Transglutaminase 2 ablation leads to mitophagy impairment associated with a metabolic shift towards aerobic glycolysis.

Macroautophagy selectively degrades dysfunctional mitochondria by a process known as mitophagy. Here we demonstrate the involvement of transglutaminase 2 (TG2) in the turnover and degradation of damaged mitochondria. In TG2-ablated cells we observed the presence of a large number of fragmented mitochondria that display decreased membrane potential, downregulation of IF1 along with increased Drp1 and PINK1 levels, two key proteins regulating the mitochondrial fission. Of note, we demonstrate that in healthy mitochondria, TG2 interacts with the dynamic proteins Drp1 and Fis1; interestingly, their interaction is largely reduced upon induction of the fission process by carbonyl cyanide m-chlorophenyl hydrazine (CCCP). In keeping with these findings, mitochondria lacking TG2 are more susceptible to CCCP treatment. As a consequence of accumulation of damaged mitochondria, cells lacking TG2 increased their aerobic glycolysis and became sensitive to the glycolytic inhibitor 2-deoxy-D-glucose (2-DG). In contrast, TG2-proficient cells are more resistant to 2-DG-induced apoptosis as the caspase 3 is inactivated through the enzyme's crosslinking activity. The data presented in this study show that TG2 plays a key role in cellular dynamics and consequently influences the energetic metabolism.

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins.

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.

53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

p53 binding protein-1 (53BP1) participates in checkpoint signaling during the DNA damage response (DDR) and during mitosis. In this study we report that 53BP1 aggregates in nuclear foci within syncytia elicited by the human immunodeficiency virus (HIV)-1 envelope. 53BP1 aggregation occurs as a consequence of nuclear fusion (karyogamy (KG)). It colocalizes partially with the promyelomonocytic leukemia protein (PML), and the ataxia telangiectasia mutated kinase (ATM), the two components of the DDR that mediate apoptosis induced by the HIV-1 envelope. ATM-dependent phosphorylation of 53BP1 on serines 25 and 1778 (53BP1S25P and 53BP1S1778P) occurs at these DNA damage foci. 53BP1S25P was also detected in syncytia present in the lymph nodes or frontal brain sections from HIV-1-infected carriers, as well as in peripheral blood mononucleated cells from HIV-1-infected individuals, correlating with viral load. Knockdown of 53BP1 caused HIV-1 envelope-induced syncytia to enter abnormal mitoses, leading to their selective destruction through mitochondrion-dependent and caspase-dependent pathways. In conclusion, depletion of 53BP1 triggers the demise of HIV-1-elicited syncytia through mitotic catastrophe.

Nitroimidazoles and hypoxia imaging: synthesis of three technetium-99m complexes bearing a nitroimidazole group: biological results.

Several Tc-99m complexes were synthesized, substituted with a nitroimidazole group, in order to visualize hypoxic tissues. The complexes were tested on rats (isolated hearts) and showed no significant uptake under hypoxic conditions.

Detection of PCR-amplified fungal DNA by using a PCR-ELISA system.

In order to speed up and standardize polymerase chain reaction (PCR)-based detection of medically important fungi in clinical samples we established a combination of commercially available kits for DNA extraction, PCR amplification and detection of the amplicons. The PCR plate assay proved to be as sensitive and specific as our previously published assay (5 cfu ml(-1) blood). Moreover, in a selected group of patients, all patients with proven and probable invasive fungal infection were found to be PCR-positive. Thus the PCR plate assay was found to be a sensitive, technically simplified and standardized method with potential for adaptation to automation.

Bloodstream infections associated with a needleless intravenous infusion system in patients receiving home infusion therapy.

