Hepatitis From Spiroplasma sp. in an Immunocompromised Patient.

A 70-year-old lung transplant recipient patient was admitted with fever, nausea, abdominal pain, peripheral edema and pronounced weakness. An initial work-up for presumed infection revealed cholestatic hepatitis, leukocytosis and thrombocytopenia, but failed to detect a pathogen. An increased glucose uptake exclusively in the liver was demonstrated by positron emission tomography. Liver biopsy showed basophilic inclusions in the cytoplasm of hepatocytes. Broad- range 16S rRNA gene PCR followed by sequence analysis yielded Spiroplasma sp. in two independent blood samples and the liver biopsy, confirming Spiroplasma sp. as the causative agent. Antibiotic treatment with doxycycline and azithromycin led to complete recovery.

Diagnostic performance of 18F-FDG-PET/CT in vascular graft infections.

OBJECTIVE: The aim of this study was to evaluate the diagnostic accuracy of positron emission tomography/computed tomography with (18)F-fludeoxyglucose (FDG-PET/CT) in a population with suspected graft infection and to validate a new diagnostic imaging score for FDG-PET/CT. METHODS: This was a prospective cohort study. FDG-PET/CT was performed prospectively in 34 patients with suspected graft infection, in 12 of them before the start of antimicrobial treatment. Diagnostic accuracy was assessed using a new five point visual grading score and by using a binary score. Maximum standardized uptake values (SUVmax) were calculated for quantitative measurements of metabolic activity, and cut off points were calculated using the receiver operator curve (ROC). The standard of reference was a microbiological culture, obtained after open biopsy or graft explantation. RESULTS: Using the new scale, FDG-PET/CT correctly recognized 27 patients with graft infection, one patient was diagnosed as false positive, six patients were correctly classified as true negative, and no patients were rated false negative. Hence, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of FDG-PET/CT for the diagnosis of graft infections were 100%, 86%, 96%, 100%, and 97%, respectively. Using a previously established binary score, sensitivity, specificity, PPV, NPV, and accuracy were 96%, 86%, 96%, 86%, and 94% respectively. ROC analysis suggested an SUVmax cut off value of ≥3.8 to differentiate between infected and non-infected grafts (p < .001). Additionally, FDG-PET/CT provided a conclusive clinical diagnosis in six of seven patients without graft infection (i.e., other sites of infections). CONCLUSIONS: The diagnostic accuracy of FDG-PET/CT in the detection of aortic graft infection is high. A newly introduced five point visual grading score and early imaging prior to antimicrobial treatment may further improve the diagnostic accuracy.

Primary non-small cell lung cancer response upon treatment with denosumab.

Here we report the case of a patient with metastatic adenocarcinoma of the lung harboring an ALK gene translocation. In this patient a response of the primary tumor and metastases has been detected upon treatment with denosumab. A possible link between ALK and RANK is postulated.

Lowering heart rate with an optimised breathing protocol for prospectively ECG-triggered CT coronary angiography.

OBJECTIVES: The aim was to prospectively characterise the effect of the level of breath-hold on heart rate in CT coronary angiography (CTCA) with prospective electrocardiogram (ECG) triggering and its impact on coronary artery attenuation. METHODS: 260 patients (86 women; mean age 59 ± 11 years) underwent 64-slice CTCA using prospective ECG triggering. Prior to CTCA, heart rates were recorded during 15 s of breath-hold at three different levels of inspiration (normal, intermediate and deep). The inspiration level with the lowest heart rate was chosen for actual CTCA scanning. Coronary artery attenuation was measured, and the presence of backflow of contrast material into the inferior vena cava (as an indicator of increased intrathoracic pressure) was recorded. RESULTS: The mean heart rate at breath-hold was significantly different for the three inspiration levels (normal, 60 ± 8 bpm; intermediate, 59 ± 8 bpm; deep, 57 ± 7 bpm; p<0.001). The maximum heart rate reduction in each patient at breath-hold averaged 5.3 ± 5.1 bpm, and was observed at a normal inspiration depth in 23 (9%) patients, at an intermediate inspiration depth in 102 (39%) patients and at deep inspiration in 135 (52%) patients. Overall, there was no association between the level of breath-hold and coronary vessel attenuation (p-value was not significant). However, the backflow of contrast material into the inferior vena cava (n = 26) was found predominantly at deep inspiration levels (p<0.001), and, when it occurred, it was associated with reduced coronary attenuation compared with patients with no backflow (p<0.05). CONCLUSION: The breath-hold level to best reduce heart rate for CTCA should be individually assessed prior to scanning because a mean heart rate reduction of 5 bpm can be achieved.

