Characterization of KIR intermediate promoters reveals four promoter types associated with distinct expression patterns of KIR subtypes.

The human killer cell immunoglobulin-like receptor (KIR) genes contain multiple promoters that control the process of gene activation and variegated expression of KIR on natural killer (NK) and T cells. Specific subfamilies of KIR genes have differences in the timing and tissue specificity of expression: however, previous studies of the proximal KIR promoters have not shown significant differences in activity between differentially expressed KIR gene subsets. The recent identification of an intermediate KIR promoter (ProI) associated with KIR2DL1 expression suggested a central role for this element in KIR expression. The current study identifies ProI elements in all of the KIR genes, revealing four classes of ProI that correspond with four distinct expression phenotypes of KIR subgroups: KIR2DL2/S2/L3 that are expressed early in reconstituting NK after transplant; KIR2DL4 that is expressed by CD56-bright NK in a non-variegated manner; KIR3DL3 that is not expressed by circulating NK cells; and the remaining KIR that are expressed by subsets of CD56-dim NK. The four classes of ProI are structurally diverse and display distinct functional properties. Altogether, these results indicate that KIR ProI elements contribute to the tissue/cell-type specificity of KIR transcription and cooperate with the probabilistic proximal promoter to control KIR expression.

The impact of human immune deficiency virus and hepatitis C coinfection on white matter microstructural integrity.

The purpose of the present study is to examine the integrity of white matter microstructure among individuals coinfected with HIV and HCV using diffusion tensor imaging (DTI). Twenty-five HIV+ patients, 21 HIV+/HCV+ patients, and 25 HIV- controls were included in this study. All HIV+ individuals were stable on combination antiretroviral therapy (cART; ≥3 months). All participants completed MRI and neuropsychological measures. Clinical variables including liver function, HIV-viral load, and CD4 count were collected from the patient groups. DTI metrics including mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), and fractional anisotropy (FA) from five subregions of the corpus callosum were compared across groups. The HIV+/HCV+ group and HIV+ group were similar in terms of HIV clinical variables. None of the participants met criteria for cirrhosis or fibrosis. Within the anterior corpus callosum, significant differences were observed between both HIV+ groups compared to HIV- controls on DTI measures. HIV+ and HIV+/HCV+ groups had significantly lower FA values and higher MD and RD values compared to HIV- controls; however, no differences were present between the HIV+ and HIV+/HCV+ groups. Duration of HIV infection was significantly related to DTI metrics in total corpus callosum FA only, but not other markers of HIV disease burden or neurocognitive function. Both HIV+ and HIV+/HCV+ individuals had significant alterations in white matter integrity within the corpus callosum; however, there was no evidence for an additive effect of HCV coinfection. The association between DTI metrics and duration of HIV infection suggests that HIV may continue to negatively impact white matter integrity even in well-controlled disease.

Characterization of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that controls KIR expression.

Members of the human KIR (killer cell immunoglobulin-like receptor) class I major histocompatibility complex receptor gene family contain multiple promoters that determine the variegated expression of KIR on natural killer cells. In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression. An individual with a single copy of the KIR2DL1 gene but a very low level of gene expression was identified. The low expression phenotype was associated with a single-nucleotide polymorphism (SNP) that created a binding site for the inhibitory ZEB1 (Zinc finger E-box-binding homeobox 1) transcription factor adjacent to a c-Myc binding site previously implicated in distal promoter activity. Individuals possessing this SNP had a substantial decrease in distal KIR2DL1 transcripts initiating from a novel intermediate promoter located 230 bp upstream of the proximal promoter start site. Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter. Reduced intermediate promoter activity revealed the existence of alternatively spliced KIR2DL1 transcripts containing premature termination codons that initiated from the proximal KIR2DL1 promoter. Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.

Identification of a KIR antisense lncRNA expressed by progenitor cells.

