Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Rapid transcriptional and metabolic adaptation of intestinal microbes to host immune activation.

The gut microbiota produces metabolites that regulate host immunity, thereby impacting disease resistance and susceptibility. The extent to which commensal bacteria reciprocally respond to immune activation, however, remains largely unexplored. Herein, we colonized mice with four anaerobic symbionts and show that acute immune responses result in dramatic transcriptional reprogramming of these commensals with minimal changes in their relative abundance. Transcriptomic changes include induction of stress-response mediators and downregulation of carbohydrate-degrading factors such as polysaccharide utilization loci (PULs). Flagellin and anti-CD3 antibody, two distinct immune stimuli, induced similar transcriptional profiles, suggesting that commensal bacteria detect common effectors or activate shared pathways when facing different host responses. Immune activation altered the intestinal metabolome within 6 hours, decreasing luminal short-chain fatty acid and increasing aromatic metabolite concentrations. Thus, intestinal bacteria, prior to detectable shifts in community composition, respond to acute host immune activation by rapidly changing gene transcription and immunomodulatory metabolite production.

Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus.

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

Commensal microbes provide first line defense against Listeria monocytogenes infection.

Listeria monocytogenes is a foodborne pathogen that causes septicemia, meningitis and chorioamnionitis and is associated with high mortality. Immunocompetent humans and animals, however, can tolerate high doses of L. monocytogenes without developing systemic disease. The intestinal microbiota provides colonization resistance against many orally acquired pathogens, and antibiotic-mediated depletion of the microbiota reduces host resistance to infection. Here we show that a diverse microbiota markedly reduces Listeria monocytogenes colonization of the gut lumen and prevents systemic dissemination. Antibiotic administration to mice before low dose oral inoculation increases L. monocytogenes growth in the intestine. In immunodeficient or chemotherapy-treated mice, the intestinal microbiota provides nonredundant defense against lethal, disseminated infection. We have assembled a consortium of commensal bacteria belonging to the Clostridiales order, which exerts in vitro antilisterial activity and confers in vivo resistance upon transfer into germ free mice. Thus, we demonstrate a defensive role of the gut microbiota against Listeria monocytogenes infection and identify intestinal commensal species that, by enhancing resistance against this pathogen, represent potential probiotics.

Cocaine-mediated impact on HIV infection in humanized BLT mice.

Cocaine abuse has been shown to have broad-ranging effects on human immunity. With regards to HIV infection, in vitro studies have shown that cocaine enhances infection of stimulated lymphocytes. Moreover, cohort studies in the pre- and post-HAART era have linked stimulant abuse with increased HIV pathogenesis. The latter data, however, have been undermined by a series of confounding factors underscoring the importance of controlled in vivo models to fully assess the impact of cocaine use and abuse on HIV infection and pathogenesis. Here, we have infected humanized mice with HIV-1 following acute cocaine exposure to assess the impact on infection. Stimulant exposure resulted in increased inflammatory cytokine expression, accelerated HIV infection, while blunting effector function of cytotoxic T lymphocytes. These data demonstrate cocaine's multifactorial impact on HIV infection that extends beyond high-risk behavior.

Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

Cocaine exposure enhances permissiveness of quiescent T cells to HIV infection.

In vivo and in vitro exposure to stimulants has been associated with increased levels of HIV infection in PBMCs. Among these lymphocyte subsets, quiescent CD4(+) T cells make up the majority of circulating T cells in the blood. Others and we have demonstrated that HIV infects this population of cells inefficiently. However, minor changes in their cell state can render them permissive to infection, significantly impacting the viral reservoir. We have hypothesized that stimulants, such as cocaine, may perturb the activation state of quiescent cells enhancing permissiveness to infection. Quiescent T cells isolated from healthy human donors were exposed to cocaine and infected with HIV. Samples were harvested at different time-points to assess the impact of cocaine on their susceptibility to infection at various stages of the HIV life cycle. Our data show that a 3-day exposure to cocaine enhanced infection of quiescent cells, an effect that appears to be mediated by σ1R and D4R. Overall, our results indicate that cocaine-mediated effects on quiescent T cells may increase the pool of infection-susceptible T cells. The latter underscores the impact that stimulants have on HIV-seropositive individuals and the challenges posed for treatment.

