Basal subtype, as approximated by triple-negative phenotype, is associated with locoregional recurrence in a case-control study of women with 0-3 positive lymph nodes after mastectomy.

PURPOSE: Basal subtype, as approximated by the triple-negative phenotype (ER-PR-Her2-), has correlated with higher LRR in recent studies. Indications for postmastectomy RT (PMRT) in women with 0-3 positive lymph nodes remain unclear. We evaluated the importance of biologic subtype in a cohort of women with LRR after mastectomy. METHODS: We identified 22 women with 0-3 positive lymph nodes at our institution who were initially treated with mastectomy (without post-mastectomy radiation), suffered LRRs, and had paraffin-embedded tissue blocks from the primary mastectomy specimen available for staining. None of these women received PMRT. We case-control matched these to 29 women with 0-3 positive nodes who had mastectomy (no PMRT) and remained without evidence of disease at last follow-up and had available primary specimens for processing. We matched controls for age (±3 years) and follow-up duration (<5 year vs. more). Paraffin-embedded specimens were used to construct a triple-redundant tissue microarray. We used conditional logistic regressions to study the association between each predictor and LRR. Results were summarized based on odds ratio (OR). RESULTS: On univariate analysis, ER+, PR+, or the combination was strongly associated with lower odds of LRR. Basal subtype, as approximated by ER-PR-Her2- (TN), was associated with higher LRR (OR 8.5, p = 0.048). Use of chemotherapy also was associated with lower LRR (OR 0.126, p = 0.0073). CONCLUSIONS: Our data are concordant with reports from others demonstrating that TN phenotype is associated with higher LRR and can be considered along with other predictors of LRR when selecting women for PMRT.

Association of vitamin A deficiency with cervical squamous intraepithelial lesions in human immunodeficiency virus-infected women.

To explore the relationship between vitamin A (retinol) deficiency and cervical squamous intraepithelial lesions (SILs) in human immunodeficiency virus (HIV)-infected women, we measured serum retinol concentrations in 1314 women enrolled in the Women's Interagency HIV Study and correlated the results with concurrent cervical cytology. At the baseline visit, 204 (15.5%) of the 1314 patients had retinol concentrations consistent with deficiency (<1.05 micromol/L). Analysis of Papanicolaou smears showed SILs in 216 (16.4%) of 1314 women. Cervical SILs were found to be associated with retinol concentrations <1.05 micromol/L (multivariate odds ratio [OR], 1.63; P=.04) in a multivariate model, which included human papillomavirus (HPV) status and markers of nutritional status and HIV disease stage. In the subset of women with genital HPV (n=774), a multivariate analysis again revealed a significant independent association between retinol <1.05 micromol/L and cervical SILs (multivariate OR, 1.75; P=.02). Our findings suggest that retinol deficiency may contribute to the development of cervical SILs in HIV-infected women.

Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor.

BACKGROUND: Mutations in the myocilin (MYOC)/TIGR gene are responsible for autosomal-dominant juvenile primary open-angle glaucoma (POAG). In patients with non-autosomal-dominant POAG, such mutations are rare, but the expression of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. We performed transfection, DNAse I footprinting, mutagenesis and electrophoretic mobility shift assays (EMSA) to identify elements responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes. RESULTS: DNAse I footprinting experiments of the human MYOC/TIGR promoter showed a major protected area between nt -106 to -77, which was not conserved in the homologous region of the mouse myoc/tigr promoter. In addition, the TATA-box was protected, as well as at least three downstream sites, including an AP-1-like sequence. Deletion of the -106 to -77 region caused a substantial loss of functional promotor activity in all cell types. Site-directed mutagenesis and EMSA experiments revealed the presence of two regulatory elements in the -106 to -77 region. Each of these cis-elements is essential for minimal promoter activity. The 5'-half of the region contains a sequence with similarities to NF-kappaB-related sites, however, binding of NF-kappaB could not be confirmed by EMSA. The 3'-half contains a canonical E-box sequence. EMSA experiments showed that the upstream regulatory factor (USF) was binding to the E-box sequence and that the binding can be supershifted by specific antibodies. CONCLUSIONS: Several DNA-protein binding elements contribute to a transcription of MYOC/TIGR, and USF is critically required for its basal transcription in trabecular meshwork cells and astrocytes.