First head-to-head comparison of effective radiation dose from low-dose 64-slice CT with prospective ECG-triggering versus invasive coronary angiography.

BACKGROUND: Reduction of radiation burden of multidetector computed tomography coronary angiography (CTCA) has remained an important task. OBJECTIVE: To compare effective radiation dose of low-dose 64-slice CTCA using prospective ECG-triggering versus diagnostic invasive coronary angiography (CA). METHODS: 42 patients referred for elective invasive CA owing to suspected coronary artery disease (CAD) were prospectively enrolled to undergo a low-dose CTCA without calcium scoring within the same day before CA. Dose-area product of diagnostic invasive CA and dose-length product of CTCA were measured, converted into effective radiation dose and compared using Mann-Whitney U tests. In addition, accuracy of CTCA to detect CAD (coronary artery narrowing > or =50%) was assessed using invasive CA as standard of reference. On an intention-to-diagnose basis all non-evaluative vessels were included in the analysis and censored as positive. RESULTS: The estimated mean effective radiation dose was 8.5 (4.4) mSv (range 1.4-20.5 mSv) for diagnostic invasive CA, and 2.1 (0.7) mSv (range 1.0-3.3 mSv) for CTCA (p<0.001). 19 patients (42.9%) had no CAD by invasive CA. 40 (95.2%) patients have been correctly classified as having CAD (23/23) or no CAD (17/19). Over 97% (551/567) of segments were evaluable. Vessel-based analysis revealed sensitivity, specificity, positive and negative predictive value of 94.2% (CI 0.8% to 1.0%), 94.8% (CI 09% to 1.0%), 89.0% (CI 0.8% to 1.0%), 97.4% (CI 09% to 1.0%) and an accuracy of 94.6%. CONCLUSIONS: Low dose CTCA allows evaluation of CAD with high accuracy, but delivers a significantly less effective radiation dose to patients compared to diagnostic invasive CA.

Impact of vessel attenuation on quantitative coronary angiography with 64-slice CT.

The aim of the study was to determine the impact of vessel attenuation on quantitative 64-slice computed tomography coronary angiography (CTCA). CTCA and invasive quantitative coronary angiography (QCA) were performed in 100 consecutive patients (42 women, 58 men; mean age 64.4+/-9.4 years; age range 39-87 years). In QCA, stenoses were quantified with dedicated software, whereas in CTCA, stenosis severity was assessed with an electronic caliper tool: stenoses were graded in 10% steps and assigned as either a calcified or non-calcified lesion. Vessel attenuation in the left main (LMA) and the proximal right coronary artery (RCA) were measured and correlated with differences in quantifications of stenosis grade between QCA and CTCA. A total of 113 coronary stenoses were detected by both methods (94 significant and 19 non-significant); 52 stenoses were rated as non-calcified and 61 as calcified lesions. The mean difference between QCA and quantitative CTCA grading was 5.1+/-16.9% (range -27 to 46%) overall; 1.9+/-14.2% (range -27 to 38%) for non-calcified lesions and 7.8+/-18.6% (range -23 to 46%) for calcified lesions. Mean vessel attenuation was 362+/-76 HU (range 191-584 HU) in the LMA and 333+/-81 HU (range 162-564 HU) in the RCA. Attenuation did not significantly correlate with differences in QCA and CTCA gradings, neither overall nor for calcified or non-calcified lesions. When 64-slice CTCA is used, coronary vessel attenuation had no impact on the quantitative grading of stenoses.

