Viral Metagenomic-Based Screening of Sugarcane from Florida Reveals Occurrence of Six Sugarcane-Infecting Viruses and High Prevalence of Sugarcane yellow leaf virus.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Ecuador.

Orange rust, Puccinia kuehnii (W. Krüger) E.J. Butler, is an important disease of sugarcane (complex hybrid of Saccharum L. species) that causes up to 53% yield loss (3), and can eliminate sugarcane clones in breeding programs. Initially confined to the Asia-Oceania region, P. kuehnii was reported in Florida in June 2007 (2) followed by confirmation in Central and South America. Orange rust pustules were observed on August 5, 2011, in commercial sugarcane fields located in the Ecuadorian Pacific coast of South America. Pustules were observed on cultivar SP79-2233 and sugarcane clones of the CINCAE breeding program (EC06-351, EC06-340, and EC01-744). Low levels of disease incidence and severity were observed in the sugarcane germplasm. Observation under a light microscope showed typical irregularly echinulate urediniospores that were pale in color with thickened apices and paraphyses inconspicuous to absent, such as those reported to be P. kuehnii (4). DNA of urediniospores were extracted and amplified using Pk1F and PK1R qPCR primers (5). Additionally, the 28s large ribosomal subunit DNA was sequenced (1), resulting in a qPCR and 100% sequence identity with a partial sequence of the P. kuehnii 28S ribosomal RNA gene, accession GU058010 (932/932 base pairs, GenBank Accession No. KF202306). Based on urediniospore morphology, DNA amplification, and sequence analysis, the causal agent of the rust observed in Ecuador was confirmed to be P. kuehnii. Commercial varieties have not yet shown symptoms of infections. However, a survey conducted in 2011 and 2012 showed an increase of disease severity from 3% to 28% in the susceptible cv. SP79-2233. Disease symptoms were evident from stalk growth to maturity (7 to 12 months), especially at the beginning of the harvesting season. To our knowledge, this is the first report of the presence, distribution, and disease spread by the sugarcane orange rust pathogen P. kuehnii in Ecuador. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) J. C. Comstock et al. Plant Dis. 92:175, 2008. (3) J. C. Comstock et al. ASSCT. 29:82, 2009. (4) L. Dixon and L. Castlebury. Orange Rust of Sugarcane - Puccinia kuehnii. Syst. Mycol. Microbiol. Lab. Retrieved from /sbmlweb/fungi/index.cfm, August 12, 2011. (5) N. C. Glynn et al. Plant Pathol. 59:703, 2010.

Plasmapheresis treatment of antibody-mediated rejection in an A2 donor to O pediatric liver transplant recipient.

It is safe to transplant kidneys from blood group A2 donors into O recipients if the latter have low titers of anti-A antibodies. However, in liver transplantation, O and B recipients of A2 donor livers are not routinely screened for anti-blood group antibodies because of the immuno-absorptive capacity of the liver and the low incidence of antibody-mediated rejection. Herein, we report a rare case of combined cell and antibody-mediated rejection in a pediatric blood group O recipient of an A2 donor liver, and rescue of the allograft using PP and IVIG.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Ivory Coast and Cameroon.

Orange rust of sugarcane caused by Puccinia kuehnii was detected in Florida in 2007 (1). It was hypothesized that the pathogen originated from Africa because brown rust of sugarcane (synonym common rust) was introduced to the Western Hemisphere from Africa (3). Requests for rust-infected sugarcane samples were made to several western and central African countries to investigate if orange rust of sugarcane was present but as yet undetected. Orange rust had not previously been reported from western or central Africa. At Zuénoula, Ivory Coast in July 2009, symptoms of sugarcane rust were observed on cvs. SP 71-6180 and Co 997 and appeared distinct to those of brown rust of sugarcane. A year later (May 2010), rust-infected specimens of SP 71-6180 and Co 997 from the same location and also from Borotou in Ivory Coast were sent to the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Also in May 2010, sugarcane rust was observed at Mbandjock and Nkoteng in Cameroon on cvs. D 88172, FR 87482, and RB 72-454 and on breeding clones RCmr 08/319 and RCmr 08/1121. All specimens had orange uredinial lesions that ranged from 0.6 to 6.5 mm × 200 to 300 µm and were ellipsoidal to elongate. Urediniospores were consistent with P. kuehnii E.J. Butler observed on specimens from Florida (1). DNA isolated from all samples was successfully amplified with P. kuehnii specific primers targeting ITS1 of rDNA (2). The nuclear large subunit region of rDNA of the rust specimens from Ivory Coast (BPI 881015-881017, GenBank Accession No. HQ666888) and Cameroon (BPI 881010-881014, GenBank Accession Nos. HQ666889-HQ666891) were sequenced. DNA sequences for all were identical to sequences of P. kuehnii and distinct from known sequences of P. melanocephala available in GenBank. To our knowledge, this is the first confirmed report of orange rust of sugarcane in western and central Africa. There is evidence that brown rust of sugarcane was introduced to the Western Hemisphere from this region of Africa (3) making it also the likely source of introduction of orange rust. Further experimentation is required to confirm this hypothesis. References: (1) J. C. Comstock et al. Plant Dis. 92:175, 2008. (2) N. C. Glynn et al. Plant Pathol. 59:703. 2010. (3) H. L. Purdy et al. Plant Dis. 69:689, 1985.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Costa Rica and Nicaragua.