Human NK cells express cell surface class I MHC receptors (killer cell immunoglobulin-like receptor, KIR) in a probabilistic manner. Previous studies have shown that a distal promoter acts in conjunction with a proximal bidirectional promoter to control the selective activation of KIR genes. We report here the presence of an intron 2 promoter in several KIR genes that produce a spliced antisense transcript. This long noncoding RNA (lncRNA) transcript contains antisense sequence complementary to KIR-coding exons 1 and 2 as well as the proximal promoter region of the KIR genes. The antisense promoter contains myeloid zinc finger 1 (MZF-1)-binding sites, a transcription factor found in hematopoietic progenitors and myeloid precursors. The KIR antisense lncRNA was detected only in progenitor cells or pluripotent cell lines, suggesting a function that is specific for stem cells. Overexpression of MZF-1 in developing NK cells led to decreased KIR expression, consistent with a role for the KIR antisense lncRNA in silencing KIR gene expression early in development.

Identification of bidirectional promoters in the human KIR genes.

Although the class I MHC receptors expressed by human and mouse natural killer (NK) cells have distinct molecular origins, they are functional analogues that are expressed in a variegated pattern. The murine Ly49 class I receptors contain bidirectional promoters that have been proposed to control the probabilistic expression of these genes. Whether similar elements are present in the human killer Ig-like receptor (KIR) genes is a fundamental question. A detailed analysis of the 2 kb intergenic region separating the KIR2DL4 gene and the adjacent KIR3DL1 gene revealed that additional promoter elements exist in the human KIR genes. Remarkably, the previously characterized KIR3DL1 proximal promoter possesses bidirectional promoter activity that maps to an 88 bp DNA fragment containing CREB, AML, Sp1 and Ets transcription factor binding sites. Individual KIR genes and alleles possess bidirectional promoters with distinct properties. Analysis of KIR(+)and KIR(-) NK cells and NK precursors indicates that reverse transcripts from the bidirectional promoter are found in cells that lack KIR protein expression, but are not present in mature KIR-expressing NK cells, suggesting that reverse transcription from the proximal promoter blocks gene activation in immature NK and precursor cells.

Identification of distal KIR promoters and transcripts.

A more complete understanding of the transcriptional control of the human and murine class I MHC receptors will help to shed light on the mechanism of selective, stochastic, gene activation that operates in these gene families. Studies of the murine Ly49 class I MHC receptor genes have revealed an important role for distal transcripts originating upstream of the proximal promoter. To date, there have been no reports of distal promoters within the functionally analogous human KIR family of class I MHC receptors. In the current study, reverse transcriptase-polymerase chain reaction (RT-PCR) and RNase protection assays were used to reveal the presence of distal KIR transcripts initiating upstream of the previously characterized proximal KIR promoter. The intergenic promoter elements detected were associated with repetitive elements of the Alu and L1 families. Unlike the proximal KIR promoter, the distal promoter regions were not NK cell-specific. KIR genes expressed in a variegated manner produced a low level of distal transcripts containing a large 5' untranslated region. In contrast, the highly expressed KIR2DL4 gene possessed a higher level of spliced distal transcripts that were capable of producing KIR2DL4 protein. The identification of distal KIR promoter elements suggests that intergenic transcripts may influence the expression of KIR genes.

Persistent pollutants urban rivers sediment survey: implications for pollution control.

The impacts of diffuse urban sources of pollution on watercourses are quantified. A survey of nine urban streams in Scotland for persistent pollutants in stream sediments is described, together with sediments from SUDS ponds. Determinands reported are: PAHs, total hydrocarbons, and toxic metals (As, Zn, Ni, Pb, Cu, Cr, Cd). Results highlight hydrocarbons as a major urban pollutant, and show significant sediment contamination by toxic metals. The metals that occurred in the highest concentrations varied across the nine streams, but Pb, Cr, Ni, Zn and Cu most frequently present exceeded sediment quality standards. The pattern of contamination by PAHs suggested that pyrolytic sources were more ubiquitous and present in greater quantities than oil spill sources in these urban catchments. Exceptions were the sites below industrial estates. The findings indicate that four levels of activity will be needed to control urban diffuse sources of pollution: reductions in quantities of toxic pollutants used by manufacturers in the motor and construction industries; housekeeping measures to minimise storage and handling risks for oil and chemicals; public engagement to minimise polluting activities such as dumping oil and chemicals, and private car use; use of SUDS technology, including retro-fits in the worst affected urban areas.

