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Cryptococcosis is a fungal disease caused by Cryptococcus neoformans and Cryptococcus gattii. By inhalation and subsequent pulmonary infection, it may disseminate to the CNS and cause meningitis or meningoencephalitis. Most cases occur in immunosuppressed hosts, including patients with HIV/AIDS, patients receiving immunosuppressing drugs, and solid organ transplant recipients. However, cryptococcosis also occurs in individuals with apparently healthy immune systems. A growing number of cases are caused by C gattii, with infections occurring in both immunosuppressed and immunocompetent individuals. In the majority of documented cases, treatment of C gattii infection of the CNS requires aggressive management of raised intracranial pressure along with standard antifungal therapy. Early cerebrospinal fluid evacuation is often needed through placement of a percutaneous lumbar drain or ventriculostomy. Furthermore, pharmacological immunosuppression with a high dose of dexamethasone is sometimes needed to ameliorate a persistently increased inflammatory response and to reduce intracranial pressure. In this Grand Round, we present the case of an otherwise healthy adolescent female patient, who, despite aggressive management, succumbed to C gattii meningoencephalitis. We also present a review of the existing literature and discuss optimum clinical management of meningoencephalitis caused by C gattii., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.

Background: O-linked N-acyl-glycosylation may regulate protein function by competing with phosphorylation of serine residues. Availability of substrate for this process is regulated, in part, by N-Acyl-D-glucosamine 2-epimerase (NAGE), which interconverts N-acetyl-glucosamine (GlcNAc) and N-acetylmannosamine (ManNAc). NAGE is also a putative renin-binding protein. This study tested the hypothesis that NAGE is present in the human heart and that NAGE expression is increased in the failing human heart., Methods and Results: Ribonuclease protection assays (RPAs) demonstrated increased NAGE gene expression in failing hearts from subjects with idiopathic dilated and ischemic cardiomyopathies compared with nonfailing hearts. In situ reverse transcriptase-polymerase chain reaction, using primers designed to localize NAGE mRNA, demonstrated that, in nonfailing hearts, NAGE gene expression was restricted to endothelial cells and not detectable in cardiac myocytes. However, in failing human hearts NAGE gene expression was selectively activated in cardiac myocytes, but not endothelial cells. Immunohistochemistry confirmed that the pattern of NAGE protein expression corresponded to the pattern of gene expression., Conclusions: NAGE gene and protein expression were selectively activated in left ventricular myocytes from end-stage failing human hearts.

Background- Vascular smooth muscle cell (VSMC) proliferation is a critical factor in the neointima formation that causes restenosis after coronary angioplasty (PTCA). Desferri-exochelin 772SM (D-EXO), a highly diffusible, lipophilic iron chelator secreted by Mycobacterium tuberculosis, inhibits proliferation of VSMCs in culture. We hypothesized that treatment with D-EXO would inhibit neointima formation in balloon-injured vessels in vivo. Methods and Results- We subjected 24 pigs to overstretch coronary artery injury with standard PTCA balloons and then administered intramural injections of either D-EXO (n=14) or vehicle (n=10) through an Infiltrator catheter. Treatments were randomized, and the investigators were blinded with regard to treatment group until data analysis was completed. One month later, we euthanized the pigs, excised the injured coronary segments, made multiple sections of each segment, and identified the site of maximal neointima formation. An injury score based on the degree of disruption of the internal or external elastic lamina or media was assigned. D-EXO reduced stenosis index (neointima area divided by the area within the internal elastic lamina), adjusted for injury score, by 47%. Neointima thickness was also reduced. Conclusions- D-EXO, injected intramurally, substantially inhibited formation of neointima in a porcine vascular injury model.

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.

