Early inflammation precedes cardiac fibrosis and heart failure in desmoglein 2 murine model of arrhythmogenic cardiomyopathy.

The study of a desmoglein 2 murine model of arrhythmogenic cardiomyopathy revealed cardiac inflammation as a key early event leading to fibrosis. Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure due to abnormalities in the cardiac desmosome. We examined how loss of desmoglein 2 (Dsg2) in the young murine heart leads to development of AC. Apoptosis was an early cellular phenotype, and RNA sequencing analysis revealed early activation of inflammatory-associated pathways in Dsg2-null (Dsg2-/-) hearts at postnatal day 14 (2 weeks) that were absent in the fibrotic heart of adult mice (10 weeks). This included upregulation of iRhom2/ADAM17 and its associated pro-inflammatory cytokines and receptors such as TNFα, IL6R and IL-6. Furthermore, genes linked to specific macrophage populations were also upregulated. This suggests cardiomyocyte stress triggers an early immune response to clear apoptotic cells allowing tissue remodelling later on in the fibrotic heart. Our analysis at the early disease stage suggests cardiac inflammation is an important response and may be one of the mechanisms responsible for AC disease progression.

Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection.

To assess directly the role of protein kinase C (PKC)epsilon in the immune system, we generated mice that carried a homozygous disruption of the PKCepsilon locus. PKCepsilon(-/-) animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. Further analysis revealed that LPS-stimulated macrophages from PKCepsilon(-/-) mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IkappaB kinase, a reduction in IkappaB degradation, and a decrease in nuclear factor (NF)kappaB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCepsilon(-/-) mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

Effect of gadolinium concentration on renal signal intensity: An in vitro study with a saline bag model.

PURPOSE: To compare the effects of gadolinium concentration on intermediate-weighted fast inversion-recovery and T2-weighted fast spin-echo magnetic resonance (MR) images by using a saline bag model of the kidney. MATERIALS AND METHODS: Normal-saline bags containing gadopentetate dimeglumine in concentrations of 0-20 mmol/L were imaged by using a variety of pulse sequences. Signal intensity was measured. RESULTS: Signal intensity loss at high gadolinium concentrations (negative enhancement) was demonstrated with all MR sequences. Increasing T2 weighting increased the negative enhancement effect and reduced the minimum gadolinium concentration at which negative enhancement was seen. The results of this study match theoretic predictions. CONCLUSION: The negative enhancement of normal renal tissue seen in intermediate-weighted fast inversion-recovery MR imaging is caused by T2 shortening at high gadolinium concentrations. Thus, gadolinium-enhanced T2-weighted fast spin-echo imaging also is expected to show negative enhancement and may prove to be a superior sequence for MR imaging of pyelonephritis. Further clinical investigation is warranted.

Co-ordination of the expression of the protein tyrosine kinase p56(lck) with the pre-T cell receptor during thymocyte development.

The protein tyrosine kinase p56(lck) is essential for the regulation of early thymocyte development by transducing pre-T cell receptor (pre-TCR) mediated-signals. Here we have investigated the developmental timing of expression of p56(lck) in the thymus. We show that p56(lck) transcripts are detectable in each CD4(-)CD8(-) double-negative (DN) thymocyte subset defined by CD25 and CD44. The steady-state level of p56(lck) mRNA with these four DN subsets is similar, varying by less than 2-fold. However, p56(lck) protein levels are undetectable in DN subsets 1 and 2, but are at least 20-fold up-regulated between DN subsets 2 and 3. Analysis of independent transgenic mouse lines in which the proximal p56(lck) promoter was used to drive expression of a green fluorescent protein (GFP) reporter gene revealed a lack of correlation between GFP mRNA expression and transgene copy number and variable expression of GFP protein in different lines. Taken together these results show that the expression of the p56(lck) gene is developmentally regulated at the post-transcriptional level at the precise developmental stage at which its upstream receptor complex, the pre-TCR, is expressed. These results also have implications for the interpretation of the phenotype of transgenic mice which utilize the p56(lck) proximal promoter.

Renal disease potentiates the injury caused by SWL.