OBJECTIVE: To determine risk factors for bloodstream infections (BSIs) in an outbreak among patients receiving home intravenous infusion therapy. DESIGN: Case-control and retrospective cohort studies. SETTING: Home health agency. PATIENTS: Patients receiving home intravenous infusion therapy from Rhode Island Home Therapeutics (RIHT) from January through December 1993. MAIN OUTCOME MEASURE: Development of primary BSI. METHODS: We compared patients with BSI (ie, case patients) with randomly selected noninfected RIHT patients receiving intravenous therapy, conducted a cohort study of all RIHT patients receiving intravenous therapy via a central venous catheter (CVC), and conducted a culture survey of injection cap luminal fluid. RESULTS: Case patients were more likely than controls to have had therapy via a CVC (11/11 vs 14/32; odds ratio [OR] undefined; P < .001) or total parenteral nutrition and intralipid therapy (TPN/IL) (9/11 vs 3/32; OR, 43.5; 95% confidence interval [CI], 4.9 to 510.0). Among RIHT patients with CVCs, risk factors for BSI were receipt of TPN/IL (9/35 vs 2/67; rate ratio [RR], 8.6; 95% CI, 2.0 to 37.7) or use of a needleless infusion system (10/41 vs 1/61; RR, 14.9; 95% CI, 2.0 to 111.8). Only the combination of both exposures was significantly associated with development of a BSI (P < .001). Luminal fluid from injection caps of needleless devices was significantly more likely to be culture positive than fluid from protected-needle devices (5/23 vs 0/18; RR undefined; P = .04). CONCLUSIONS: Our data suggest that a needleless device used for TPN/IL was associated with increased risk of BSI when injection caps were changed every 7 days.

Progress on the childhood immunization initiative.

President Clinton submitted the Comprehensive Childhood Immunization Initiative Act to Congress in April 1993. The objective of the legislation is to protect all children in the United States by their second birthday against nine vaccine-preventable infectious diseases. As originally introduced in the Congress the initiative called for (a) Federal purchase and distribution of recommended childhood vaccines for all children, (b) improving the public health capacity to deliver vaccine, (c) establishing a State-based national immunization information and tracking system, and (d) expanding immunization education and mobilization efforts directed to health care providers and parents. The authors review the progress and current status of the initiative, updating a previous progress report. The President's legislative proposal, modified by Congress, was enacted August 10, 1993. Several key provisions of the original legislation, deferred by Congress, may be incorporated in subsequent legislation or implemented through existing authorities. Therefore, the evolving framework for the initiative derives not from a single legislative mandate, but expands current immunization program activities and adds important new and complementary activities. As mentioned in the original title of the legislation, this is a "comprehensive" effort to address the problem of under-immunization in U.S. preschool children.

The president's child immunization initiative--a summary of the problem and the response.

After only 24 days in office, President Bill Clinton announced a comprehensive childhood immunization initiative designed to assure that all children in the United States lead healthier lives by receiving age-appropriate immunizations against preventable diseases such as polio, mumps, measles, whooping cough, and diphtheria. As part of his economic stimulus proposal, the President requested $300 million for Fiscal Year 1993 to reinforce the nation's immunization infrastructure by providing funding for communities to extend clinic hours, provide more staff, and increase information and education efforts and for the planning and implementation of a national immunization tracking system. In its Fiscal 1994 budget request, the Administration asked for a doubling of the Centers for Disease Control and Prevention's immunization program funding to $667 million. In cooperation with key congressional committees, the Administration has also prepared legislation that would provide recommended childhood vaccines to States for free distribution to health care providers who serve children enrolled in Medicaid or who don't have health insurance that covers immunization services. Providers could not charge for the vaccine but could charge a fee for administration. State Medicaid programs would also be required to reimburse providers reasonably for vaccine administration. This measure is designed to improve universal access to immunization services by helping to remove financial barriers that impede children from being immunized at the appropriate age.

Surveillance for diabetes mellitus--United States, 1980-1989.