Symptoms and signs of orange rust on sugarcane (a complex hybrid of Saccharum L. species) were observed from July 2007 on cv. SP 71-5574 in Costa Rica at the Coopeagri Sugar Mill located in Pérez Zeledón, San José and on multiple cultivars (CP 72-2086, Pindar, Q 132, Q 138, SP 71-5574, and SP 79-2233) at the Providencia Sugar Mill near Muelle, San Carlos and Cutris Sugar Mill near Los Chiles during August 2007. The same symptoms and signs were observed on cv. CP 72-2086 during September 2007 in Nicaragua at Ingenio San Antonio, located near Chinandega, and Ingenio Monte Rosa near El Viejo, Nicaragua. Disease symptoms and uredinia appeared different from brown rust caused by Puccinia melanocephala, and brown rust usually does not occur on these cultivars. Uredinia and urediniospores were similar to those described for orange rust (1,2). Cvs. SP 71-5574 and SP 79-2233 are susceptible and cv. CP 72-2086 is moderately susceptible to orange rust in Costa Rica and cvs. ISACP 00-1075, ISA 00-1000, and CP 72-2086 are moderately susceptible in Nicaragua. Samples from both locations (Costa Rica BPI No. 878816 and Nicaragua BPI No. 878817) examined at the USDA-ARS Mycology and Microbiology Laboratory in Beltsville, MD showed morphological characteristics consistent with those of P. kuehnii. Analysis of ITS1, 5.8S, and ITS2 rDNA sequences of the rust infecting cv. CP 72-2086 (GenBank Accession No. FJ532477) from Costa Rica and cv. ISA 00-1000 from Nicaragua (GenBank Accession No. FJ532476) confirmed the identity as P. kuehnii, the causal agent of sugarcane orange rust. Beside the cultivars already mentioned, orange rust also was confirmed on cvs. RB 73-9735 and CPCL 02-2130 in Costa Rica. To our knowledge, this is the first report of orange rust of sugarcane in Costa Rica and Nicaragua and the third confirmation of the disease in the Western Hemisphere and Caribbean Basin. References: (1) J. C. Comstock et al. Plant Dis. 92:175, 2008. (2) W. Ovalle et al. Plant Dis. 92:973, 2008.

First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Mexico, El Salvador, and Panama.

Symptoms of sugarcane orange rust were observed on July 17, 2008 on sugarcane cvs. Mex 57-1285, Mex 61-230, and Co 301 (a clone received in Mexico in 1953) at the Centro de Investigación y Desarrollo de la Caña de Azúcar en Tuxtla Chico, Chiapas, Mexico. In El Salvador, from August 2008 through January 2009, rust symptoms were observed on cv. CP 72-2086 (previously resistant to brown rust caused by Puccinia melanocephala Syd. & P. Syd.) in 117 dispersed sugarcane-production fields in various localities of El Salvador. Likewise, rust symptoms were first observed on sugarcane cv. SP 74-8355 (more than 25% severity and considered resistant to brown rust) at Natá, Coclé Province in Panama from January to February 2008. Dried herbarium leaf samples of sugarcane rust-infected leaves collected in El Salvador and Mexico were sent to the ARS, USDA Systematic Mycology and Microbiology Laboratory in Beltsville MD for identification. Panamanian samples were collected similarly and analyzed at the CALESA Biotechnology Laboratory. Morphological features of uredinial lesions and urediniospores were distinct from those of P. melanocephala and consistent with P. kuehnii E. J. Butler observed previously on specimens from Florida, Guatemala, Costa Rica, and Nicaragua (1-3). Analysis of the ITS1, 5.8S, and ITS2 and 28S large subunit rDNA sequences of the rust on infected cvs. Mex 57-1285, Mex 61-230, and Co 301 (BPI 878930, 879139, and 879140; GenBank Accession Nos. GO283006, GO283004, and GO283005, respectively) from Mexico and cv. CP 72-2086 from three locations in El Salvador (BPI 879135, 879136, and 879137; GenBank Accession Nos. GO283009, GO283007, and GO283008, respectively) all confirmed the identification of P. kuehnii. Similar analysis of the ITS1, 5.8S, and ITS2 rDNA sequence for the rust infecting cv. SP 74-8355 (GenBank Accession No. GO281584) confirmed the identification of P. kuehnii in Panama. To our knowledge, this is the first report of P. kuehnii causing orange rust disease of sugarcane in El Salvador, Mexico, and Panama. These findings also confirm the wider distribution of orange rust in the Western Hemisphere. References: (1) E. Chavarria et al. Plant Dis. 93:425, 2009. (2) J. C. Comstock et al. Plant Dis. 92:175, 2008. (3) W. Ovalle et al. Plant Dis. 92:973, 2008.