Interrater Variability in Diagnosis of Cervical Biopsies from Women with HIV-1: Results from the Women's Interagency HIV Study.

OBJECTIVES: To determine interrater variability in classifying cervical biopsies from women with human immunodeficiency virus (HIV). MATERIALS AND METHODS: Cervical biopsies performed on women participating in the Women's Interagency HIV Study (WIHS) were read at the six participating sites. A 10% random sample was retrieved and reviewed using standardized terminology by pathologists with a special interest in gynecologic pathology. Results were compared with kappa values and Mantel-Haentzel tests. RESULTS: Biopsies from 288 HIV-seropositive and 24 HIV-seronegative women were reviewed. The weighted kappa value of 0.67 indicated moderate to strong agreement between original and review diagnoses, with a range of 0.54 to 0.84 across sites. No cancers were identified. Significantly more specimens showing cervical intraepithelial neoplasia (CIN) grade 2 or 3 were identified by review pathologists (p = .02). CIN2 or CIN3 was graded less severely by local pathologists in 18 (51%) of 35 cases, all from HIV-seropositive women. Local pathologists' diagnoses of CIN2 or CIN3 were downgraded by reviewers in 4 of 21 cases (19%). Discrepancies were more common among women with lower CD4 lymphocyte counts. CONCLUSIONS: Although discrepancies occur, interrater correlation in the interpretation of cervical biopsies from women with HIV is moderate to strong.

Alterations in blood-brain barrier glucose transport in SIV-infected macaques.

The neurological manifestations of HIV infection may be in part due to alterations in the blood-brain barrier. These may be caused by structural changes in the barrier or may consist of subtle metabolic or biochemical disturbances in barrier function. In the CNS, the family of glucose transporter proteins plays a key role in controlling movement of glucose across cell membranes. The 55 kDa isoform of glucose transporter 1 (GLUT1) regulates import of glucose from blood to brain across the endothelial cells of the blood-brain barrier (BBB), whereas the 45 kDa form of GLUT1 predominantly regulates nonvascular glial glucose uptake. In this study, expression of 55 and 45 kDa forms of GLUT1 in different regions of the brain from 18 SIV-infected macaques was measured by quantitative immunoblot and then compared with the severity of SIV encephalitis to determine whether neurologic disease is related to altered glucose metabolism at the BBB and in brain parenchyma. An inverse relationship was found between severity of SIV encephalitis and expression of the endothelial 55 kDa isoform of GLUT1 at the BBB in cortical grey matter, caudate nucleus, and cerebellum. A similar relationship also was found for the glial 45 kDa GLUT1 isoform in cortical grey matter. In addition, a significant increase in 55 kDa GLUT1 expression was found in caudate nucleus during the early stages of infection. In the brains of macaques with moderate to severe encephalitis, 55 kDa GLUT1 expression had declined to pre-infection levels. These GLUT1 alterations at the BBB and in glial cells may reflect severe disturbances in the CNS microenvironment that contribute to CNS dysfunction.

TbetaR-I(6A) is a candidate tumor susceptibility allele.

