Assessment of quality of life of the patients with diabetic retinopathy using National Eye Institute Visual Functioning Questionnaire (VFQ-25).

BACKGROUND: Diabetes affects most of the organs causing macrovascular and microvascular complications. Diabetic retinopathy (DR) results from the prolonged uncontrolled hyperglycemia which causes impairment of vision. Quality of life (QoL) of patients with DR is affected due to vision loss. National Eye Institute Visual Functioning Questionnaire (NEI-VFQ-25) is the questionnaire used to study the effect of DR on QoL. OBJECTIVES: To assess the QoL of the patients with DR. MATERIALS AND METHODS: The study enrolled 149 (male-104 and female 45) patients with DR. The previous translated and validated version of NEI-VFQ-25 questionnaire was used in the study. RESULTS: Cronbach alpha for internal consistency was between 0.6 and 0.8. The male patient showed significantly higher (p<0.05) QoL scores (60.73±1.63) as compared to the female patients (53.15±2.84). Hypertensive patients showed poor QoL as compared to non-hypertensive patients. The patients with a history of diabetes for 16-30 yrs. showed better QoL as compared to other patients. CONCLUSION: DR affects the QoL of life of patients. Routine assessment of QoL using NEI-VFQ-25 questionnaire would be useful for physicians and health care team.

Detection of cytochrome P450 and other drug-metabolizing enzyme mRNAs in peripheral blood mononuclear cells using DNA arrays.

The purpose of this study was to determine whether the expression of cytochrome P450 (CYP) enzyme mRNAs, other drug-metabolizing enzyme mRNAs, and transporter mRNAs can be detected using DNA arrays. Total RNA was isolated from peripheral blood mononuclear cells of 10 multiple sclerosis patients and 10 age- and sex-matched controls. The mRNA was reverse transcribed to radiolabeled cDNA, and the resultant cDNA was used to probe a DNA array containing several thousand known human genes. The signals corresponding to several CYPs, drug-metabolizing, and transporter mRNAs was substantially above background. The results demonstrate that the DNA array technique has the sensitivity and the selectivity for applications in the pharmaceutical sciences. The mean values for mRNAs of specific CYPs and drug-metabolizing enzymes in peripheral blood cells were compared with reported values for liver. The capabilities of DNA arrays may prove useful for characterizing CYP expression in a variety of clinical samples.

Transient monocyte release of interleukin-10 in response to traumatic brain injury.

A significant component of the immune response to trauma results in the systemic presence of cytokines which have the potential to suppress the patient's immune response to infection and contribute to post-injury complications. We assayed peripheral blood leukocytes obtained from 10 patients with head trauma to determine their production of interleukin (IL). Serum was assayed for the presence of IL-10, TGFbeta1, and IFNgamma by ELISA. Peripheral blood leukocytes were screened for intracellular IL-10 and IFNgamma by fluorescence-activated flow cytometry, and cytokine-specific mRNA was detected by the polymerase chain reaction. We detected an immediate, but transient, presence of IL-10 in the sera of all 10 patients who suffered head trauma. IL-10-specific intracytoplasmic immunofluorescence was also detected immediately after injury in peripheral blood monocytes, but not in lymphocytes or granulocytes. IL-10-specific mRNA was detected in peripheral blood leukocytes in only 50% of patients immediately after injury, when the highest serum levels of IL-10 were observed. Our data indicates that release of pre-formed IL-10 by monocytes contributes to the presence of IL-10 found in patient peripheral blood immediately after head injury.

Differential surface expression of MICA by endothelial cells, fibroblasts, keratinocytes, and monocytes.