First Report of Puccinia kuehnii, Causal Agent of Orange Rust of Sugarcane, in Guatemala.

In September 2007 at Masagua, Escuintla Department, Guatemala, uredial lesions that appeared different from those of brown rust were observed on a sugarcane (a complex hybrid of Saccharum L. species) cultivar (CP 72-2086) considered resistant to brown rust caused by Puccinia melanocephala Syd. & P. Syd. Samples were sent to the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Observed morphological features were consistent with P. kuehnii E.J. Butler and appeared similar to orange rust samples obtained from Florida in July (2). Uredinial lesions were hypophyllous, orange, and variable in size measuring 650 to 850 × 26 to 32 µm. Urediniospores were mostly obovoid to pyriform or broadly ellipsoidal, variable in size, 32 to 45 × 25 to 30 µm, and moderately echinulate with spines evenly distributed, 3 to 5 µm apart. Urediniospore walls were orange-to-light cinnamon brown, 1 to 2.5 µm thick with a pronounced apical wall and four to five equatorial pores. Telia and teliospores were not observed. The nuclear large subunit rDNA region of the rust infecting cv. CP 72-2086 (BPI 898289, GenBank Accession No. EU344904) and the ITS1, 5.8S, and ITS2 rDNA regions (GenBank Accession No. EU543434) were sequenced (1,3). DNA sequences matched sequences of P. kuehnii in GenBank and were distinct from known sequences of P. melanocephala available in GenBank (3). Thirteen cultivars were rated as to their relative resistance using severity of orange rust symptoms; CG 96-59, CG 96-135, CP 72-1312, CP 73-1547, and CP 88-1165 were resistant; CG 96-40, CG 98-121, CP 72-2086, CP 88-1508, and CP 89-2143 were intermediate; and CG 96-52, CG 98-0115, and SP 79-2233 were susceptible. Orange rust was previously reported in Florida (2), but to our knowledge, this is the second report of its occurrence in the Western Hemisphere. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) J. C. Comstock et al. Plant Dis. 92:175, 2008. (3) E. V. Virtudazo et al. Mycoscience 42:447, 2001.

First Report of Puccinia kuehnii, Causal Agent of Orange Rust of Sugarcane, in the United States and Western Hemisphere.

In June 2007, approximately 8 km east of Belle Glade, FL, a rust disease was observed on a sugarcane (a complex hybrid of Saccharum L. species) cultivar (CP 80-1743) considered resistant to brown rust caused by Puccinia melanocephala Syd. & P. Syd. Approximately 10 km south of Canal Point, FL, another cultivar (CP 72-2086), also considered resistant to P. melanocephala, was found to be infected with a rust. Samples were sent to the USDA-APHIS National Mycologist and the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Observed morphological features were consistent with P. kuehnii E.J. Butler. Uredinial lesions were orange and variable in size, measuring 650 to 850 × 26 to 32 µm, hypophyllous, ellipsoidal to fusiform in shape, and distinctly lighter than pustules of P. melanocephala that were present in the area along with P. kuehnii. Urediniospores were mostly obovoid to pyriform or broadly ellipsoidal, variable in size, 32 to 45 × 25 to 30 µm, and moderately echinulate with mostly evenly distributed spines 2 to 4.5 µm apart. Walls were orange-to-light cinnamon brown, 1 to 2.5 µm thick with a pronounced apical wall thickening as much as 7 µm, and 4 to 5 equatorial pores. Similar orange uredinial lesions were subsequently observed on the same two cultivars and several other cultivars, including CPCL99-1777 and CPCL01-1055, at different locations in South Florida. Telia and teliospores were not observed. The nuclear large subunit rDNA region of the rust infecting cv. CP 80-1743 (BPI 878243, GenBank Accession No. EU164549) and the ITS1, 5.8S, and ITS2 rDNA regions of the rust infecting CP 80-1743 (GenBank Accession No. EU176009) and CP 72-2086 (GenBank Accession No. EU176008) were sequenced (1,4). All sequences were identical to sequences of P. kuehnii and distinct from known sequences of P. melanocephala (4). To our knowledge, this is the first confirmed record of P. kuehnii infecting sugarcane in the Western Hemisphere, and the disease appears to be distributed widely in the South Florida sugarcane-growing area. Although listed by P. Holliday (3) as occurring in Cuba, the Dominican Republic, and Mexico, CMI map no. 215 ed. 4 (2) does not include these three countries in the known distribution of P. kuehnii. P. kuehnii has also been reported in the literature as present in Hawaii (4). However, examination of the specimen label found that the specimen cited in those papers (BPI 079624) was actually collected in Tahiti. Therefore, the report from Hawaii is erroneous. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) CMI. Distribution Maps of Plant Diseases. No. 215, ed. 4. CAB International, Wallingford, UK, 1981. (3) P. Holliday. Fungus Diseases of Tropical Crops. Cambridge University Press, Cambridge, 1980. (4) E. V. Virtudazo et al. Mycoscience 42:447, 2001.