[Influence of body mass index on coronary artery opacification in 64-slice CT angiography]. / Einfluss des Bodymass-Index auf den Kontrast in den Koronararterien mit der 64-Schicht-CT.

PURPOSE: To evaluate the influence of the body mass index (BMI) on coronary artery opacification in 64-slice CT. MATERIAL AND METHODS: Sixty-two patients retrospectively underwent ECG-gated 64-slice CT coronary angiography (tube potential 120 kV, tube current time product 650 mAs) after intravenous injection of 80 ml of iodinated contrast agent (320 mg/ml, 5 ml/s). Attenuation values (HU) were measured and contrast-to-noise ratios (CNR) were calculated in the right coronary artery (RCA) and left main artery (LMA). The CNR was defined as the difference between the mean attenuation in the vessel and the mean attenuation in the perivascular fat tissue divided by the image noise in the ascending aorta. The height and weight of the patients at the time of the CT scan were recorded and the BMI was calculated. RESULTS: The mean BMI was 26.2 +/- 3.2 kg/m (2) (range 19.7 - 32.2 kg/m (2)), the mean attenuation in the LMA was 330 +/- 64 HU, and the mean attenuation in the RCA was 309 +/- 68 HU. The CNR in the LMA was 16.7 +/- 3.8, and the CNR in the RCA was 15.9 +/- 3.6. The image noise in the ascending aorta significantly correlated with the BMI (r = 0.36, p < 0.01). A weak negative correlation was found between the BMI and LMA attenuation (r = - 0.28, p < 0.05), whereas no significant correlation was found for the RCA (r = - 0.21, p = 0.12). A significant negative correlation was found between the BMI and the CNR in the RCA (r = - 0.41, p < 0.05) and the LMA (r = - 0.47, p < 0.001). CONCLUSION: With constant scan parameters and a constant contrast medium amount, the CNR in both coronary arteries decreases while the BMI increases. This implies a modification of previously standardized and fixed examinations with respect to individually adapted protocols with variable parameters for CT coronary angiography.

Role of putative virulence factors of Streptococcus pyogenes in mouse models of long-term throat colonization and pneumonia.

To investigate the role of putative virulence factors of Streptococcus pyogenes (group A streptococcus; GAS) in causing disease, we introduced specific mutations in GAS strain B514, a natural mouse pathogen, and tested the mutant strains in two models of infection. To study late stages of disease, we used our previously described mouse model (C3HeB/FeJ mice) in which pneumonia and systemic spread of the streptococcus follow intratracheal inoculation. To study the early stages of disease, we report here a model of long-term (at least 21 days) throat colonization following intranasal inoculation of C57BL/10SnJ mice. When the three emm family genes of GAS strain B514-Sm were deleted, the mutant showed no significant difference from the wild type in induction of long-term throat colonization or pneumonia. We inactivated the scpA gene, which encodes a complement C5a peptidase, by insertion of a nonreplicative plasmid and found no significant difference from the wild type in the incidence of throat colonization. However, there was a small but statistically significant decrease in the incidence of pneumonia caused by the scpA mutant. Finally, we demonstrated a very important effect of the hyaluronic acid capsule in both models. Following intranasal inoculation of mice with a mutant in which a nonreplicative plasmid was inserted into the hasA gene, which encodes hyaluronate synthase, we found that all bacteria recovered from the throats of the mice were encapsulated revertants. Following intratracheal inoculation with the hasA mutant, the incidence of pneumonia within 72 h was significantly reduced from that of the control strain (P = 0.006). These results indicate that the hyaluronic acid capsule of S. pyogenes B514 confers an important selective advantage for survival of the bacteria in the upper respiratory tract and is also an important determinant in induction of pneumonia in our model system.