MICA is a new, highly divergent and polymorphic HLA-related gene that has a similar intron-exon organization as the HLA class I genes. It functions as a restriction element for intestinal gammadeltaT cells and it behaves as a cell stress molecule. It is likely that the polymorphic MICA molecule may be target for specific antibodies and T cells in solid organ grafts or in graft versus host disease (GVHD). Previously, we generated three MICA-specific sera in rabbits, which were used for Western blot, flow cytometry and immunoprecipitation. We demonstrated that MICA is expressed in endothelial cells, keratinocyes and monocytes, but not in CD4+, CD8+ or CD19+ lymphocytes. We also found that MICA is expressed on the cell surface in HeLa cells. In the present work, performing peptide neutralization assays, we further confirmed the specificity of the reactivity of these sera against MICA. Also, by Western blot we demonstrate that freshly isolated human skin-derived fibroblasts express MICA. We also investigated the surface expression of MICA in different, freshly-isolated cells. The results show that endothelial cells and fibroblasts express MICA at the cell surface. Although expressing the 62 kDa MICA band, as detected by Western blots, keratinocytes and monocytes do not seem to express this antigen on the cell membrane. This differential surface expression of MICA by endothelial cells and fibroblasts vs. keratinocytes and monocytes, may indicate that the levels of surface MICA are differentially regulated in different cells. Moreover, the expression of MICA on the surface of endothelial cells makes this polymorphic molecule a possible target during the immune response of graft rejection in organ transplantation.

Multiple epitopes of HLA-DRB1*0411 are recognized by T-cell clones originated from individuals carrying other DR4 subtypes.

HLA polymorphism dictates the binding and recognition of specific peptides, leading to variations in individual immune responses and may contribute to autoimmune disorders and outcome in organ transplantation. We have studied the molecular basis for the cellular recognition of DRB1*0411 in individuals carrying other sequence-related DR4-alleles by characterization of T-cell clones (TLC). A set of 166 TLC were raised by priming cells from DRB1*0401,0402 and DRB1*0405,0901 individuals and 52 of them recognized DRB1*0411. Five distinct patterns of T-cell allorecognition were found: DRB1*0411 alone, DRB1*0411 and 0405, DRB1*0411 and 0406, DRB1*0411 and 0407 and DRB1*0411, 0406 and 0407, depending on responder phenotypes and epitopes recognized by their T cells. A stretch of 30 amino acids on DRB1*0411 from positions 57 to 86 behaves as a functional domain and residues S57, R71, E74 and V86 seem to be crucial in forming immunogenic determinants recognized by these TLC. The knowledge of shared amino acid residues between closely related DR4 alleles, which show similar patterns of recognition by T cells could also be useful in the selection of prospective donors for clinical transplantation of solid organs or bone marrow.

Crystallization and preliminary X-ray analysis of the class 1 alpha 1,2-mannosidase from Saccharomyces cerevisiae.

The alpha 1,2-mannosidase from Saccharomyces cerevisiae catalyzes the conversion of Man9GlcNAc2 to Man8GlcNAc2 during the formation of N-linked oligosaccharides and is a member of the Class 1 alpha 1,2-mannosidases conserved from yeast to mammals. The enzyme is a type II membrane protein and a recombinant form of the alpha 1,2-mannosidase from S. cerevisiae, lacking the transmembrane domain, has been expressed in Pichia pastoris and crystallized using the hanging drop vapor diffusion technique. The crystals grow as flat plates, with unit cell dimensions a = 57.5 A, b = 84.1 A, c = 107.1 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and diffract to a minimum d-spacing of 3.5 A resolution. On the basis of density calculations one monomer is estimated to be present in the asymmetric unit (Vm = 2.08 A3 Da-1). This is the first report of the crystallization of any glycosidase involved in N-glycan biosynthesis.

Crystallization and preliminary X-ray analysis of human placental S-adenosylhomocysteine hydrolase.

A recombinant form of human placental S-adenosylhomocysteine (AdoHcy) hydrolase expressed in E. coli, which was inactivated by a type-I mechanism-based inhibitor, has been crystallized using the hanging-drop vapour-diffusion technique. The crystals grow as flat plates, with unit-cell dimensions a = 96.2, b = 173.6, c = 142.9 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group C222 and diffract to a minimum spacing of approximately 2.0 A resolution at the Cornell High Energy Synchrotron Source. On the basis of density calculations two monomers of the tetrameric protein are estimated to be present in the asymmetric unit (V(m) = 2.99 A(3) Da(-l)). The self-rotation function clearly indicates the location of the non-crystallographic twofold axis.

Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families.

The hereditary breast cancer gene BRCA2 was recently cloned and is believed to account for almost half of site-specific breast cancer families and the majority of male breast cancer families. We screened 49 site-specific breast cancer families for mutations in the BRCA2 gene using single strand conformation analysis (SSCA) followed by direct sequencing. We found mutations in eight families, including all four families with male breast cancer. The eight mutations were small deletions with the exception of a single nonsense mutation, an all were predicted to interrupt the BRCA2 coding sequence and to lead to a truncated protein product. Other factors which predicted the presence of a BRCA2 mutation included a case of breast cancer diagnosed at age 35 or below (P = 0.01) and a family history of pancreatic cancer (P = 0.03). Two mutations were seen twice, including a 8535delAG, which was detected in two French Canadian families. Our results suggest the possibility that the proportion of site-specific breast cancer families attributable to BRCA2 may be overestimated.

Synthesis of O-glycan core 3: characterization of UDP-GlcNAc: GalNAc-R beta 3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines.

UDP-GlcNAc: GalNAc-R beta 3-GlcNAc-transferase (core 3 beta 3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine, GalNAc is N-acetyl-D-galactosamine and T is transferase) is expressed in a tissue-specific fashion and is high in normal colonic tissue, but downregulated in colon cancer. To further study the control of this enzyme, we examined the activity in pig, rat and human colonic tissues, and several human cancer cell lines. The enzyme was difficult to solubilize by detergents and was extremely unstable in the solubilized form. Using synthetic derivatives of the GalNAc-R substrate, we showed that the specificity of the enzyme in normal rat and human colonic mucosa requires all the substituents of the GalNAc-sugar ring of substrates for maximal activity. Core 3 beta 3-GlcNAc-T was significantly influenced by the structure of the aglycon group. None of the inactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine alpha-benzyl was a weak substrate and significantly inhibited the incorporation of GLcNAc into GalNAc alpha-benzyl by human colonic homogenates. Surprisingly, none of the colonic cancer cell lines or any other cancer and leukaemia cells examined exhibited detectable activity of the enzyme, although a number of other glycosyltransferase activities involved in O-glycan biosynthesis were active. Mixing experiments did not reveal an endogenous inhibitor in HL60 cells or an activator of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T activity in cancer cell lines may be due to cell transformation or cell culturing.

Crystallization and preliminary X-ray analysis of a mutant duck delta II crystallin.

A duck delta II crystallin mutant, where histidine 178 has been replaced by an aspartic acid residue, has been purified from a bacterial expression system and subsequently crystallized. The crystals grow as flat plates, with unit cell dimensions a = 94.1 A, b = 99.9 A, c = 108.7 A and beta = 102 degrees. The crystals exhibit the symmetry of space group P2(1) and diffract to a minimum d-spacing of 2.8 A resolution. On the basis of density calculation four monomers (one tetramer) are estimated to be present in the asymmetric unit (Vm = 2.5 A3/Da). Self-rotation functions clearly show the presence of 222 non-crystallographic symmetry.

O-glycan biosynthesis in human colorectal adenoma cells during progression to cancer.