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.

Silent myocardial infarction and diabetic cardiovascular autonomic neuropathy.

Seventy-three consecutive diabetic adults with symptomatic peripheral neuropathy were evaluated for the presence of cardiovascular autonomic neuropathy and electrocardiographic evidence of myocardial infarction (MI). Twenty-five (34.2%) patients demonstrated cardiovascular autonomic neuropathy, and ten (13.7%) patients had electrocardiographic evidence of MI. Of the ten MI identified, seven were asymptomatic (silent) by history. The incidence of silent MI was significantly higher (P less than .04) in patients with cardiovascular autonomic neuropathy. It is postulated that sudden death in diabetic patients with cardiovascular autonomic neuropathy may be due to silent MI.

Cardiovascular events and correlates in the Veterans Affairs Diabetes Feasibility Trial. Veterans Affairs Cooperative Study on Glycemic Control and Complications in Type II Diabetes.

BACKGROUND: The risks and benefits of intensive therapy in non-insulin-dependent diabetes mellitus (NIDDM) need to be defined. In preparation for a long-term trial, a feasibility study of 153 men in 5 medical centers compared standard vs intensive insulin therapy. OBJECTIVE: To assess the rate of development of new cardiovascular events and their correlates. METHODS: Patients with a mean +/- SD age of 60 +/- 6 years and diagnosis of NIDDM for 7.8 +/- 4.0 years were randomly assigned to a standard (1 insulin injection every morning) or to an intensive treatment arm (stepped plan from 1 evening injection of insulin, alone or with glipizide, to multiple daily injections) designed to attain near-normal glycemia levels. A 2.07% separation of glycosylated hemoglobin (HbA1c) was sustained for a mean follow-up of 27 months (P < .001). Predefined cardiovascular events were assessed by a committee unaware of treatment assignment. RESULTS: Mild and moderate hypoglycemic events were more frequent in the intensive than in the standard treatment arm (16.5 vs 1.5 per patient per year, respectively). Mean insulin dose was 23% lower in the standard treatment arm (P < .001). There were 61 new cardiovascular events in 24 patients (32%) in the intensive treatment arm and in 16 patients (20%) in the standard treatment arm (P = .10). There was no difference in total and cardiovascular mortality (n = 5 and n = 3 in the intensive and standard treatment arms, respectively) or in new events in patients with cardiovascular history (n = 10 in each arm). In Cox regression analysis, the only significant correlate for new cardiovascular events was previous cardiovascular disease (P = .04). Entering in the analysis any baseline cardiovascular abnormality, the regression model indicated a lower HbA1c level prior to the event as the only correlate for new cardiovascular events (P = .05). CONCLUSION: A long-term prospective trial is needed to assess the risk-benefit ratio of intensive insulin therapy for NIDDM in patients who require it.

Effect of intensive glycemic control on fibrinogen, lipids, and lipoproteins: Veterans Affairs Cooperative Study in Type II Diabetes Mellitus.