PURPOSE: The present study tested the hypothesis that renal disease potentiates the structural/functional changes induced by a clinical dose of shockwaves. MATERIALS AND METHODS: Experimental pyelonephritis was induced in 6- to 8-week-old pigs before treatment with 2,000 shocks at 24 kV. These pigs were divided into two groups according to whether they were infected with a highly virulent (Group 1) or less virulent (Group 2) inoculation of E. coli. All animals were imaged by MR prior to SWL as a means of documenting the extent of pyelonephritis and immediately after SWL to examine the lesion produced by the shockwaves. The glomerular filtration rate (GFR), renal plasma flow (RPF) and para-aminohippurate (PAH) extraction were determined bilaterally on day 30 (Group 1) or day 80 (Group 2). RESULTS: In group 2, urine flow and sodium excretion were reduced by 50% from baseline in the shocked kidneys at both 1 and 4 hours post-SWL. A sustained reduction in RPF through 4 hours post-SWL was noted in the shocked kidneys in Group 1, but RPF was significantly reduced only at the 1-hour determination in Group 2. Large, consistent reductions in GFR were evident at 1 and 4 hours post-SWL in shocked and unshocked kidneys of Group 2 and in the shocked kidneys of Group 1. No significant changes were noted in PAH extraction. CONCLUSION: Acute pyelonephritis exaggerated the effect of a clinical dose of shockwaves on renal hemodynamics. This effect suggests that renal disease may be risk factor for SWL-induced injury.

Pulmonary disease in the immunocompromised child.

Immune deficiency states in children may be related to primary immunodeficiency syndromes or secondary disorders of the immune system. The secondary immunodeficiencies in children include human immunodeficiency virus-associated acquired immunodeficiency, as well as immunosuppression secondary to antineoplastic chemotherapeutic agents, bone marrow transplantation, and drugs given to prevent transplant rejection. This article discusses the common primary and secondary immunocompromised states of childhood, with emphasis on their attendant infectious, lymphoproliferative, and neoplastic complications.

Childhood pyelonephritis: comparison of gadolinium-enhanced MR imaging and renal cortical scintigraphy for diagnosis.

PURPOSE: To compare gadolinium-enhanced inversion-recovery magnetic resonance (MR) imaging with renal cortical scintigraphy in the diagnosis of childhood pyelonephritis. MATERIALS AND METHODS: Thirty-seven patients with fever-producing urinary tract infection underwent gadolinium-enhanced inversion-recovery MR imaging and technetium-99m renal cortical scintigraphy. Each study was read in double-blind fashion by two radiologists. The kidney was divided into three zones, and each was graded as positive, equivocal, or negative for pyelonephritis. RESULTS: Seventy kidneys (210 zones) were imaged. Twenty-six kidneys (54 zones) had evidence of pyelonephritis at both MR imaging and scintigraphy. Twenty-four kidneys (100 zones) were negative on both studies. Twelve kidneys (42 zones) were positive at MR imaging but negative at scintigraphy, and four kidneys (seven zones) were negative at MR imaging but positive at scintigraphy. The results of MR imaging for pyelonephritis were not equivalent to the results of scintigraphy (P = .001 for renal zones). The proportion of positive agreement between readers for the presence of pyelonephritis was 0.85 and 0.57 for MR imaging and scintigraphy, respectively. The proportion of negative agreement was 0.88 and 0.80 for MR imaging and scintigraphy, respectively. CONCLUSION: Gadolinium-enhanced inversion-recovery MR imaging enabled detection of more pyelonephritic lesions than did renal cortical scintigraphy and had superior interobserver agreement.

HIV- I Nef severely impairs thymocyte development and peripheral T-cell function by a CD4-independent mechanism.

Nef is a regulatory protein of the human and simian immunodeficiency viruses (HIV and SIV) whose role in infection and the viral life cycle are not fully understood. In T-lymphocytes Nef down-regulates cell-surface CD4, and has been implicated in an increase in infectivity at low primary viral isolate titres. Additionally, the SIV nef gene is necessary for viraemia and AIDS-like pathogenesis in rhesus macaques. We report here in an in vivo murine transgenic model that thymocyte and T-cell-specific nef gene expression results in a marked decrease in thymic cellularity from 16 days post coitus. This reduction in thymocyte cell number is independent of CD4 expression and Nef-induced CD4 down-regulation, but can be restored by expressing a constitutively active p56lckF505 gene. Functional analyses have revealed a severe decrease in thymocyte and T-cell proliferation in response to both T-cell-receptor- and mitogen-mediated stimuli. In addition, a significant proportion of Nef-expressing peripheral T-cells display cell-surface characteristics associated with cellular activation. These results suggest that Nef expression in developing thymocytes can severely reduce the regeneration capacity of the immune system, whereas expression in mature T-cells dramatically decreases their potential to respond to antigen. With the recent recognition of a persistently high viral load in HIV-infected individuals, these findings have important implications for the mechanism of the progressive deterioration of the immune system that leads to AIDS.