Multidimensional assessment of anger in Vietnam veterans with posttraumatic stress disorder.

This study examined the effects of combat exposure and posttraumatic stress disorder (PTSD) on dimensions of anger in Vietnam veterans. Vietnam combat veterans were compared with Vietnam era veterans without war zone duty on the Multidimensional Anger Inventory (MAI). Combat veterans were not significantly more angry than their veteran peers who did not serve in Southeast Asia. Additionally, various parameters of war zone duty were not highly associated with anger scores. However, combat veterans with PTSD scored significantly higher than veterans without PTSD on measures of anger arousal, range of anger-eliciting situations, hostile attitudinal outlook, and tendency to hold anger in. These results suggest that PTSD, rather than war zone duty, is associated with various dimensions of angry affect.

Structure, properties, and tissue localization of apoplastic alpha-glucosidase in crucifers.

Apoplastic alpha-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three alpha-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic alpha-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli alpha-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside revealed that the acidic alpha-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

T cell- and NK cell-independent inhibition of hepatic metastases by systemic administration of an IL-12-expressing recombinant adenovirus.

IL-12 is a potent immunoregulatory cytokine that has been shown to mediate tumor regression in a variety of tumor models. We describe the construction of AdCMV-IL-12, a recombinant adenovirus that encodes both subunits of IL-12 under transcriptional control of the CMV promoter. This recombinant virus efficiently infects a wide variety of cell types leading to the production of high levels of biologically active IL-12. Because the liver is a primary site of infection after i.v.-administered adenovirus, we tested the therapeutic efficacy of this virus in a murine hepatic metastasis tumor model. Systemic administration of AdCMV-IL-12 dramatically inhibited the formation of 3-day Renca hepatic metastases (mean of 16 metastases per liver) compared with the control virus AdCMV-betagal (mean of 209) or vehicle alone (mean of 272). Histologic analysis indicated that metastatic growth inhibition was accompanied by a dramatic perivascular infiltrate consisting of T cells, macrophages, and neutrophils. Therapeutic efficacy was not diminished in animals depleted of CD4+ or CD8+ T cells, or in SCID mice, even after NK cell ablation. In the latter case, a hepatic perivascular infiltrate composed of macrophages and neutrophils was observed after AdCMV-IL-12-treatment, while numerous activated Kupffer cells were noted in the hepatic parenchyma. Analysis of therapy-induced changes in hepatic gene expression demonstrated increased levels of IP-10 and Mig RNAs, but no increase in iNOS, Fas, or FasL RNA levels was observed. Our data suggest a model of metastatic growth inhibition mediated by nonlymphocyte effector cells including macrophages and neutrophils and that may involve anti-angiogenic chemokines.

Sensitivity of fluorochrome microscopy for detection of Mycobacterium tuberculosis versus nontuberculous mycobacteria.

The results for 6,532 consecutive mycobacterial respiratory specimens collected from 1,040 patients from 1993 to 1995 in a Texas hospital were studied to determine the sensitivity of fluorescence microscopy for detection of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Smears were positive for acid-fast bacilli (AFB) in 63% (677 of 1,082) of specimens growing M. tuberculosis and 56% (638 of 1,148) of specimens growing the four most common species of NTM. Smear positivity by species was 58% (446 of 776) for M. avium complex, 51% (154 of 300) for rapidly growing mycobacteria (98% were M. abscessus), 78% (29 of 37) for M. kansasii, and 26% (9 of 35) for M. gordonae. Definite or probable disease by clinical criteria was present in 79% of patients with M. avium complex, 93% of patients with rapidly growing mycobacteria, 100% of patients with M. kansasii, and 0% of patients with M. gordonae. Patients with M. avium complex had a low incidence of AIDS (7%), and approximately 50% of non-AIDS patients had upper-lobe cavitary disease and 50% had nodular bronchiectasis. Only 23 of 6,532 (0.35%) of AFB smears were positive with a negative culture excluding patients on therapy for established mycobacterial disease. These studies suggest that NTM are as likely as M. tuberculosis to be detected by fluorescent microscopy in specimens from patients from areas endemic for NTM lung disease and at low risk for AIDS.