BACKGROUND: The Veterans Affairs Cooperative Study in Type II Diabetes Mellitus prospectively studied insulin-treated patients with type 2 (non-insulin-dependent) diabetes mellitus, achieving 2.1% glycosylated hemoglobin separation between intensive- and standard-treatment arms (P<.001) for 2 years. OBJECTIVE: To assess the effect of intensive therapy on serum fibrinogen and lipid levels, compared with standard treatment. METHODS: One hundred fifty-three male subjects with type 2 diabetes mellitus and who required insulin treatment were recruited from 5 Veterans Affairs medical centers. The subjects were divided into intensive- and standard-treatment arms for a randomized prospective study. Dyslipidemia was managed identically in both arms (diet, drugs). Fibrinogen levels and lipid fractions were measured in the full cohort. Lipid fractions are separately reported in patients not treated with hypolipidemic agents. RESULTS: There were no baseline differences between arms. Fibrinogen levels rose in the intensive-treatment arm at 1 year (from 3.34+/-0.12 to 3.75+/-0.15 g/L; P<.001) but returned to baseline at 2 years (3.47+/-0.12 g/L). There was no change in the standard-treatment arm. Triglyceride levels decreased in the intensive-treatment arm from 2.25+/-0.27 to 1.54+/-0.14 mmol/L (199+/-24 to 136+/-12 mg/ dL) at 1 year (P = .004) and to 1.74+/-0.18 mmol/L (154+/-16 mg/dL) at 2 years (P = .03); there was no change in the standard-treatment arm. Cholesterol levels decreased in the intensive-treatment arm at 1 year from 5.4+/-0.21 to 4.99+/-0.13 mmol/L (207+/-8 to 193+/-5 mg/dL) (P = .02); there was no change in the standard-treatment arm. Levels of low- and high-density lipoprotein cholesterol decreased in the standard-treatment arm only by 2 years, from 3.44+/-0.13 to 3.16+/-0.10 mmol/L (133+/-5 to 122+/-4 mg/ dL) (P =.02) and from 1.10+/-0.03 to 1.00+/-0.03 mmol/L (42+/-1 to 38+/-1 mg/dL) (P<.001) for low-density and high-density lipoprotein cholesterol, respectively. Levels of apolipoprotein B decreased in both treatment arms (P<.001), and apolipoprotein A1 levels decreased in the standard-treatment arm (P<.01). Lipoprotein (a) levels did not change in either treatment arm. Lipid results were essentially identical whether examined in the full cohort or excluding those patients receiving hypolipidemic agents. CONCLUSIONS: Intensive insulin therapy led to a potentially beneficial reduction in serum triglyceride levels and preservation of high-density lipoprotein cholesterol and apolipoprotein A1 levels. However, it caused transient elevation in plasma fibrinogen levels, a possible thrombogenic effect.

Safety and efficacy of normalizing fasting glucose with bedtime NPH insulin alone in NIDDM.

OBJECTIVE: To examine the safety and overall clinical effects of normalizing the fasting plasma glucose (FPG) level with bedtime NPH insulin alone in patients with non-insulin-dependent diabetes mellitus (NIDDM) that is poorly controlled with maximal doses of sulfonylureas. RESEARCH DESIGN AND METHODS: Twelve obese male NIDDM subjects were treated for 16 weeks with bedtime insulin after a 4-week sulfonylurea washout. The insulin dosage was increased until the FPG level was normalized. The 24-h plasma glucose profiles and lipid and HbA1c levels were measured at the beginning and end of the study, and the incidence and severity of hypoglycemic episodes were closely monitored. In addition, hyperglycemic clamp studies were performed to assess insulin secretion and provide an indirect measurement of insulin sensitivity. RESULTS: FPG (14.6 +/- 0.9 mmol/l at week 0) was normalized ( < 6.4 mmol/l) within 6 weeks (5.9 +/- 0.6 mmol/l) and remained at target levels until the end of the study (4.0 +/- 0.03 mmol/l at week 16, P < 0.001). The insulin dose was 80 +/- 9 U/day (0.86 +/- 0.10 U/kg). Improved glycemic control was confirmed by a reduction in HbA1c (10.9 +/- 0.05 vs. 7.2 +/- 0.2%, P < 0.001) and mean 24-h glucose (17.2 +/- 0.2 vs. 7.4 +/- 0.2 mmol/l, P < 0.001). The incidence of mild or moderate hypoglycemic episodes was 3.4 +/- 1/patient for the entire 16-week study, and no patient experienced severe hypoglycemia. Bedtime insulin significantly improved total cholesterol, low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and triglyceride levels (P < 0.01). Weight gain was 2.4 +/- 0.7 kg, and blood pressure was unchanged. During the hyperglycemic clamp, there was an improvement in the first phase (P < 0.001) and in the second phase (P < 0.01) of insulin secretion. There also was an increase in the rate of exogenous glucose infused (M) (P < 0.01) and in the M/C-peptide ratio (P < 0.02), suggesting enhanced insulin sensitivity. CONCLUSIONS: NPH insulin given at bedtime in amounts sufficient to achieve a normal FPG level does not cause excessive or severe hypoglycemia and does lead to good glycemic and lipid control in NIDDM. Bedtime insulin therapy also is accompanied by improved insulin secretion and insulin sensitivity. We conclude that a single dose of insulin alone at bedtime merits consideration as a therapeutic strategy in patients with poorly controlled NIDDM.

