Pancreatic beta cell function persists in many patients with chronic type 1 diabetes, but is not dramatically improved by prolonged immunosuppression and euglycaemia from a beta cell allograft.

AIMS/HYPOTHESIS: We measured serum C-peptide (at least 0.167 nmol/l) in 54 of 141 (38%) patients with chronic type 1 diabetes and sought factors that might differentiate those with detectable C-peptide from those without it. Finding no differences, and in view of the persistent anti-beta cell autoimmunity in such patients, we speculated that the immunosuppression (to weaken autoimmune attack) and euglycaemia accompanying transplant-based treatments of type 1 diabetes might promote recovery of native pancreatic beta cell function. METHODS: We performed arginine stimulation tests in three islet transplant and four whole-pancreas transplant recipients, and measured stimulated C-peptide in select venous sampling sites. On the basis of each sampling site's C-peptide concentration and kinetics, we differentiated insulin secreted from the individual's native pancreatic beta cells and that secreted from allografted beta cells. RESULTS: Selective venous sampling demonstrated that despite long-standing type 1 diabetes, all seven beta cell allograft recipients displayed evidence that their native pancreas secreted C-peptide. Yet even if chronic immunosuppression coupled with near normal glycaemia did improve native pancreatic C-peptide production, the magnitude of the effect was quite small. CONCLUSIONS/INTERPRETATION: Some native pancreatic beta cell function persists even years after disease onset in most type 1 diabetic patients. However, if prolonged euglycaemia plus anti-rejection immunosuppressive therapy improves native pancreatic insulin production, the effect in our participants was small. We may have underestimated pancreatic regenerative capacity by studying only a limited number of participants or by creating conditions (e.g. high circulating insulin concentrations or immunosuppressive agents toxic to beta cells) that impair beta cell function.

Human tumor-associated antigens detected by serological techniques: analysis of autologous humoral immune responses to primary and metastatic human sarcomas by an enzyme-linked immunoabsorbent solid-phase assay (ELISA).

An enzyme-linked immunoabsorbent solid-phase assay was developed for measuring humoral immune responses to human sarcoma-associated antigens, and binding of autologous sera to 1-butanol extracts of fresh sarcomas was determined. Binding of autologous sarcoma patients' sera was reproducible and significantly greater than normal sera. Extensive quantitative competitive binding inhibition tests with normal tissue were unable to completely remove binding to autologous tumor extracts by sera from five of eight patients, thus demonstrating a quantitatively distinct specificity present on tumor tissue. An antibody in autologous sera recognizing a determinant on normal adult tissue was also identified. Autologous humoral immune responses to soluble 1-butanol extracts of paired primary and metastatic human sarcomas from six patients from this group were assessed. Antibody binding was observed to both primary and metastatic tumor extracts; however, binding was significantly greater to the primary extracts in 14 of 16 autologous serum-tumor combinations tested. Absorption and competitive binding inhibition studies demonstrated the presence of common sarcoma-associated antigens in extracts of both the primary tumor and its metastasis from all patients studied. Two individual metastases from the same patient also possessed antigens recognized in that patient's primary tumor extract in two cases. Autologous sarcoma patients' sera recognize sarcoma-associated antigens common to both the primary tumor and its metastasis.

Interleukin-8 priming of human neutrophils is not associated with persistently altered calcium fluxes but is additive with lipopolysaccharide.

Interleukin-8 (IL-8) priming was studied in neutrophils to examine its dependency on altered calcium fluxes and for similarity to lipopolysaccharide (LPS). IL-8 caused a rapid rise in [Ca2+]i that returned to baseline values by 20 min. Peak [Ca2+]i transients in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) were unaltered in IL-8-primed compared with unprimed cells. In comparison to LPS and tumor necrosis factor (TNF), IL-8 was a much weaker priming agent as measured by either O2- or H2O2 production. Despite their large disparity in potency, IL-8 and LPS printing were additive using fMLP, a receptor-dependent stimulator, and synergistic using the post-receptor, protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) to trigger the respiratory burst. In contrast, IL-8 and TNF priming were synergistic for fMLP (P = 0.05), but completely nonadditive when PMA was used as the neutrophil stimulant (P = 0.05 for subadditivity). Thus, lasting alterations in [Ca2+]i are not a necessary characteristic of IL-8-primed cells. IL-8 and LPS appear to prime by non-overlapping pathways, whereas IL-8 and TNF appear to share mechanisms distal to protein kinase C activation. IL-8 and LPS may independently contribute to neutrophil-mediated host defense or injury by priming through distinct pathways.

A rapid, single step technique for determination of B and T cell surface markers in malignant lymphoid cells.

The immunological classification of lymphocytic subpopulations has become increasingly important in the treatment of hematologic malignancy. In the present study, the standard techniques for the identification of subsets of both normal and malignant lymphocytes were compared to a new one step procedure for the simultaneous assessment and identification of cells with T or B cell surface markers. With this new technique, cells with T lymphocyte markers form SRBC rosettes and cells with B lymphocyte markers form bead rosettes after incubation with microspheres coated with an anti-immunoglobulin antibody. In the present study, analysis of patient material using this simultaneous marker method produced results similar to standard techniques. In addition, the simultaneous E-rosette and immunobead method for detection of lymphocyte subpopulations permits direct examination of cell morphology, is easier to perform, and does not require fluorescence microscopy.