A human colonic adenoma cell line PC/AA derived from a familial polyposis coli patient was passaged in culture to form an intermediate premalignant clonogenic variant AA/C1 and, upon treatment with differentiating and carcinogenic agents, a cell line AA/C1/SB10 which is tumourigenic in nude mice. These three mucin-secreting cell lines have been used as a model to study the changes in O-glycan biosynthesis during the progression to cancer. Several glycosyltransferases involved in the synthesis, elongation and termination of the common O-glycan core structures were found to decrease in the progression sequence towards adenocarcinoma. Higher activity of a number of enzymes was seen in the intermediate cell line. O-glycan biosynthesis in the original PC/AA cell line was closest to the normal human colonic phenotype, since all four common mucin O-glycan cores and their extended structures could be synthesized; core 3 beta 3-GlcNAc-transferase and alpha 6-sialytransferase acting on GalNAc-mucin were still detectable and core 2 beta 6-GlcNAc-transferase activity was accompanied by core 4 and I beta 6-GlcNAc-transferase activities. During progression towards adenocarcinoma, the expression of alpha 6-sialyltransferase, core 3 beta 3-GlcNAc-transferase, core 4 and I beta 6-GlcNAc-transferases were turned off. Using monoclonal antibodies, Tn antigen, sialyl-Tn antigen, O-acetyl-sialomucin and sialyl-Lea determinants were not detected in secreted or cellular mucin isolated from any of the cell lines. The exposure of MUC1 epitopes was seen in the malignant line, whereas sialyl-Lex determinants were found only in the premalignant PC/AA line. Sulfotransferase activities using core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl, were high in PC/AA cells and progressively decreased upon development to adenocarcinoma, and this decrease correlated with mucin sulfation. In summary, the synthesis of less abundant, sialylated, fucosylated and extended, unbranched core 1 structures should be facilitated in the malignant cells. This is the first report of glycosyltransferase changes in human premalignant cells developing to tumourigenic cells. The data demonstrate that these cell lines are an excellent model to study the changes and regulation of mucin oligosaccharide biosynthesis during progression to cancer.

The health of children adopted from Romania.

OBJECTIVE: To determine the medical condition of Romanian adoptees and the effects of the Romanian orphanage system on their health. DESIGN: Case series. SETTING: The international adoption clinics at the University of Minnesota, Minneapolis, and the New England Medical Center, Tufts University, Boston, Mass. PARTICIPANTS: Sixty-five Romanian adoptees who were brought to the United States during a 12-month period beginning in October 1990. MAIN OUTCOME MEASURES: Incidence of hepatitis B, intestinal parasites, tuberculosis, syphilis, human immunodeficiency virus type 1, growth failure, and developmental delay. RESULTS: Although the adopted children were presumably chosen from the most vital and attractive adoptees, only 15% were judged to be physically healthy and developmentally normal. Fifty-three percent had serological evidence of past or present hepatitis B infection, and 20% of screened children tested positive for hepatitis B surface antigen. In children aged 7 months or older, the overall prevalence of chronic hepatitis B was 23%. Intestinal parasites were found in 33% of subjects, and 45% of infected children had two or more pathogens identified. All the children tested for human immunodeficiency virus type 1 were negative. Two patterns of growth failure were observed that resembled the two subtypes of psychosocial short stature that occur in association with prolonged psychological harassment or emotional deprivation. Infants' length, weight, head circumference, and weight-for-height were adversely affected by institutionalization. Older children's height was reduced. Only 10% of children older than 12 months were developmentally normal. CONCLUSION: Romanian adoptees are an extraordinarily high-risk pediatric group as a consequences of decades of government-sanctioned child neglect and abuse.

Alterations in Alzheimer's disease-associated protein in Alzheimer's disease frontal and temporal cortex.

Alzheimer's disease (AD)-associated protein is present in brain and cerebrospinal fluid of patients with AD but not in adult, nondemented, normal controls. This protein may represent an abnormal epitope of the "tau" microtubule-associated protein and has been detected before the appearance of senile plaques and neurofibrillary tangles. The amount of AD-associated protein in the frontal and temporal cortices in 93 cases of neuropathologically confirmed AD was compared with the amount that was present in 20 cases without AD. The amount of AD-associated protein was significantly increased in the cases of AD for both brain regions compared with that in the cases without AD. The presence of high levels of this protein is a useful adjunct, postmortem marker of the presence of AD and may eventually lead to tests that allow early detection of individuals at risk for this disease.

Medical evaluation of internationally adopted children.