Glycemic control and complications in type II diabetes. Design of a feasibility trial. VA CS Group (CSDM)

OBJECTIVE: To determine, after 1 yr of follow-up in type II diabetes patients, whether a statistically and clinically significant difference can be achieved in HbA1c between a standard therapy group and an intensively treated group, while maintaining HbA1c levels in both groups within ranges acceptable in regular community practice. Secondary objectives include assessment of patient adherence to protocol, side effects, and accuracy of data collection. RESEARCH DESIGN AND METHODS: This is a prospective, randomized, controlled VA CS conducted with 151 patients at five VAMCs. Patients are males, age 40-69 yr, treated at entry with a maximum dose of sulfonylurea or with insulin, exhibiting an HbA1c level > 3 SDs above the normal mean (5.05 + 3 x 0.50 = > 6.55%). Standard control is achieved with insulin and intensive control with a step-up regimen including insulin alone or insulin/glipizide combinations. Education and management of cardiovascular risk factors are handled similarly in both groups. Primary macrovascular end points are nonfatal myocardial infarction, congestive heart failure, stroke, amputation, and cardiovascular death. Primary microvascular end points are appearance and progression of retinopathy, documented by centrally read seven-field-stereo fundus photographs. Other measured indicators include resting and ambulatory ECGs, ventricular function (MUGA scan), serum lipid and apolipoprotein levels, plasma fibrinogen, nonsymptomatic peripheral vasculopathy, neuroautonomic status by heart-beat variation on Valsalva maneuver, and microalbuminuria. CONCLUSIONS: This study may be the basis for a long-term trial, involving 1400 patients, to assess the long-term effects of metabolic control on macro- and microvascular end points.

Veterans Administration Cooperative Study on antiplatelet agents in diabetic patients after amputation for gangrene: II. Effects of aspirin and dipyridamole on atherosclerotic vascular disease rates.

We report the results of a randomized multicenter clinical trial on the effects of aspirin plus dipyridamole versus placebo on major vascular end points in 231 non-insulin-dependent diabetic men with either a recent amputation for gangrene or active gangrene. Primary end points were death from atherosclerotic vascular disease plus amputation of the opposite extremity for gangrene. There were 24 atherosclerotic deaths in the drug treatment group (21.8%) and 23 in the placebo group (19.0%). There were 22 patients in the drug treatment group (20.0%) and 29 patients in the placebo group (24.0%) with opposite-side amputations. Survival curve analyses revealed little difference between these groups for major vascular end points, total mortality, all amputations, or myocardial infarctions. The most noteworthy group difference was observed for cerebrovascular end points (strokes and transient ischemic attacks), with an incidence of 8.2% (9 patients) in the drug treatment group and 19.0% (23 patients) in the placebo group. We conclude from this study that antiplatelet agents have no effect on the primary vascular end points, vascular deaths and/or amputation of the opposite extremity, in this population. Similarly, no effects were seen on secondary vascular end points, except for a suggestion of protection versus strokes and transient ischemic attacks. However, this finding must be interpreted with caution, since it is a secondary end point and was found only after multiple analyses of the data.

Response to intensive therapy steps and to glipizide dose in combination with insulin in type 2 diabetes. VA feasibility study on glycemic control and complications (VA CSDM).

OBJECTIVE: The feasibility study for the VA Cooperative Study on Glycemic Control and Complications in Type 2 Diabetes (VA CSDM) prospectively studied 153 insulin-requiring type 2 diabetes patients, randomized between an intensively treated arm and a standard treatment arm during a mean follow-up of 27 months. The glycemic response to each of the progressive, sequential phases of insulin treatment was assessed, along with the incidence of hypoglycemic reactions and the relative efficacy of different doses of glipizide in combination with fixed doses of insulin. RESEARCH DESIGN AND METHODS: Five medical centers participated; half of the patients were assigned to the intensive treatment arm aiming for normal HbA1c levels. Age of patients was 60 +/- 6 years, duration of diabetes 8 +/- 3 years, and BMI 30.7 +/- 4 kg/m2. A four-step management technique was used, with patients moving to the next step if the operational goals were not met: Phase I, evening intermediate or long-acting insulin; phase II, added day-time glipizide; phase III, two injections of insulin alone; and phase IV, multiple daily insulin injections. Home glucose monitoring measurements were done twice daily and at 3:00 A.M. once a week. Hypoglycemic reactions and home glucose monitoring results were recorded and counted in each of the treatment phases. RESULTS: Baseline HbA1c was 9.3 +/- 1.8%, and fasting plus serum glucose was 11.4 +/- 3.3 mmol/1. Fasting serum glucose fell to near normal in phase I, and remained so in the other treatment phases. An HbA1c separation of 2.1% between the arms was maintained during the course of the study, while the intensive arm kept HbA1c levels below 7.3% (P = 0.001). Most of the decrease in HbA1c occurred with one injection of insulin alone (phase I, -1.4%) or adding day-time glipizide (phase II, -1.9% compared with baseline). HbA1c did not decrease further after substituting two injections of insulin alone, with twice the insulin dose. Multiple daily injections resulted in an additional HbA1c fall (-2.4% compared with baseline). However, two-thirds of the patients were still on one or two injections a day at the end of the study. Changes in home glucose monitoring levels paralleled those of the HbA1c, as did the increments in number of reported hypoglycemic reactions, virtually all either "mild" or "moderate" in character. For the combination of glipizide and insulin (phase II), the only significant effect was obtained with daily doses up to 10 mg a day; there were no significant additional benefits with up to fourfold higher daily doses, and HbA1c levels had an upward trend with doses > 20 mg/day. CONCLUSIONS: A simple regime of a single injection of insulin, alone or with glipizide, seemed sufficient to obtain clinically acceptable levels of HbA1c for most obese, insulin-requiring type 2 diabetes patients. Further decrease of HbA1c demanded multiple daily injections at the expense of doubling the insulin dose and the rate of hypoglycemic events. In combination therapy, doses of glipizide > 20 mg/day offered no additional benefit.