We have previously described a type I transforming growth factor (TGF)-beta receptor (TbetaR-I) polymorphic allele, TbetaR-I(6A), that has a deletion of three alanines from a nine-alanine stretch. We observed a higher than expected number of TbetaR-I(6A) homozygotes among tumor and nontumor DNA from patients with a diagnosis of cancer. To test the hypothesis that TbetaR-I(6A) homozygosity is associated with cancer, we performed a case-control study in patients with a diagnosis of cancer and matched healthy individuals with no history of cancer and who were identical in their gender and their geographical and ethnic background to determine the relative germ-line frequencies of this allele. We found nine TbetaR-I(6A) homozygotes among 851 patients with cancer. In comparison, there were no TbetaR-I(6A) homozygotes among 735 healthy volunteers (P < 0.01). We also observed an excess of TbetaR-I(6A) heterozygotes in cancer cases compared to controls (14.6% versus 10.6%; P = 0.02, Fisher's exact test). A subset analysis revealed that 4 of 112 patients with colorectal cancer were TbetaR-I(6A) homozygotes (P < 0.01). Using mink lung epithelial cell lines devoid of TbetaR-I, we established stably transfected TbetaR-I and TbetaR-I(6A) cell lines. We found that, compared to TbetaR-I, TbetaR-I(6A) was impaired as a mediator of TGF-beta antiproliferative signals. We conclude that TbetaR-I(6A) acts as a tumor susceptibility allele that may contribute to the development of cancer, especially colon cancer, by means of reduced TGF-beta-mediated growth inhibition.

Effects of anticoagulant, processing delay, and assay method (branched DNA versus reverse transcriptase PCR) on measurement of human immunodeficiency virus type 1 RNA levels in plasma.

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

Pathogenesis of simian immunodeficiency virus pneumonia: an immunopathological response to virus.

Although many human immunodeficiency virus-infected individuals develop lymphocytic interstitial pneumonia, the roles of host and viral factors in the pathogenesis of pneumonia are not well defined. Human immunodeficiency virus-infected children with lymphocytic interstitial pneumonia have human immunodeficiency virus-specific cytotoxic T cells in pulmonary infiltrates, increased survival time, and a reduced incidence of opportunistic infections, suggesting that lymphocytic interstitial pneumonia may reflect an effective antiviral immune response. In this study, 20 macaques were inoculated with related macrophage-tropic simian immunodeficiency viruses and examined for pulmonary lesions and virus gene expression. Ten macaques developed moderate to severe pneumonia characterized by perivascular, peribronchial, and interstitial infiltrates of lymphocytes and macrophages. Large numbers of pulmonary cytotoxic lymphocytes were demonstrated in macaques with moderate to severe pneumonia (P < 0.05) by immunostaining for TIA-1. There was no difference in viral load between macaques with moderate to severe pneumonia and those with mild to no pulmonary lesions. In five macaques inoculated with the same virus swarm, there was a significant (P < 0.05) inverse correlation between the percentage decline in CD4+ T-cell counts and the severity of pulmonary lesions. Pulmonary infiltrates of cytotoxic lymphocytes, the lack of correlation between severity of pulmonary lesions and virus gene expression, and the inverse relationship between pneumonia and inmune status suggest that simian immunodeficiency virus pneumonia may represent an immunopathological response to macrophage-tropic virus.

Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS.

Studies of circulating T (CD3(+)) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5-2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3(+) cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3(+) lymphocyte counts before the inflection point was close to zero for HIV- and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3(+) cell counts relative to HIV- men and HIV+ men not developing AIDS. A CD3(+) cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.

Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.

Enzyme electrodes and their application.

Starting from the state of the art, principles for improving the analytical characteristics of enzyme electrodes are discussed. Coupling of appropriate amperometric electrode processes with enzyme systems, e.g. urease or aminopeptidases, results in a simplification of operation. Optimal sample frequencies are realized on the basis of enzyme membranes, with both a small characteristic diffusion time and a high enzyme activity, applied in a well-designed sample-processing system. Coupled enzyme reactions of the sequence or competition type are successfully used for extension to new analytes, e.g. inhibitors, cofactors or alternative substrates. Cyclization of the analyte enhances the sensitivity of enzyme electrodes to the nanomolar concentration range. Enzymic anti-interference layers are a tool for improving the sensor specificity. The operational characteristics of enzyme electrodes are thus adaptable to any given analytical problem.

Enzyme electrode for urea with amperometric indication: Part I--Basic principle.