Experimental pyelonephritis in piglets: diagnosis with MR imaging.

PURPOSE: To compare findings at magnetic resonance (MR) imaging with those at histopathologic examination in the detection of experimentally induced pyelonephritis in piglets. MATERIALS AND METHODS: MR imaging was performed in 23 piglets with and nine piglets without experimentally induced pyelonephritis. Escherichia coli were injected into the bladder of the 23 piglets with surgically created vesicoureteral reflux. Imaging was performed with unenhanced and contrast material-enhanced T1-weighted and fast multiplanar inversion-recovery (IR) and fast spinecho T2-weighted sequences. MR images and pathologic findings were reviewed independently by two pediatric radiologists and a pathologist, respectively, in a blinded fashion. RESULTS: Sixty-four kidneys and 192 renal zones were evaluated. Coronal gadolinium-enhanced fast multiplanar IR imaging was the only sequence that was sensitive and specific for the diagnosis of pyelonephritis. For the two reviewers, respectively, sensitivity was 85% (n = 75) and 92% (n = 81) of 88 histopathologically positive zones and specificity was 95% (n = 99) and 94% (n = 98) of 104 pathologically negative zones. Findings at gadolinium-enhanced fast multiplanar IR imaging were not statistically different from findings at histopathologic examination in the detection of pyelonephritis. Interobserver reproducibility for the contrast-enhanced fast multiplanar IR sequence was excellent (kappa statistic = 0.82 and 0.90, respectively, for interpretation of a renal zone and of a kidney). CONCLUSION: Contrast-enhanced fast multiplanar IR imaging is a sensitive and specific test for detection of experimental pyelonephritis in this piglet model.

Hepatocellular carcinoma in a child with Niemann-Pick disease: imaging findings.

Niemann-Pick disease (NPD) is a metabolic disease in which cirrhosis is relatively common; however, there is no known association of NPD with hepatocellular carcinoma. We present a case of metastatic, multifocal hepatocellular carcinoma in a 4-year-old boy with NPD and onset of cirrhosis in infancy.

Altered cytokine expression in T lymphocytes from human immunodeficiency virus Tat transgenic mice.

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.

Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses.

The identification of human immunodeficiency virus (HIV) as the etiologic agent of AIDS has led to the proposal of novel intervention strategies to block HIV infection and viral replication or eliminate HIV-infected cells. We have produced recombinant retroviruses for a molecular ablation system, whereby a toxin gene can be delivered to hematopoietic cells for the specific elimination of HIV Tat-expressing cells. For this cell-specific ablation, we have coupled the conditional toxin herpes simplex virus type 1 thymidine kinase (tk) gene to the HIV-2 promoter and Tat responsive region (TAR) in order that transcriptional activity be under the absolute control of HIV and simian immunodeficiency virus Tat trans-activator proteins. Since the HIV-2 promoter has a considerable level of basal expression in the absence of Tat, we constructed a number of modifications in the HIV-2 promoter to minimize the risk of cytotoxicity to cells not containing HIV Tat. We demonstrate that certain promoter modifications reduce basal transcription while maintaining high trans-activated levels of expression when transfected or transduced by retroviral vectors into several different cell lines. In mouse and human cells infected with HIV-2 tk retroviruses, we show that Tat-induced expression from the HIV-2 promoter results in differential ablation and a massive reduction in Tat-positive cells after ganciclovir treatment. Thus, the retroviruses produced in these studies may be applicable to HIV ablative therapy.

CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.

Studies of HIV-2 promoter activity and cell specific ablation.

We have a developed a retroviral mediated molecular ablation method to specifically eliminate HIV Tat-expressing cells. This approach utilizes the Tat-inducible HIV-2 promoter and a conditional toxin gene. The Herpes Simplex Virus thymidine kinase gene product is toxic to mammalian cells only after treatment with Ganciclovir (GCV) or other nucleoside analogues. We demonstrate here that certain promoter modifications can decrease basal expression while retaining the ability to be transactivated. Furthermore, we show that a HIV-2 promoter thymidine kinase gene cassette transduced via retroviral vectors into tissue culture cells can specifically promote the ablation of HIV-Tat expressing cells in the presence GCV. We also show that there is a large differential in HIV-thymidine kinase gene transcription and lethal drug dose between Tat-expressing cells and Tat-negative cells.