Should balloon angioplasty and stents have any role in operative intervention for lower extremity ischemia?

Balloon angioplasty has been combined with open vascular surgery to treat lower extremity ischemia due to multilevel occlusive disease. The purposes of this study were: (1) to compare staged and simultaneous approaches to determine the optimal method for combining endovascular and open techniques and; (2) to assess the role of stents in intraoperative balloon angioplasty. Among 274 patients undergoing lower extremity revascularization over 30 months, 38 (13.9%) required a combination of endovascular and open techniques; 17 were staged (endovascular followed at an interval by distal open surgery) and 21 were simultaneous (intraoperative balloon angioplasty with or without stent placement at the time of open surgery). Groups were similar with respect to demographics, lesions treated with endovascular intervention, incidence and location of stent placement, and results of surgery. Additional operating time required for intraoperative endovascular intervention was 41.0 +/- 30.7 min., fluoroscopic time was 3.9 +/- 2.4 min. and contrast administered was 58.8 +/- 28.1 ml. There was no perioperative mortality. Length of stay was longer in the staged than in the simultaneous group (p < 0.01). Cumulative combined primary patency at 1 year by life-table methods was 82 +/- 10% in the staged group and 83 +/- 9% in the simultaneous group (p = 0.79). Mean follow-up was 13 +/- 6 months. There is a role for balloon angioplasty and stent placement in operative revascularization of ischemic limbs in selected patients: patency was similar to that produced with the staged approach while the length of stay was shorter. Intraoperative balloon angioplasty is safe and effective and stents permit a measure of control in assuring an optimal intraoperative postangioplasty result.

Selection of skin test antigens to evaluate PPD anergy.

BACKGROUND: A purified protein derivative (PPD) tuberculin skin test may be nonreactive because of cutaneous anergy, technical problems with the test, or absence of tuberculosis infection. This study investigated the sensitivity and specificity of five test agents in measuring cutaneous anergy when the PPD test is nonreactive. Agents evaluated include antigens for Candida, mumps, histoplasmin, tetanus, and Trichophyton. METHODS: Delayed-type hypersensitivity skin test records were analyzed in 1113 patients admitted to the University of Texas Health Center at Tyler from December 1988 through June 1993. These patients were admitted with initial diagnoses of diseases other than active tuberculosis or human immunodeficiency virus infection. RESULTS: Patients with a negative PPD test reacted most often to the control skin test Candida (63.5%), followed by mumps (52.2%), histoplasmosis (37.2%), tetanus (35.7%), and Trichophyton (6.1%). Analysis of these data indicates that the use of more than three of the four most commonly reactive control tests (Candida, mumps, and histoplasmin or tetanus) yielded minimal additional precision in the determination of skin test anergy compared with using all five control skin tests. This finding remained constant whether the PPD was considered negative at < 5 mm, < 10 mm, or < 15 mm of induration. CONCLUSIONS: In controlling for false-negative PPD tests, the use of three skin test antigens, Candida, mumps, and tetanus, should provide reliable control for delayed-type hypersensitivity anergy.

A case of recurrent typhoid fever in the United States: importance of the grandmother connection and the use of large restriction fragment pattern analysis of genomic DNA for strain comparison.

An 8-year old girl was infected for a second time with Salmonella typhi by contact with her grandmother, a known typhoid carrier. The S. typhi from both patient and grandmother had closely related genomic pulsed field gel electrophoresis patterns that differed from epidemiologically unrelated strains. The girl responded well to a 14-day course of oral trimethoprimsulfamethoxazole. The grandmother was treated successfully with a 28-day regimen of oral ciprofloxacin. Typhoid fever remains an endemic disease in the United States, largely because of recognized chronic stool carriers. Most of these carriers had typhoid in the preantibiotic era and remain potential sources of disease when they provide meals for others, not uncommonly grandchildren. The importance of this "grandmother" connection to endemic typhoid fever is reviewed, as is the potential use of pulsed field gel electrophoresis pattern analysis for comparison of strains of S. typhi.