Streptococcus pyogenes infection in mice.

We inoculated 5 to 7-week-old female C3HeB/FeJ mice with Streptococcus pyogenes strain B514-Sm (type M50) by both an intranasal and intratracheal route and characterized the resulting illness. Following intranasal inoculation, the animals developed signs of illness within 1 to 8 days post-inoculation which correlated with acute, suppurative, bronchopneumonia during histopathologic analysis; however, the relationship of response to dose was non-linear, as seen previously in a small group of mice. Intratracheal inoculations were then performed to increase the quantitative reliability of the model. Following intratracheal inoculation, the animals succumbed to an illness that was indistinguishable from that seen after intranasal inoculation, and the incidence of pneumonia followed a steep dose response curve. The dose at which 50% of the animals exhibited signs of respiratory illness within 72 h was 1.0 x 10(7) colony forming units. All of the animals that appeared ill had lung lesions as determined by gross and histopathologic examination. Bacteraemia followed pneumonia in two-thirds of the intratracheally inoculated animals, indicating that the S. pyogenes had crossed tissue barriers. We hope that this model will be useful in future studies concerning the role of suspected streptococcal virulence factors in the later stages of pathogenesis of invasive S. pyogenes infection.

Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of Streptococcus pyogenes.

Many Streptococcus pyogenes immunoglobulin-binding proteins have structural similarities to the antiphagocytic M protein, including the well-known C repeats. One of these molecules is the immunoglobulin A-binding protein Arp, which is expressed by a serotype 4 strain for which no antiphagocytic M protein has yet been described. We expressed Arp4 in an S. pyogenes strain from which the structural gene for the M protein has been deleted and found that Arp4 is not sufficient to inhibit phagocytosis.

Regulation of rns, a positive regulatory factor for pili of enterotoxigenic Escherichia coli.

Attachment of enterotoxigenic Escherichia coli to the human gut is considered an important early step in infection that leads to diarrhea. This attachment is mediated by pili, which belong to a limited number of serologically distinguishable types. Many of these pili require the product of rns, or a closely related gene, for their expression. We have located the major promoter for rns and found that although its sequence diverges significantly from a sigma-70 promoter consensus sequence, it is very strong. Transcription of rns is negatively regulated both at a region upstream of this promoter and at a region internal to the rns open reading frame. In addition, rns positively regulates its own transcription, probably by counteracting these two negative effects.

Cytotoxicity of extracellular Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaire's disease and Pontiac fever, is known to produce a cytopathic effect on macrophages. The capacity of extracellular L. pneumophila to mediate toxicity for guinea pig peritoneal macrophages and J774 mouse macrophages was assessed. Extracellular organisms were found to be capable of mediating toxicity; however, toxic activity appeared to require close proximity with the mononuclear cell surface. Serogroup 1 strains grown on supplemented Mueller-Hinton agar exhibited variable expression of toxic activity. One strain positive on supplemented Mueller-Hinton agar was cytotoxic and unable to replicate in J774 macrophages but remained virulent for guinea pigs at high doses.

Virulence of two Streptococcus pyogenes strains (types M1 and M3) associated with toxic-shock-like syndrome depends on an intact mry-like gene.

The major virulence factor of Streptococcus pyogenes, the M protein, is positively regulated at the transcriptional level by mry in the M type 6 strain studied. We show here that in two S. pyogenes strains isolated from cases of toxic-shock-like syndrome, a type M1 strain and a type M3 strain, an mry-like gene is also required for resistance to phagocytosis.

Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen of alveolar macrophages. Although previous studies have demonstrated that specific antibody facilitates uptake of L. pneumophila by phagocytic cells, the role of complement has been unclear. Thus, we have examined the relative contributions of Fc gamma- and complement receptor-mediated adherence to guinea pig peritoneal macrophages, U937 human monocytic cells, and J774 mouse macrophage cells. Opsonization of L. pneumophila (Philadelphia 2) with polyclonal immunoglobulin G promoted maximum adherence to guinea pig macrophages. In contrast, incubation in the presence of 20% fresh nonimmune human serum from a single donor did not promote adherence. The results obtained with U937 and J774 cells paralleled those obtained with guinea pig macrophages. In the absence of specific antibody, opsonization with guinea pig complement did not enhance adherence of the Philadelphia 1, Philadelphia 2, or Knoxville strain. However, when complement was added to heat-inactivated, specific antiserum, a fourfold increase in the number of adherent organisms was observed. Blocking studies utilizing membrane receptor-specific monoclonal antibodies demonstrated that both Fc and complement receptors mediated adherence of organisms treated with complement in the presence of specific antibody. These results suggest that complement augments adherence of L. pneumophila only when acting in concert with specific antibody.

Thymocyte subpopulations during early fetal development in the BALB/c mouse.

Phenotypic analysis of thymocytes during murine fetal development may be of use in determining the pathways of thymocyte differentiation. The expression of the functionally significant molecules Lyt-2 (CD8), L3T4 (CD4), and the TCR has already been described. However, mAb specific for several other murine lymphocyte surface markers are now available and, although these have been used to characterize adult thymocytes, a detailed analysis of fetal thymocytes with these antibodies has not previously been undertaken. In this study, we have used mAb specific for Thy-1, J11d, Pgp-1, and the IL-2R, in addition to those for Lyt-2 and L3T4, to identify subpopulations of early fetal thymocytes. By using two-color flow cytometric analysis of cells obtained from fetal thymuses on sequential days of gestation, we have been able to follow the development of various subpopulations through early fetal ontogeny. Our data indicate that the earlier thymocytes are found in the J11d+/Pgp-1+ subset which is abundant at fetal day 14 but constitute a numerical minority by day 16.

Cooperation between helper T cells and cytotoxic T lymphocyte precursors.

We have investigated the requirement for the presence of L3T4-positive T cells in the in vivo priming of Lyt-2-positive cytotoxic T-lymphocyte precursors. The antigens used were the male-specific antigen H-Y and autosomal minor histocompatibility antigens. In some experiments, responder mice were depleted of L3T4-positive cells by repeated intraperitoneal injections of the anti-L3T4 antibody, GK1.5. In other experiments, lymphoid cells from normal mice were fractionated in vitro by antibody-plus-complement treatment and the populations primed in irradiated adoptive hosts. In these antigen systems, depletion of L3T4-positive helper cells decreases the level of priming of cytotoxic T lymphocytes. With regeneration of the L3T4-positive subpopulation, the CTL response to antigen increases. To some extent, the reliance on L3T4-positive cells can be overcome by increasing the antigen dose. We conclude that in most physiological responses, L3T4-positive T cells enhance the cytotoxic T-cell response.

Expression of T cell antigen receptor beta-chains on subsets of mouse thymocytes. Analysis by three-color flow cytometry.

The expression on adult mouse thymocytes of a T cell antigen receptor beta-chain epitope, recognized by the antibody F23.1, has been studied by three-color flow cytometry. Low density F23.1 staining was found mainly on CD4+8+ thymocytes. High density staining was mainly on CD4+8- and CD4-8+ cells. Variable proportions of CD4-8- cells were also F23.1+. Among CD4-8+ cells, F23.1 was expressed only on the J11d- subset with mature T cell function. We conclude that many subpopulations of thymocytes express antigen receptors and are candidates for the population subject to thymic selection, but at present no single subpopulation makes a convincing claim.

Transient expression of IL-2 receptor precedes the differentiation of immature thymocytes.