BACKGROUND: Despite many reports of medical illness in children adopted from abroad, there are currently no accepted guidelines for medical evaluation of this population. METHODS: Two hundred ninety-three children adopted from 15 countries (mean age, 14.0 months; 55 percent girls) were evaluated by history taking, physical examination, and screening tests for hepatitis B virus (HBV), human immunodeficiency virus type 1, tuberculin reactivity, intestinal parasites, syphilis, excretion of cytomegalovirus, renal disease, and anemia. All but four were seen within one month of their arrival in the United States. RESULTS: Fifty-seven percent of the children (168 of 293) were found to have at least one important medical condition. Eighty-one percent of the diagnoses were established by screening test, rather than by history taking or physical examination. Infectious diseases made up the majority of the medical conditions (73 percent). Serologic testing for hepatitis B surface antigen was positive in 5 percent of the children. Characteristics associated with the acquisition of HBV infection included arrival within the first three years of the study (P = 0.017), Asian origin (P = 0.011), and receipt of a blood transfusion abroad (P = 0.008). Ten children (3 percent) had positive Mantoux skin tests, and four of these had active pulmonary tuberculosis. Tuberculin reactivity was significantly associated with older age (P less than 0.001) and lower weight (P = 0.037). Intestinal parasites were isolated from 14 percent of the international adoptees. Non-Korean adoptees were 16 times more likely to be harboring at least one intestinal parasite than were Korean adoptees (P = 0.005). CONCLUSIONS: Directed screening tests should be a routine component of the medical evaluation of all children adopted from abroad, regardless of age, sex, or country of origin.

Unsuspected infectious diseases and other medical diagnoses in the evaluation of internationally adopted children.

Seven simple screening tests--hepatitis B profile, urine culture for cytomegalovirus, Mantoux test for tuberculosis, stool examination for ova and parasites, VDRL, complete blood cell count, and vision and hearing screening--were used to evaluate 52 consecutive children at a pediatric clinic for international adoptees. In 63% of these children, unsuspected medical diagnoses were made by a combination of history, physical examination, and appropriate screening tests. When only those children previously examined by a physician in the United States were included in our analysis, the rate of unsuspected diagnosis remained high (67%). Omission of screening tests was the single most frequent cause of missed diagnoses, of which the majority were infectious diseases. More than 50% of our newly established diagnoses carried the potential for long-term sequelae without proper treatment. These data emphasize that internationally adopted children should receive a thorough screening evaluation for medical problems that may adversely affect their growth and development.

A critical evaluation of cysteamine as a tool to deplete somatostatin in the rat central nervous system.

The wide central nervous system (CNS) distribution of somatostatin (SRIF) as well as the well documented reduction in SRIF concentration in the cerebral cortex in patients with Alzheimer's disease have served as an impetus for studies of this peptide's neurobiological role in the brain. These studies were designed to evaluate the efficacy of centrally administered cysteamine (CYS) as a tool to deplete SRIF in the hypothalamus (HYP) and extrahypothalamic brain areas. Somatostatin was measured by RIA in the frontal cortex (COR), hippocampus (HIP), and HYP in rats after seven daily infusions of CYS into unilateral cannulae stereotaxically positioned into either the lateral ventricle (LV; 300 micrograms/2 microliters) or the dorsal HIP (100 micrograms/2 microliters), and after single (300 mg/kg) or daily (100 mg/kg) sc injections; rats were killed 4 or 24 h after the last injection. After LV infusions, the SRIF concentration was significantly reduced only in the HYP (35% at 4 h and 27% at 24 h). After HIP infusions, the SRIF concentration was significantly reduced only in the HYP at 4 h (23%); no reductions were observed at 24 h. Both a single and repeated sc administrations of CYS reduced SRIF in the HYP only 24 h after treatment (54% and 50%, respectively). Acute sc CYS reduced SRIF in the COR (23%) and the HYP (29%) 4 h after treatment; repeated sc CYS reduced SRIF in the COR (25%) and the HYP (63%). Although the reduction of SRIF in the HYP was increased by repeated sc dosing, the reduction of extrahypothalamic SRIF by sc CYS was relatively small in magnitude and was not enhanced by repeated dosing. These results suggest that CYS is not an ideal tool for depletion of extrahypothalamic SRIF after sc or CNS administration and, moreover, raise serious questions about studies in which behavioral or endocrine alterations after CYS treatment were attributed to specific actions on SRIF-containing neurons.