Veterans Affairs Cooperative Study on glycemic control and complications in type II diabetes (VA CSDM). Results of the feasibility trial. Veterans Affairs Cooperative Study in Type II Diabetes.

OBJECTIVE: It is not clear whether intensive pharmacological therapy can be effectively sustained in non-insulin-dependent diabetes mellitus (NIDDM). The relative risks and benefits of intensive insulin therapy in NIDDM are not well defined. Accordingly, we designed a feasibility study that compared standard therapy and intensive therapy in a group of NIDDM men who required insulin due to sustained hyperglycemia. RESEARCH DESIGN AND METHODS: A prospective trial was conducted in five medical centers in 153 men of 60 +/- 6 years of age who had a known diagnosis of diabetes for 7.8 +/- 4 years. They were randomly assigned to a standard insulin treatment group (one morning injection per day) or to an intensive therapy group designed to attain near-normal glycemia and a clinically significant separation of glycohemoglobin from the standard arm. A four-step plan was used in the intensive therapy group along with daily self-monitoring of glucose: 1) an evening insulin injection, 2) the same injection adding daytime glipizide, 3) two injections of insulin alone, and 4) multiple daily injections. Patient accrual and adherence, glycohemoglobin (HbA1c), side effects, and measurements of endpoints for a prospective long-term trial were assessed. RESULTS: Accrual goals were met, mean follow-up time was 27 months (range 18-35 months), and patients kept 98.6% of scheduled visits. After 6 months, the mean HbA1c in the intensive therapy group was at or below 7.3% and remained 2% lower than the standard group for the duration of the trial. Most of the decrease in the mean HbA1c in the intensive group was obtained by a single injection of evening intermediate insulin, alone or with daytime glipizide. By the end of the trial, 64% of the patients had advanced to two or more injections of insulin a day, aiming for normal HbA1c. However, only a small additional fall in HbA1c was attained. Severe hypoglycemia was rare (two events per 100 patients per year) and not significantly different between the groups, nor were changes in weight, blood pressure, or plasma lipids. There were 61 new cardiovascular events in 40 patients and 10 deaths (6 due to cardiovascular causes). CONCLUSIONS: Intense stepped insulin therapy in NIDDM patients who have failed glycemic control on pharmacological therapy is effective in maintaining near-normal glycemic control for > 2 years without excessive severe hypoglycemia, weight gain, hypertension, or dyslipidemia. Cardiovascular event rates are high at this stage of NIDDM. A long-term prospective trial is needed to assess the risk-benefit ratio of intensified treatment of hyperglycemia in NIDDM patients requiring insulin.

Evaluations of retinopathy in the VA Cooperative Study on Glycemic Control and Complications in Type II Diabetes (VA CSDM). A feasibility study.

OBJECTIVE: The main goal of the study of 153 male veterans was to determine whether a statistically and clinically significant difference in HbA1c could be achieved between a standard therapy and an intensively treated group of patients with type II diabetes. A second major goal was to assess the feasibility of collecting reliable high-quality endpoint data, including microvascular and macrovascular events. Retinopathy was defined as a key microvascular endpoint. RESEARCH DESIGN AND METHODS: This was a randomized prospective trial of 153 men between the ages of 40 and 69 years, with type II diabetes for 15 years or less. Of the patients, 78 were assigned to the standard therapy arm and 75 to the intensive therapy arm. The goal of standard therapy was good general medical care and well-being and avoiding excessive hyperglycemia, glycosuria, ketonuria, or hypoglycemia. This was generally accomplished with one shot of insulin per day. The goal of intensive therapy was to obtain an HbA1c within two standard deviations of the mean of nondiabetic subjects (4.0-6.1%). This was obtained by a four-step management technique, with patients moving to the next step only if operational goals were not met. The steps were as follows: step 1: evening intermediate or long-acting insulin only; step 2: evening insulin with daytime glipizide; step 3: insulin, twice a day, no glipizide; and step 4: more than two injections of insulin, no glipizide. Retinopathy was assessed at baseline, 12, and 24 months by seven-field stereo fundus photography done at each of the five participating VA medical centers and read at the Central Reading Center at the Department of Ophthalmology, University of Wisconsin Medical School, Madison. Visual acuity was determined by ophthalmologists at each of the participating hospitals. RESULTS: After the 6th month of the 24-month study, an average HbA1c of approximately 7.1% in the intensively treated group was sustained for the full study and was significantly lower than that seen in the standard group (9.2%, P < 0.001). Compliance in obtaining fundus photographs was excellent. Near normalization of glycemia did not cause transient worsening of retinal morphology nor did it prevent the onset or delay the progression of retinopathy. There was no effect on visual acuity. CONCLUSIONS: 1) A glycemic control intervention study in people with type II diabetes is feasible and safe; 2) intensive control did not cause transient deterioration of retinopathy; and 3) although no improvement was seen in retinopathy, the follow-up was 24 months, an interval shorter than the 3 years or more of intensive therapy before improvement is seen in type 1 diabetic studies. This does not rule out the possibility that longer periods of intensive therapy would have improved retinopathy. A full-scale intervention trial in type II diabetes is needed to resolve this issue.