PROBLEM/CONDITION: In the United States, diabetes mellitus is the most important cause of lower-extremity amputation and end-stage renal disease; the major cause of blindness among working-age adults; a major cause of disability, premature mortality, congenital malformations, perinatal mortality, and health-care costs; and an important risk factor for the development of many other acute and chronic conditions (e.g., diabetic ketoacidosis, ischemic heart disease, stroke). Surveillance data describing diabetes and its complications are critical to increasing recognition of the public health burden of diabetes, formulating health-care policy, identifying high-risk groups, developing strategies to reduce the burden of this disease, and evaluating progress in disease prevention and control. REPORTING PERIOD COVERED: In this report, data are summarized from CDC's diabetes surveillance system; trends in diabetes and its complications are evaluated by age, sex, and race for the years 1980-1989. DESCRIPTION OF SYSTEM: CDC has established an ongoing and evolving surveillance system to analyze and compile periodic, representative data on the disease burden of diabetes and its complications in the United States. Data sources currently include vital statistics, the National Health Interview Survey, the National Hospital Discharge Survey, and Medicare claims data for end-stage renal disease. RESULTS AND INTERPRETATION: In 1989, approximately 6.7 million persons in the United States reported that they had diabetes mellitus, and a similar number probably had this disabling chronic disease without being aware of it. The disease burden of diabetes and its complications is large and is likely to increase as the population grows older. Effective primary, secondary, and tertiary prevention strategies are needed, and these efforts need to be intensified among groups at highest risk, including blacks. Important gaps exist in periodic and representative data for describing the disease burden. ACTIONS TAKEN: CDC is assisting diabetes control programs in 26 states and one territory. These programs attempt to reduce the burden of diabetes by preventing blindness, lower-extremity amputations, cardiovascular disease, and adverse outcomes of pregnancy among persons with diabetes. Because of important limitations in measuring the burden of diabetes, CDC is exploring sources of surveillance data for blindness, adverse outcomes of pregnancy, and the public health burden of diabetes among minority groups.

Causes of death in persons with human immunodeficiency virus infection.

PURPOSE: Pneumocystis carinii pneumonia (PCP) was reported to be the predominant cause of human immunodeficiency virus (HIV)-related deaths prior to 1988, the year that effective prophylaxis against PCP entered routine use. Our study was performed to study the causes of HIV-related death since January 1988 in a region where patient tracking is virtually complete. PATIENTS AND METHODS: We surveyed physicians associated with the Brown University Acquired Immunodeficiency Syndrome (AIDS) Program who cared for greater than 95% of known HIV-positive patients in Rhode Island. These physicians identified all those HIV-infected persons who had died under their care between January 1988 and July 1990, and determined these patients' causes of death by chart review. For comparison, death certificates of identified persons were also reviewed at the Rhode Island Department of Vital Statistics. RESULTS: Among 126 deaths since January 1988, bacterial infections were the most common cause of death (30%), whereas PCP was responsible for only 16% of deaths. Persons not receiving any form of PCP prophylaxis were more likely to die from PCP than were those who received prophylaxis (26% versus 11% [p = 0.04]). Cause of death as recorded on actual death certificates was imprecise, although bacterial infections were again the most common cause indicated. Only one death occurred in a patient with a CD4 count greater than 200/mL, and this was not HIV-related. CONCLUSION: PCP has not been the leading cause of death in our region since January 1988. Bacterial infections contribute substantially to mortality, and this may influence future prophylactic regimens. HIV-related deaths in patients with CD4 counts greater than 200/mL are unusual.

Trends in diabetes and diabetic complications, 1980-1987.

OBJECTIVE: Although diabetes is a major source of morbidity and mortality in the United States, only recently has a unified national surveillance system begun to monitor trends in diabetes and diabetic complications. RESEARCH DESIGN AND METHODS: We established a diabetes surveillance system using data for 1980-1987 from vital records, the National Health Interview Survey, the National Hospital Discharge Survey, and the Health Care Financing Administration's records to examine trends in the prevalence and incidence of diabetes, diabetes mortality, hospitalizations, and diabetic complications. RESULTS: From 1980 through 1987, the number of individuals known to have diabetes increased by 1 million--to 6.82 million. Age-standardized prevalence for diabetes increased 9% during this period, from 25.4 to 27.6/1000 U.S. residents (P = 0.03). The incidence of diabetes increased among women (P = 0.003), particularly among those greater than 65 yr old (P = 0.02). Age-standardized mortality rates (for diabetes as either an underlying or contributing cause) per 100,000 individuals with diabetes declined 12%, from 2350 to 2066. Annual mortality rates from stroke (as an underlying cause and diabetes as a contributing cause) and diabetic ketoacidosis declined 29% (P = 0.003) and 22% (P less than 0.001), respectively. During these 8 yr, hospitalization rates for major CVD and stroke (as the primary diagnoses and diabetes as a secondary diagnosis) increased 34% (P = 0.006) and 38% (P = 0.01), respectively. Also during this period, hospitalization rates increased 21% for diabetic ketoacidosis (P = 0.01) and 29% for lower-extremity amputations (P = 0.06). From 1982 through 1986, treatment for end-stage renal disease related to diabetes increased greater than 10% each year (P less than 0.001). The prevalence of diagnosed diabetes was nearly twice as high in blacks as in whites (P = 0.04). Blacks also had increased rates of lower-extremity amputation (P = 0.02), diabetic ketoacidosis (P less than 0.001), and end-stage renal disease (P = 0.01). CONCLUSIONS: Diabetes surveillance data will be useful in planning, targeting, and evaluating public health efforts designed to prevent and control diabetes and its complications.