HIV-1 infection of hematopoietic progenitor cells in vivo in humanized mice.

HIV infection has been associated with defective hematopoiesis since the earliest days of the HIV/AIDS epidemic. Generation of all hematopoietic lineages suffers in the face of infection. The mechanisms by which HIV impairs normal blood cell development remain unclear, and direct infection of intermediate hematopoietic progenitors has not been established as a source of HIV-associated hematopoietic pathology. Here, we demonstrate infection of multiple subsets of highly purified intermediate hematopoietic progenitors by wild-type HIV both in vitro and in vivo. Although direct infection is clearly cytotoxic, we find that some infected progenitors can survive and harbor proviral DNA. We report intermediate hematopoietic progenitors to be a novel target of infection and their permissivity to infection increases with development. Further, the nonobese diabetic severe combined immunodeficiency common γ chain knockout-bone marrow-liver-thymus humanized mouse provides a unique model for studying the impact of HIV infection on bone marrow-based human hematopoiesis.

HIV restriction in quiescent CD4⁺ T cells.

The restriction of the Human Immunodeficiency Virus (HIV) infection in quiescent CD4⁺ T cells has been an area of active investigation. Early studies have suggested that this T cell subset is refractory to infection by the virus. Subsequently it was demonstrated that quiescent cells could be infected at low levels; nevertheless these observations supported the earlier assertions of debilitating defects in the viral life cycle. This phenomenon raised hopes that identification of the block in quiescent cells could lead to the development of new therapies against HIV. As limiting levels of raw cellular factors such as nucleotides did not account for the block to infection, a number of groups pursued the identification of cellular proteins whose presence or absence may impact the permissiveness of quiescent T cells to HIV infection. A series of studies in the past few years have identified a number of host factors implicated in the block to infection. In this review, we will present the progress made, other avenues of investigation and the potential impact these studies have in the development of more effective therapies against HIV.

Introduction of exogenous T-cell receptors into human hematopoietic progenitors results in exclusion of endogenous T-cell receptor expression.

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

Using the BLT humanized mouse as a stem cell based gene therapy tumor model.

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) (1, 2) or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) (3). Both models were mainly utilized for the study of HIV infection. One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant (4). The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency (5-8). We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC (7, 9). This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9). The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (http://jaxmice.jax.org/research/immunology/005557-housing.html).

Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.

The goal of cancer immunotherapy is the generation of an effective, stable, and self-renewing antitumor T-cell population. One such approach involves the use of high-affinity cancer-specific T-cell receptors in gene-therapy protocols. Here, we present the generation of functional tumor-specific human T cells in vivo from genetically modified human hematopoietic stem cells (hHSC) using a human/mouse chimera model. Transduced hHSC expressing an HLA-A*0201-restricted melanoma-specific T-cell receptor were introduced into humanized mice, resulting in the generation of a sizeable melanoma-specific naïve CD8(+) T-cell population. Following tumor challenge, these transgenic CD8(+) T cells, in the absence of additional manipulation, limited and cleared human melanoma tumors in vivo. Furthermore, the genetically enhanced T cells underwent proper thymic selection, because we did not observe any responses against non-HLA-matched tumors, and no killing of any kind occurred in the absence of a human thymus. Finally, the transduced hHSC established long-term bone marrow engraftment. These studies present a potential therapeutic approach and an important tool to understand better and to optimize the human immune response to melanoma and, potentially, to other types of cancer.

Preoperative and postoperative evaluation of intra-articular fractures of the calcaneus based on computed tomography scanning.

OBJECTIVE: To define the relationship between postoperative congruency of the posterior facet of the subtalar joint based on computed tomography (CT) and clinical results. DESIGN: Preoperative and postoperative CT scans were taken prospectively and analyzed. SETTING: University medical center. PATIENTS: Twenty-nine displaced fractures in twenty-five patients. INTERVENTION: All patients were treated with open reduction and internal fixation without bone graft. MAIN OUTCOME MEASURES: Postoperative CT findings were classified into three groups, according to the degree of displacement: anatomic, no displacement; nearly anatomic, displaced less than two millimeters; and approximate, displaced more than two millimeters. RESULTS: The reduction state after operative treatment for the cases with more comminution showed worse results when analyzed in both preoperative and postoperative CT scans. Fifteen of seventeen fractures (88 percent) with anatomic reduction and seven of eight fractures (87 percent) with nearly anatomic reduction had excellent or good clinical results. In contrast, no fracture with an approximate reduction had an excellent result. CONCLUSION: An excellent or good clinical result can be expected when the postoperative displacement of the posterior facet of the subtalar joint is less than two millimeters.