Effect of intensive glycemic control on microalbuminuria in type 2 diabetes. Veterans Affairs Cooperative Study on Glycemic Control and Complications in Type 2 Diabetes Feasibility Trial Investigators.

OBJECTIVE: Microalbuminuria can reflect the progress of microvascular complications and may be predictive of macrovascular disease in type 2 diabetes. The effect of intensive glycemic control on microalbuminuria in patients in the U.S. who have had type 2 diabetes for several years has not previously been evaluated. RESEARCH DESIGN AND METHODS: We randomly assigned 153 male patients to either intensive treatment (INT) (goal HbA(1c) 7.1%) or to standard treatment (ST) (goal HbA(1c) 9.1%; P = 0.001), and data were obtained during a 2-year period. Mean duration of known diabetes was 8 years, mean age of the patients was 60 years, and patients were well matched at baseline. We obtained 3-h urine samples for each patient at baseline and annually and defined microalbuminuria as an albumin:creatinine ratio of 0.03-0.30. All patients were treated with insulin and received instructions regarding diet and exercise. Hypertension and dyslipidemia were treated with similar goals in each group. RESULTS: A total of 38% of patients had microalbuminuria at entry and were evenly assigned to both treatment groups. INT retarded the progression of microalbuminuria during the 2-year period: the changes in albumin:creatinine ratio from baseline to 2 years of INT versus ST were 0.045 vs. 0.141, respectively (P = 0.046). Retardation of progressive urinary albumin excretion was most pronounced in those patients who entered the study with microalbuminuria and were randomized to INT. Patients entering with microalbuminuria had a deterioration in creatinine clearance at 2 years regardless of the intensity of glycemic control. In the group entering without microalbuminuria, the subgroup receiving ST had a lower percentage of patients with a macrovascular event (17%) than the subgroup receiving INT (36%) (P = 0.03). Use of ACE inhibitors or calcium-channel blockers was similarly distributed among the groups. CONCLUSIONS: Intensive glycemic control retards microalbuminuria in patients who have had type 2 diabetes for several years but may not lessen the progressive deterioration of glomerular function. Increases in macrovascular event rates in the subgroup entering without albuminuria who received INT remain unexplained but could reflect early worsening, as observed with microvascular disease in the Diabetes Control and Complications Trial.

Ethnic differences in the glycemic response to exogenous insulin treatment in the Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus (VA CSDM).

OBJECTIVE: The Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus was conducted in NIDDM patients to determine if a significant difference in HbA1c could be achieved between groups receiving standard and intensive treatment. We observed differences in the response to exogenous insulin between African-Americans and other intensively treated patients. Therefore, we assessed the variations of response and correlated factors that might explain such differences. RESEARCH DESIGN AND METHODS: One hundred fifty-three men aged 40-69 years with NIDDM for < or = 15 years were randomized to either the standard therapy (n = 78) or the intensive therapy (n = 75) arm. Of the 75 patients in the intensive therapy group, 57 completed the study on insulin therapy alone. Of these, 18 were African-Americans and 39 were non-African-Americans. We conducted an analysis of the data collected to determine differences in baseline characteristics, glycemic response, insulin requirement, body weight, exercise, and basal C-peptide level, factors that may explain a difference in response to insulin therapy. RESULTS: Glycemic control improved in all patients with intensive insulin therapy. African-Americans achieved a greater improvement in HbA1c compared with non-African-Americans with a similar increment in insulin. This difference could not be explained by differences in body weight, activity, concomitant use of other medicines, or insulin-secretory capacity of the pancreas. CONCLUSIONS: We conclude that ethnic differences may exist in the response to insulin therapy. A knowledge of such differences may aid in achieving good glycemic control, especially since minorities have a greater prevalence of and burden from the microvascular complications of diabetes.