Evidence that anti-basement membrane zone antibodies in bullous eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen.

Circulating and tissue-deposited IgG antibodies to the cutaneous basement membrane zone (BMZ) were detected in 3 patients with the clinical, pathologic, and immunologic features of bullous eruption of systemic lupus erythematosus (SLE). The antibodies were present in sera and IgG fractions in all cases and in eluates of cutaneous immune deposits from one of the cases. The antibodies were easily detected in sera by indirect immunofluorescence on adult human thigh skin separated through the lamina lucida by incubation in 1.0 M NaCl but were less easily detected on intact neonatal foreskin. The antibodies had features of epidermolysis bullosa acquisita (EBA) anti-BMZ antibodies including binding to the dermal side of the BMZ in separated skin, binding to the cutaneous but not vascular or glomerular basement membranes, binding to and just below the lamina densa, and binding to 290 or 290 and 145 kD dermal proteins previously identified as components of the EBA autoantigen. The antibodies were relatively specific for SLE patients with features of bullous eruption of SLE since they were detected in 3 of 4 of those cases and in only 1 of 20 SLE patients without blisters. These results show anti-BMZ antibodies with features of EBA antibodies are present in patients with bullous eruption of SLE and suggest there may be a close relationship between that disease and EBA. The results also suggest that EBA antibodies may be part of the autoantibody spectrum of SLE and that separated skin is more sensitive than intact skin for their detection.

Hemodynamic responses to anesthetic induction with midazolam or diazepam in patients with ischemic heart disease.

Hemodynamic responses to induction of anesthesia with midazolam maleate and diazepam were compared in patients with ischemic heart disease. While breathing 100% oxygen, 10 patients (group M) received midazolam maleate, 0.2 mg/kg, and 10 patients (group D) received diazepam, 0.5 mg/kg. In addition, 10 patients (group MN) breathing 50% nitrous oxide in oxygen received midazolam, 0.2 mg/kg. Patients in group M had a small but statistically significant (p less than 0.05) decrease (vs awake control values) in systemic and pulmonary arterial blood pressure, pulmonary arterial occluded pressure, stroke index, and left and right ventricular stroke work indices. Patients in group D experienced statistically significant decreases in systemic blood pressure. The only statistically significant differences between groups M and D occurred 5 minutes followed drug administration: heart rates were higher and systemic pressures and left ventricular stroke work indices were lower following midazolam. Hemodynamic changes following midazolam and nitrous oxide were similar to those observed in patients given midazolam and 100% oxygen. Patients in all three groups responded to endotracheal intubation with transient increases in blood pressure, heart rate, and systemic vascular resistance, but the hemodynamic values spontaneously returned toward control levels within 2 to 5 minutes. Although differing somewhat, midazolam, like diazepam, provided rapid, hemodynamically stable induction of anesthesia in patients with ischemic heart disease.

Hemodynamics during diazepam induction of anesthesia for coronary artery bypass grafting.

The hemodynamics during induction of anesthesia were studied in ten patients with ischemic heart disease about to have coronary artery bypass grafting. Intravenous diazepam, 0.5 mg/kg (with 50% N2O in oxygen inspired and pancuronium IV), was used to induce anesthesia. Compared to awake baseline, induction caused statistically significant decreases in the mean arterial pressure, rate pressure product, stroke index, and left and right ventricular stroke work indexed. Although statistically significant, the hemodynamic changes were small and transient and required no modifying treatment. This anesthetic induction technic is safe, efficient, and well tolerated by patients having myocardial revascularization surgery.