R-phycoerythrin as a fluorescent label for immunolocalization of bound atrazine residues.

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.

Fluorescence immunolocalization of bound atrazine residues in plant tissue.

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material.

[Initial studies of 3-dimensional imaging using ultrasound]. / Erste Untersuchungen zur dreidimensionalen Darstellung mittels Ultraschall.

In the study reported here three-dimensional sonographic imaging of organs was achieved for the first time. To make this possible it was first necessary to ensure, by appropriate guidance of the probe, that the sequence of sonographic sections was coordinated in their spatial arrangement. This was accomplished by constructing a probe guide with which parallel sonographic sections could be demonstrated. The distance between these sections was known, so that with the aid of suitable computer programs three-dimensional reconstruction of a kidney examined in a water bath was possible. Since, however, due to the uneven surface of the body, it will hardly be possible to obtain parallel sonographic sections of an organ, a new solution had to be found to ensure the necessary coordinated sequence of sections. The solution lay in rotating the probe. A further device was constructed in which the probe could be rotated farther, by known angles, from section to section. The pivotal point was at the center of the probe tip. The computer knew the angular distance between these sections and reconstruction to a three-dimensional image was therefore possible. Prior the three-dimensional reconstruction the ultrasonographic sections had to be contoured, since only the surface of the organ was available for three-dimensional image construction. Three-dimensional imaging of an organ can be achieved on the one hand by binary representation and on the other with a continuous organ surface. The advantage of binary representation is that the original sonographic data are incorporated in the image-producing process without any computer manipulation; with a continuous surface the distance between the individual sections has to be interpolated.(ABSTRACT TRUNCATED AT 250 WORDS)

[Three-dimensional imaging in ultrasonic diagnosis. Initial results]. / Dreidimensionale Dartstellung in der Ultraschalldiagnostik. Erste Ergebnisse.

A new method of three-dimensional (3-D) reconstruction of 2-D ultrasound images of the kidney is described. It is based on a coordinated spatial reconstruction of sequential cross-sectional images. The ultrasound head is moved longitudinally between two rails (parallel sections) and rotated. With a suitable computer program and contouring of each cross-section (so that the organ limits are defined for the computer) these cross-sectional pictures can be reconstructed into 3-D organ images. The kidney can then be presented spatially either as a binary picture or with closed surface. Ultrasound investigators are still unaccustomed to colour reproduction of 3-D reconstructed organs. It remains to be seen whether the method is valuable in routine clinical use.

[Fetal functional atrioventricular blocks in pregnancies at risk]. / Fetale funktionelle AV-Blockierungen bei Risikoschwangerschaften.

Because antepartal and subpartal CTG monitoring is now widespread, fetal cardiac arrhythmias are being diagnosed ever more frequently. In a high-risk group of 148 pregnancies with placental insufficiency and preterm births, 14 CTGs manifested a rigid rectangular pattern, with alternating normal-frequency and slightly bradycardial cycles. The change in frequency occurred suddenly. All the children of the high-risk group were delivered by cesarean section and weighed less than 2500 g at birth. There is no description in the literature of similar CTG curves with sligthly bradycardial phases. However, there are some descriptions of cases of fetal AV blocks with severe bradycardias. These frequently occur in combination with fetal cardiac abnormalities and collagenoses of the mother. In the present authors' group these underlying conditions were not diagnosed. The fixed numerical ratio of the output frequency to slight bradycardia in the CTGs described here supports the tentative diagnosis of an atrioventricular conduction disorder, with relatively high conduction from the atrium to the ventricle. Since the arrhythmias did not continue in the newborns post partum it may be assumed that they were functional events. Birthweight, Apgar score, and the placental weight of the 14 births with the rectangular CTG curve described were lower than in the control group, i.e., the other 134 births. The CTG patterns described appear to have been caused by a chronic supply deficiency, resulting in functional AV conduction disorders. This CTG course should therefore be reason enough for intensive fetal monitoring.