Previous studies have demonstrated that environmentally or genetically induced changes in the intracellular proteins that compose the cytoskeleton can contribute to heart failure. Because neonatal right ventricular myocytes are immature and are in the process of significant cytoskeletal change, we hypothesized that they may be particularly susceptible to pressure stress. Newborn calves exposed to hypobaric hypoxia (barometric pressure = 430 mmHg) for 14 days developed severe pulmonary hypertension (pulmonary arterial pressure = 101 +/- 6 vs. 27 +/- 1 mmHg) and right heart failure compared with age-matched controls. Light microscopy showed partial loss of myocardial striations in the failing neonatal right but not left ventricles and in neither ventricle of adolescent cattle dying of altitude-induced right heart failure. In neonatal calves, immunohistochemical analysis of the cytoskeletal proteins (vinculin, metavinculin, desmin, vimentin, and cadherin) showed selectively, within the failing right ventricles, patchy areas characterized by loss and disorganization of costameres and intercalated discs. Within myocytes from the failing ventricles, vinculin and desmin were observed to redistribute diffusely within the cytosol, metavinculin appeared in disorganized clumps, and vimentin immunoreactivity was markedly decreased. Western blot analysis of the failing right ventricular myocardium showed, compared with control, vinculin and desmin to be little changed in total content but redistributed from insoluble (structural) to soluble (cytosolic) fractions; metavinculin total content was markedly decreased, tubulin content increased, particularly in the structural fraction, and cadherin total content and distribution were unchanged. We conclude that hypoxic pulmonary hypertensive-induced neonatal right ventricular failure is associated with disorganization of the cytoskeletal architecture.

Modulation of gene expression at the level of mRNA stability has emerged as an important regulatory paradigm. In this context, differential expression of numerous mRNAs in normal versus neoplastic tissues has been described. Altered expression of these genes, at least in part, has been demonstrated to be at the level of mRNA stability. Two ubiquitously expressed mRNA binding proteins have recently been implicated in the stabilization (Hu antigen R/HuR) or destabilization (AU-rich element mRNA binding protein [AUF1]/heterogeneous nuclear ribonucleoprotein D) of target mRNAs. Further, their functional activity appears to require cytoplasmic localization. In the present study, we demonstrate a strong correlation between increased cytoplasmic expression of both AUF1 and HuR with urethane-induced neoplasia and with butylated hydroxytoluene-induced compensatory hyperplasia in mouse lung tissue. In addition, when compared with slower growing cells, rapidly growing neoplastic lung epithelial cell lines expressed a consistently higher abundance of both AUF1 and HuR proteins. Moreover, in nontumorigenic cell lines, both AUF1 and HuR protein abundance decreased with confluence and growth arrest. In contrast, in spontaneous transformants, AUF1 and HuR abundance was unaffected by changes in cell density. We suggest that growth-regulated alterations in AUF1 and HuR abundance may have pleiotropic effects on the expression of a number of highly regulated mRNAs and that this significantly impacts the onset, maintenance, and progression of the neoplastic phenotype. Mol. Carcinog. 28:76-83, 2000., (Copyright 2000 Wiley-Liss, Inc.)

The beta(1)-adrenergic receptor (AR) is the dominant subtype in non-failing and failing myocardium. beta(1)-AR signaling, by the endogenous neurotransmitter norepinephrine, is central to the regulation of myocardial contractility. In heart failure, the beta(1)-AR undergoes subtype-selective downregulation which may protect against the increased cardiac adrenergic drive associated with this pathophysiological state. To examine the hypothesis that chronically increased beta(1)-AR mediated signaling has adverse myocardial effects, transgenic mice overexpressing the human beta(1)-AR in a cardiac-selective context were produced, utilizing an alpha-myosin heavy chain (MHC) promoter. In these mice, beta(1)-AR protein abundance was approximately 24-46-fold (1-2 pmol/mg protein) that of wild-type mice. Histopathological examination of young (4 months old) and old (approximately 9 months old) transgenic mouse hearts consistently demonstrated large areas of interstitial replacement fibrosis, marked myocyte hypertrophy and myofibrilar disarray. In addition, increased expression of the pre-apoptotic marker, Bax, was observed coincident with regions of fibrosis accompanied by an increased apoptotic index, as measured by TUNEL assay. Older non-transgenic mice exhibited a slight tendency towards a decreased fractional shortening, whereas older beta(1)-AR transgenic mice had a marked reduction in fractional shortening (%FS approximately 30) as determined by echocardiography. Additionally, older beta(1)-AR transgenic mice had an increased left ventricular chamber size. In summary, cardiac-directed overexpression of the human beta(1)-AR in transgenic mice leads to a significant histopathological phenotype with no apparent functional consequence in younger mice and a variable degree of cardiac dysfunction in older animals. This model system may ultimately prove useful for investigating the biological basis of adrenergically-mediated myocardial damage in humans., (Copyright 2000 Academic Press.)