The growth of mature T lymphocytes after activation by antigen is regulated by the binding and endocytosis of interleukin-2 (IL-2). In the thymus, approximately 50% of adult thymocytes that carry neither the CD4 nor the CD8 antigen and day 14-15 fetal CD4-8- thymocytes express receptors for IL-2(IL-2R). The CD4-8- (double-negative) subpopulation of thymocytes contains the precursors of cells that can differentiate along an unknown pathway into thymocytes bearing either CD8 or CD4, with the characteristics of mature T lymphocytes. The basis for IL-2R expression by double-negative thymocytes is unclear as they appear to lack a functional T-cell receptor/CD3 complex through which activation of peripheral T cells is mediated. The argument for a role for IL-2 in thymocyte differentiation has also been complicated by conflicting reports on the inability or capability of double-negative thymocytes to respond to IL-2 in vitro. At present, both the nature of the stimuli within the thymic micro-environment which induce IL-2R expression and its relevance to thymocyte differentiation are not known. We show here that the IL-2R-bearing subset has a greater potential to differentiate into phenotypically mature T lymphocytes than do IL-2R-negative thymocytes. In addition, progeny of IL-2R-negative donor cells transiently express IL-2R in the thymuses of adoptive hosts before generating CD8 and/or CD4-positive thymocytes. These results identify the IL-2R-positive cells as a more differentiated double-negative thymocyte subset on the pathway to mature T lymphocytes.

Differentiation potential of subsets of CD4-8- thymocytes.

Precursor T cells in the thymus are contained within a subpopulation of thymocytes that lack the markers CD4 and CD8. We have examined the heterogeneity of these cells by flow cytometric analysis, and defined four subpopulations using the cell surface markers Thy-1, J11d and the IL-2 receptor (IL-2R). The J11d+ subset of CD4-8- cells all bear the antigen Thy-1, and some express the IL-2R. Staining and RNA analysis of J11d+ cells suggest that some express receptors of the CD3 gamma delta type, but none express CD3 alpha beta receptors. In fetal thymus organ culture, the J11d+ cells diversify to form 'cortical type' CD4+8+ cells and 'medullary type' cells expressing either CD4 or CD8; in vivo they repopulate the thymus of an irradiated host and seed the periphery with T cells. In contrast, the J11d- subset of CD4-8- thymocytes do not all bear Thy-1 and none express the IL-2R, but some express antigen receptors of the CD3 alpha beta type. They have more limited diversification potential in organ culture, and in vivo fail to recolonize the irradiated host in a homing-independent assay. We conclude that they are not precursor T cells, but rather a side-branch from the main line of T cell differentiation.

T cell receptor expression and receptor-mediated induction of clonal growth in the developing mouse thymus. High surface beta-chain density is a requirement for functional maturity.

The ontogeny of T cell antigen receptor expression and function in the mouse thymus has been studied using a monoclonal antibody, F23.1, which recognizes a determinant on the beta chain of the receptor, and stains 25% of mature T cells and around 7-15% of adult thymocytes from most mouse strains. The same monoclonal antibody selectively activates Lyt-2+ peripheral T cells. Receptors are detectable by staining and fluorescence-activated cell sorter analysis from fetal day 17, and thereafter the overall frequency increases steadily towards adult levels. However, late fetal thymocytes express all of their antigen receptor beta chain at a very low level, visible by staining as a "shoulder" on the peak of negative cells. Thymocytes with high-density surface beta chain, visible by staining as a distinct peak, appear only after birth and are a prominent feature at neonatal day 4. In the late fetus, expression of beta chain can be detected on thymocytes with the "mature" L3T4-, Lyt-2+ phenotype. Despite this, F23.1-responsive precursors are not found in the fetal thymus, and appear in two waves, the first during day 1 of postnatal life and the second between days 3 and 4. These data suggest that high-density surface expression of T cell receptor beta chain occurs in parallel with functional maturation.