paris Distinguished Service Award. He was recognized for service to the Orthopaedic Section in the area of manual therapy and for the formation of the American Academy of Orthopaedic Manual Physical Therapists (AAOMPT). Clinical excellence is a journey that every clinician should commence early in his/her career. The purpose of this paper is to discuss clinical excellence and its importance to the physical therapy profession. To address this topic, the following items will be discussed: 1) clinical competency and clinical expertise; 2) communication and patient management in the current era of health care reform; 3) politics relating to manual therapy; 4) the role of manual therapy in our profession; 5) clinical residency educational programs; and 6) the academic and clinician role in education. The author concludes that if physical therapy is a profession of excellent clinicians, then the physical therapy profession can survive evolving changes in the health care system.

Successful chemotherapy in experimental leishmaniasis is influenced by the polarity of the T cell response before treatment.

Little is known about the influence of host factors on successful chemotherapy in leishmaniasis. Although successfully treated patients will convert from a delayed-type hypersensitivity (DTH)-negative to a DTH-positive state, the importance of the immune status of the host before treatment remains largely unexplored. In experimental murine cutaneous leishmaniasis, it was found that increased polarization towards a Th2 cytokine profile before the onset of drug therapy leads to an increased frequency of relapse after treatment. Whereas >50% of mice with established Leishmania major infections were cured when treated with the antileishmanial drug sodium stibogluconate, <10% of mice were cured when the animals had been pretreated with anti-interferon-gamma antibody to polarize the response toward a Th2 cytokine pattern before therapy. With successful drug therapy, cytokine profiles were found to switch from a Th2 to Th1 pattern, and resistance to reinfection was observed.

The influence of the site of parasite inoculation on the development of Th1 and Th2 type immune responses in (BALB/c x C57BL/6) F1 mice infected with Leishmania major.

Although inbred strains of mice are classified as genetically resistant or susceptible to Leishmania major based upon their ability to control infection, other factors such as the strain, dose, and site of parasite inoculation can also affect the outcome of the disease. Here we used the F1 progeny of BALB/c (susceptible) and C57BL/6 (resistant) mice (designated CB6F1) to investigate whether mice or intermediate susceptibility to infection differed from the parental strains in their ability to control infections at different cutaneous sites. CB6F1 mice developed progressive disease when inoculated in the dorsal skin, but healed infections in the footpad. Consistent with these observations, mice inoculated in the footpad ultimately developed Th1 responses, known to be required for healing, while Th2 responses developed in mice inoculated in the dorsal skin. However, IL-4 and IFN-gamma production during the first few weeks of infection was similar in CB6F1 mice inoculated at either site, suggesting that factors in addition to the relative levels of these cytokines produced early in infection may influence the nature of the antileishmanial immune response, and the eventual disease outcome. Infection in CB6F1 mice provides a model for the study of immunity to L. major in genetically identical animals, in which a prolonged mixed Th1/Th2 cytokine pattern initially develops, but ultimately diverges into more defined Th1 and Th2 type responses.

Making change pay. Survey: more hospitals tap compensation as a tool of change.

No matter how much you say you want your health care organization's performance levels to change, you've got to put your money where your wish list is. That, in a nutshell, is the bottom-line finding of this year's Hay Hospital Compensation Survey, exclusively in this issue. But while the concept is simple, the execution is not, as the authors point out in their analysis.

Physician executive contracts: negotiating the future.

Once viewed as a matter of standard protocol, physician executive contracts have become as complex as the health care industry itself. Historically, hospital administration and physicians negotiated a few key points, then sent the ideas to an attorney for insertion of standard legalize and boilerplate. Today, physician executive contracts are an important part of the changes in health care. They not only cover traditional hospital and physician relations, but increasingly apply to new types of relations (such as employment) between hospitals and physicians, physicians and physicians, and health plans and physicians. In this article, we will explore both the "content" and the "context" of physician executive contracts. Content will deal with the specific provisions typically included in contracts. Context will address issues associated with preparing for and negotiating a contract.

Activity of pentostam (sodium stibogluconate) against cutaneous leishmaniasis in mice treated with neutralizing anti-interferon-gamma antibody.

Studies with Leishmania donovani in the mouse have demonstrated that an intact T cell compartment is required for effective anti-leishmanial therapy using pentavalent antimony compounds such as Pentostam (sodium stibogluconate), suggesting that the in vivo efficacy of drug treatment is at least partially immune-based. Similarly, Leishmania-infected, immunodeficient human patients including those with acquired immunodeficiency syndrome (AIDS) generally relapse following therapy with antimonials. However, sodium stibogluconate is directly parasiticidal in vitro, in the absence of T cells or T cell products. Using a model of a cutaneous form of leishmaniasis, in which susceptible BALB/c mice were infected with Leishmania major, we investigated whether the antileishmanial activity of the drug demonstrated a requirement for interferon-gamma (IFN-gamma), a cytokine produced during a T helper cell type 1 (Th1) immune response and known to contribute to resistance to infection, and whether drug therapy affected the nature of the antileishmanial response. Lesion development was suppressed in mice treated from the onset of infection with sodium stibogluconate alone, and in animals treated with sodium stibogluconate plus a neutralizing anti-IFN-gamma antibody, and tissue parasite burdens were approximately 10,000-fold less at the end of therapy in both groups compared with controls. Lesion development was similarly suppressed in mice with established lesions treated with either sodium stibogluconate alone, or sodium stibogluconate plus anti-IFN-gamma antibody. The production of IFN-gamma by cells from infected animals was somewhat increased immediately following therapy with sodium stibogluconate, an effect that was not long-lasting, while interleukin-4 (IL-4) production was not affected by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)