Background: Excessive lengthening of cardiac myocytes attributed to series addition of sarcomeres is a consistent feature of left ventricular dilation in chronic heart failure. Currently, it is not feasible to assess myocyte dimensions, particularly myocyte length, in a manner that is of potential diagnostic usefulness., Methods and Results: Isolated myocytes from three groups of normal rats (100, 200, and 300 g) were obtained by using two different methods: (1) digestion of formalin-fixed myocardial tissue using potassium hydroxide (KOH) and (2) retrograde aortic perfusion of fresh hearts with collagenase. There was no difference in mean cell length between the two methods. The KOH method was also used to isolate intact, rod-shaped myocytes from formalin-fixed human cadaver left ventricles (control, n = 3; heart failure, n = 3) and from human right ventricle biopsy specimens (n = 6). Confirming our previous work using collagenase-isolated myocytes from fresh human explants, left ventricular myocytes from failing hearts showed longer mean cell length compared with control hearts. Data from human right ventricle biopsy specimens confirmed our previous finding in rats that myocyte lengthening is less pronounced in this chamber in heart failure., Conclusions: The KOH method can be used to obtain reliable measurements of myocyte length and other cellular parameters from myocardial biopsies and autopsy material. Such data may be useful in the diagnostic assessment of remodeling associated with heart failure.

Background: The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT1) receptor in the failing human heart are not understood., Methods and Results: Radioligand binding with 3H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). 125I-Ang II formation was measured in a subset of hearts. Saralasin (125I-(Sar1,Ile8)-Ang II) was used to measure total Ang II receptor density. AT1 and AT2 receptor binding were determined with the AT1 receptor antagonist losartan. Maximal ACE binding (Bmax) was 578+/-47 fmol/mg in IDC left ventricle (LV), 713+/-97 fmol/mg in PPH LV, and 325+/-27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE Bmax was 737+/-78, 638+/-137, and 422+/-49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). 125I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT1 receptor subtype in failing PPH ventricles: 6.41+/-1.23 fmol/mg in PPH LV, 2.37+/-0.50 fmol/mg in PPH RV, 5.38+/-0.53 fmol/mg in NF LV, and 7.30+/-1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV)., Conclusions: ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT1 receptor is downregulated only in the failing RV.

Increased ventricular expression of several genes, including atrial natriuretic factor (ANF), has been documented in experimental models of cardiac hypertrophy. It remains to be clarified whether altered expression of these genes is a consistent marker of the hypertrophy itself or a marker of some parallel pathogenetic process. Using a transgenic mouse model of hypertrophic cardiomyopathy as a tool, we assessed the relationship between the amount of ventricular ANF gene expression and the degree of hypertrophy as well as the relationship between the cells expressing ANF and tissue pathology. We determined that hypertrophy is not always associated with increased ventricular expression of ANF and that cells expressing ANF are found in regions of tissue pathology. We propose that alteration in the ventricular expression of this gene is a sensitive indicator of cardiac pathogenesis and may result from a number of different stimuli that include, among others, abnormal tissue architecture and hemodynamic load.

Existing evidence supports the hypothesis that human papillomavirus (HPV) may play an etiologic role in the malignant transformation of squamous epithelial cells. Although HPV DNA has been identified in a high proportion of squamous cell carcinomas (SCC) of the cervix, anorectum, skin, and upper airways, few studies have tested for HPV in SCC of the lung. To confirm the presence of HPV in lung SCC, we tested for HPV DNA extracted from formalin-fixed tissues of 34 patients by polymerase chain reaction (PCR). DNA amplification was performed using HPV L1 consensus sequence primers (MY11 and MY09; Perkin-Elmer Cetus, Norwalk, CT) which recognize a broad spectrum of HPV types including 6, 11, 16, 18, 31, and 33, among many other known types, as well as at least 20 other unidentified types. PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization with [32P]-labeled generic HPV probes. HPV DNA positive cases were subsequently analyzed by slot-blot hybridization of the PCR products with specific probes for HPV types 6, 11, 16, 18, and 33. HPV type 18 was detected in two cases, including one case from a 44-year-old female and one from a 64-year-old male, with the remaining 32 cases negative. In situ hybridization for HPV DNA failed to detect HPV types 6/11, 16/18, or 31/33/35 in any of the cases. We conclude that a small proportion of cases of primary pulmonary SCC test positive for HPV type 18 but that the great majority of cases are not associated with HPV.