Glial cells of the oligodendrocyte lineage express both kainate- and AMPA-preferring subtypes of glutamate receptor.

mRNAs for AMPA- and kainate-preferring glutamate receptor subunits are expressed abundantly in the CNS, yet functional studies of neurons and glia from brain suggest selective expression of AMPA receptors. We now show that glial cells of the O-2A lineage express rapidly desensitizing responses to kainate, mRNAs for GluR6, GluR7, KA-1, and KA-2, rapidly desensitizing responses to AMPA, and mRNAs for GluR-B, -C, and -D. Analysis of glutamate receptor currents in single cells reveals two receptor populations with high and low affinity for kainate and different sensitivity for potentiation by concanavalin A and for block of desensitization by cyclothiazide. Our experiments describe the characterization of native kainate-preferring receptors in glia and reveal coexpression in single cells of functional AMPA- and kainate-preferring receptors.

Initial clinical assessment of the comatose patient: cerebral malaria vs. meningitis.

One hundred twenty-one Liberian children were admitted in coma to the ELWA Hospital, Monrovia, Liberia. Admitting diagnoses, before lumbar puncture, were compared with discharge diagnoses. Ninety-four children were discharged with a final diagnosis of cerebral malaria and 27 with a diagnosis of meningitis. The admitting diagnosis was correct in 76.6% (72 of 94) of patients with cerebral malaria and 59.3% (16 of 27) of patients with meningitis. The cerebrospinal fluid leukocyte count was the single most significant factor in determining the correct diagnosis. Without the cerebrospinal fluid analysis, the discriminant accuracy (77%), i.e. definitive separation of the two illnesses, was comparable to the physician's admission diagnosis (73%). Other data contributing to the differential diagnosis of cerebral malaria and meningitis included the number of days of fever before admission, the presence or absence of nuchal rigidity, fontanelle fullness and peripheral blood malaria smear. Mortality rates for cerebral malaria and meningitis were 14.9 and 29.6%, respectively. These data suggest that physicians cannot reliably discriminate between cerebral malaria and meningitis without cerebrospinal fluid analysis.

Photothrombotic lesions of the frontal cortex impair the performance of the delayed non-matching to position task by rats.

The effects of photochemically induced lesions of the frontal cortex on the short-term memory capacity of the rat have been investigated using the delayed non-matching to position task. Pretrained animals received lesions and were tested 4 days after surgery and twice per week for 3 weeks. The lesions produced a profound impairment of performance of this task which was still evident 3 weeks after surgery. Spontaneous locomotor activity was recorded 7 days after surgery and no difference was found between the control and lesion group. These effects indicated a generalized disruption of performance of this task in the absence of motor dysfunction. These results suggest that photothrombotic lesions of the frontal cortex can produce reliable, long-term behavioural deficits.

Effect of cyclooxygenase inhibition on amphotericin B-induced lung injury in awake sheep.

Amphotericin therapy in humans has been reported to cause severe pulmonary dysfunction in some patients, and these abnormalities have been reproduced in unanesthetized sheep. To determine the role of cyclooxygenase products in this response, paired, random-order experiments in 11 sheep were done using the cyclooxygenase inhibitor ibuprofen. Ibuprofen blunted increases in pulmonary artery pressure (Ppa) after amphotericin (peak Ppa 38 +/- 3 cm H2O in amphotericin-alone group vs. 30 +/- 1 cm H2O in ibuprofen + amphotericin group, P less than .05) and reduced peak lung lymph flow to approximately 170% of baseline compared with 350% of baseline in amphotericin-alone group (P less than .05). In addition, the increase in airflow resistance across the lung and the decrease in partial pressure of oxygen seen after amphotericin was blocked by ibuprofen. Therefore, amphotericin-induced lung dysfunction is produced in part through the generation of cyclooxygenase products of arachidonic acid metabolism and can be ameliorated by pretreatment with the cyclooxygenase inhibitor ibuprofen.