A novel method for amperometric determination of substrates of hydrolytic enzymes has been developed. As an example the pH dependence of electrochemical oxidation of hydrazine in the Tafel region was combined with the urease catalysed splitting of urea to construct an amperometric membrane electrode for urea. The characteristic features of this sensor are: a linear dependence of the current of hydrazine oxidation on hydrogen ion concentration (as opposed to the logarithmic response of potentiometric sensors), linear urea concentration-signal relationship between 0.8 and 35 mmol/litre sample concentration, a response time of about 20 s, a relative standard deviation of 1% and measuring frequency up to 40 samples/h with an electrode operational stability of 2 weeks. Calibration curves for aqueous urea solutions are given as functions of starting pH, urease and hydrazine concentration and the potential dependence of the signal was determined.

Neuroendocrine dysfunction and response to tricyclic antidepressants.

Patients with DSM-III unipolar major depression (N = 26) were treated with tricyclic antidepressants after 2 weeks of inpatient drug-free observation and neuroendocrine testing. Medication was prescribed with individualized dosages and was monitored with blood levels. Treatment response (greater than or equal to 50% decrease in Hamilton depression score after 4 weeks) was seen in 6 of 13 patients given desipramine and 7 of 13 given nortriptyline. Of the 13 nonresponders, 9 accepted treatment with another drug and/or ECT; 5 responded. Thus, 18 of the 26 patients responded to treatment. Abnormal pretreatment DST was correlated with increased responsiveness to treatment, although all patients had a high probability of response.

The TRH test and urinary MHPG in unipolar depression.

Twenty-five men and 26 women with major unipolar depression were evaluated by the TRH test and urinary MHPG excretion. A significant positive correlation between TSH response to TRH and urinary MHPG was found in the men, though not in the women. These findings suggest that at least for depressed men, central norepinephrine deficiency may be the neurobiological substrate of blunted TSH responses to TRH.

Clinical correlates of the TRH infusion test in primary depression.

Relationships between clinical measures, diagnosis and neuroendocrine findings were examined in a group of 25 primary depressives maintained drug free on a Neuropsychiatric Evaluation Unit. The TSH response to TRH infusion curves for unipolar and bipolar depressives were significantly different. Agitated patients but not psychomotor retarded patients demonstrated a blunted TSH response curve. No relationships were noted for cortisol hypersecretion and/or loss of diurnality and either diagnosis or psychomotor activity levels in depression. Biochemical and diagnostic implications of these findings are discussed.

The dexamethasone suppression and thyrotropin-releasing hormone tests and response to treatment in unipolar depression.

We examined retrospectively the response to tricyclic antidepressants and/or electroconvulsive therapy (ECT) in twenty-seven inpatients with major depressive disorder, primary unipolar subtype who had a thyrotropin-releasing hormone (TRH) test and dexamethasone suppression test (DST) prior to treatment. Thirteen failed to suppress on the DST and had a blunted thyroid stimulating hormone (TSH) response to TRH; nine had one test abnormality; and five had neither abnormality. Physicians selected ECT for patients with abnormalities on both tests significantly more frequently than for patients with one or neither test abnormalities. Fourteen of nineteen tricyclic trials and nine of ten courses of ECT resulted in unequivocally positive clinical response. There were no statistically significant relationships between tricyclic response and either test abnormality.

Thyrotropin-releasing hormone test and male unipolar depression.

Change in maximal TSH response to a TRH infusion were measured in 18 unipolar depressed males and 2 control groups matched for age, sex, and base-line thyroid status. The mean maximal change in TSH response was significantly lower for the unipolar than the control groups. Clinical and research implications of these findings are discussed. By using a deltaTSH cutoff of 7.0 rather than 5 microIU/ml, 14 rather than 7 depressed patients were correctly identified as having a blunted deltaTSH; 29 rather than 32 to 36 control patients were correctly identified as having a normal deltaTSH score.

Diagnostic issues in primary anorexia nervosa.

A literature review and clinical case presentation approach are employed to highlight unresolved diagnostic issues in Primary Anorexia Nervosa. The material is examined and discussed along a multidimensional list of variables considered important for the diagnosis, including definitions of weight loss, associated biological symptoms, eating patterns and body image disturbances.