5'-mutations in the dystrophin gene can result in cardiomyopathy without clinically-apparent skeletal myopathy. The effect of dystrophin mutations on the assembly and stability of the dystrophin associated protein (DAP) complex in human heart are not fully understood. The molecular defect in the dystrophin complex was explored in a family with an X-linked pedigree and severe dilated cardiomyopathy. Dystrophin gene analysis demonstrated a 5' duplication involving exons 2-7, which encodes the N-terminal actin binding domain of dystrophin. Ribonuclease protection and PCR assays demonstrated a reduction in muscle promoter transcribed dystrophin mRNA in the heart compared to skeletal muscle. A deficiency of cardiac dystrophin protein was observed by Western blot and lack of membrane localization by immunocytochemistry. The cardiac expression of the dystrophin related protein utrophin was increased, and the 43 kDa (beta-dystroglycan), 50 kDa (alpha-sarcoglycan) and 59 kDa (syntrophin) dystrophin associated proteins (DAPs) were co-isolated and present in nearly normal amounts in the membrane. However, cardiac dystrophin deficiency and increased utrophin expression were associated with loss of extracellular 156 kDa dystrophin associated glycoprotein (alpha-dystroglycan) binding to the cardiomyocyte membrane. alpha-Dystroglycan is responsible for linkage of the dystrophin complex to the extracellular matrix protein laminin. Therefore, 5' dystrophin mutations can reduce cardiac dystrophin mRNA, protein expression, and dystrophin function in X-linked cardiomyopathy (XLCM). The presence of membrane-associated beta-dystroglycan, alpha-sarcoglycan, syntrophin, and utrophin are insufficient to maintain cardiac function. This XLCM family has a 5' dystrophin gene mutation resulting in cardiac dystrophin deficiency and a loss of alpha-dystroglycan membrane binding., (Copyright 1997 Academic Press Limited.)

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.

Background: The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined., Methods and Results: Angiotensin II receptors were measured by saturation binding of 125I-[Sar1,Ile8]angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes., Conclusions: AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.

Plasma CK concentrations have been widely used as the primary muscle enzyme marker for diagnosis and progression of myositis. Recently, total CK and CK-MB serum concentrations have been compared to, and used in conjunction with, serum concentrations of aspartate aminotransferase in diagnosis of myositis. The algorithmic use of CK, AST, and aldolase plasma concentrations to diagnose and categorize patients with myopathy may be a useful method of diagnosing specific muscle disease without invasive procedures. CAIII, as a specific marker for skeletal muscle damage, may replace CK as the enzyme of choice in diagnosis and progression of myositis and other muscle disease. Additional studies are required to determine the usefulness of carbonic anhydrase for the diagnosis and assessment of myositis.

A full length cDNA for human M creatine kinase has been isolated and sequenced. The cDNA contains 77 bp of 5' untranslated, 338 bp of 3' untranslated sequence and the entire coding region (1146 bp) for human M creatine kinase. The M creatine kinases from different species share considerable sequence homology within the coding region (77-91%) and in amino acid sequence (82-97%). Little or no sequence homology is observed in the 3' untranslated sequence of the mammalian M creatine kinases, although canine and human creatine kinase share overall 80% sequence homology in 5' untranslated sequence. A unique 8 bp sequence was identified in the 5' untranslated regions of mammalian M creatine kinase but is not present in B creatine kinase cDNA. The degree of sequence conservation observed implies an evolutionary constraint on M creatine kinase structure beyond that which would be expected for the maintenance of enzymatic function.