Epidemiology and prevention of periodontal disease in individuals with diabetes.

OBJECTIVE: This article reviews the epidemiological evidence of the relationship between diabetes and periodontal disease, possible physiological mechanisms for the association, and effects of interventions on the occurrence and severity of periodontal disease among individuals with diabetes. RESEARCH DESIGN AND METHODS: A comprehensive qualitative review of published literature in the area was performed. RESULTS: Much of the research in this area was found to contain methodological problems, such as failing to specify the type of diabetes, small sample sizes, and inadequate control of covariates such as age or duration of diabetes. CONCLUSIONS: Trends indicate that periodontal disease is more prevalent and more severe among individuals with diabetes. This trend may be modified by factors such as oral hygiene, duration of diabetes, age, and degree of metabolic control of diabetes. Generally, poor oral hygiene, a long history of diabetes, greater age, and poor metabolic control are associated with more severe periodontal disease. The association of diabetes and periodontal disease may be due to numerous physiological phenomena found in diabetes, such as impaired resistance, vascular changes, altered oral microflora, and abnormal collagen metabolism. With some modifications, the same prevention and treatment procedures for periodontal disease recommended for the general population are appropriate for those with diabetes. People with diabetes who appear to be particularly susceptible to periodontal disease include those who do not maintain good oral hygiene or good metabolic control of their diabetes, those with diabetes of long duration or with other complications of diabetes, and teenagers and pregnant women.

Evaluating outcomes of pregnancy in diabetic women. Epidemiologic considerations and recommended indicators.

Many programs have been applied in various settings to reduce adverse outcomes of pregnancy in women with diabetes. Efforts to standardize criteria and methods for evaluating these programs are relatively recent. Without such standardization, evaluation of the impact of many programs and comparisons among programs have not been possible. We review the suitability of available data sources for monitoring adverse outcomes of pregnancy in women with diabetes in light of epidemiological considerations relevant to selection of indicators of program impact. This article is intended to be a resource to help evaluate in a standardized fashion the impact of programs at a regional, state, or local level. We conclude that primary data (information collected by programs themselves) collected in a standardized manner are necessary for evaluation of programs for diabetes in pregnancy. Secondary data sources alone are of limited value for monitoring outcomes because of underreporting of maternal diabetes, especially in the absence of identified complications. Ultimately, the ability to rigorously assess the impact of efforts to improve outcomes of diabetes in pregnancy may depend on the creation of comprehensive statewide systems to identify women of childbearing age who have diabetes.

Gestational diabetes. Incidence, maternal characteristics, and perinatal outcome.

Accurate estimates of the incidence of abnormal glucose tolerance during pregnancy are virtually nonexistent. Screening select populations of women with risk factors for the condition and the nonrandom, non-population-based nature of most studies have given rise to wide variances in reported incidence. We analyzed data from the states of Mississippi and Washington and from the National Natality and Fetal Mortality Surveys conducted in 1980 in an attempt to provide more accurate population-based estimates of the incidence of gestational diabetes mellitus (GDM). In the national surveys GDM was noted (screening and diagnostic criteria were unavailable) as a complication in 0.38% of all sampled pregnancies; overt (type I and type II) diabetes was noted in 0.78%. Mean maternal age for the GDM group was 28.4 yr; 85% were white (81% controls) and 15% non-white (19% controls). Prepregnancy weights were higher in the GDM group by an average of 20 lb. However, mean weight gain was less in this group than in controls (23 versus 29 lb). Perinatal mortality was noted in approximately 2.8% (1.3% in controls) of the offspring in GDM-complicated pregnancies and congenital malformations in 6.4% (7.9% in controls). Methodologic problems were encountered and included lack of screening and diagnostic criteria, underreporting, and underrecording.

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.