Assessing the effects of thyroid suppression on benign solitary thyroid nodules. A model for using quantitative research synthesis.

Systematic review of the available information with a modified, largely quantitative method of research synthesis disclosed that an initial trial of thyroid hormone suppression therapy leads to clinically significant (> or = 50%) reduction of nodule size or arrest of nodule growth in a subset of patients with benign solitary thyroid nodules. In fact, in addition to objective improvements due to decreasing nodule size, L-T4 suppression therapy may benefit patients by reducing perinodular thyroid volume. Consequently, both pressure symptoms and cosmetic complaints may improve (9, 68). Additional studies for the assessment of the risks versus benefits of supraphysiologic doses of L-T4, the optimal level of thyroid suppression and the dose needed to achieve this magnitude of reduction, the optimal length of the initial trial, and the conditions for the continuation of L-T4 thyroid suppression therapy, as well as the identification of markers for patients most likely to respond to this therapy, are warranted. Finally, quantitative assessment of available evidence as described here may be applicable to the review of other controversial issues as well.

The effect of total parenteral nutrition on chemotherapy-induced myelosuppression. A randomized study.

We studied the effect of total parenteral nutrition on recovery from myelosuppression in patients receiving intensive chemotherapy. Twenty-seven patients (ages 11 to 33 years) with locally recurrent or metastatic Ewing's sarcoma, rhabdomyosarcoma, or osteosarcoma were randomly selected to receive either conventional oral nutrition or total parenteral nutrition concurrently with intensive chemotherapy. The control group (15 patients) received significantly fewer calories (range 380 to 880/m2 per day, median 685 versus range 1,020 to 2,100 median 1,650) and less nitrogen (0-3.7 g/m2 per day, median 1.5 versus range 5.3 to 12.4, median 8.9) than the group receiving total parenteral nutrition (12 patients). Assessment of recovery from myelosuppression was based on the length of time the absolute granulocyte count was below 500/mm3, the length of time the platelet count was below 40,000/mm3, the number of days the platelet count was below 20,000/mm3, and the number of blood transfusions required. There was no statistical difference in any of the parameters evaluated between the group that received total parenteral nutrition and the control group (p less than 0.05); granulocyte and platelet recovery and the difference in transfusion requirements favored the control group with marginal statistical significance (p = 0.05). The frequency of clinical infections was similar in the patients receiving total parenteral nutrition (five of 12) and in those receiving conventional oral nutrition (five of 15). Thus, although total parenteral nutrition could be safely administered in this severely myelosuppressed population, no benefit could be defined in recovery from bone marrow suppression or frequency of clinical infections.

Cytokine polymorphic analyses indicate ethnic differences in the allelic distribution of interleukin-2 and interleukin-6.

BACKGROUND: Polymorphisms in the regulatory regions of cytokine genes affect protein production and are associated with allograft outcome. Ethnic origin has been identified as a significant prognostic factor for several immune-mediated diseases and for outcome after allotransplantation. A clear relationship between cytokine polymorphisms and ethnicity has not been shown. METHODS: One hundred sixty subjects including 102 whites and 43 African-Americans were studied. Using polymerase chain reaction-based assays and, in some cases, restriction enzyme digestion, we determined genetic polymorphisms for the cytokines interleukin (IL) -2, IL-6, IL-10, tumor necrosis factor-alpha, transforming growth factor-beta, and interferon-gamma (IFN-gamma). Genetic polymorphism frequencies were then compared to ethnicity using chi-square analysis and Fisher's exact two-tailed tests. RESULTS: For both the IL-2 and IL-6 genes, we found that whites and African-Americans differed significantly (P <0.05) in their allelic distribution and genotype frequency. A trend toward ethnic distribution was noted among the alleles and genotypes for the IL-10 and IFN-gamma genes. We found no correlation between ethnicity and either allelic distribution or genotype frequency for the tumor necrosis factor-alpha or transforming growth factor-beta genes. When comparisons were made between patients with or without a history of kidney failure, the allelic or genotypic distributions for the IL-6 and IFN-gamma genes were found to significantly differ. CONCLUSIONS: Our work demonstrates a correlation between ethnicity and polymorphisms in several cytokine genes. In addition, we found that patients requiring renal transplantation differ from the general population with regard to certain cytokine gene polymorphisms. These findings may have relevance in making prognostic determinations or tailoring immunomodulatory regimens after renal transplantation.

Endotoxemia in human septic shock.

To evaluate the incidence, pattern and clinical importance of endotoxemia in septic shock, frequent, serial endotoxin determinations were made prospectively in patients with shock. Detectable endotoxin occurred in 43 of 100 patients with septic shock, but in only one of ten patients with shock due to nonseptic causes. During septic shock, endotoxemia frequently occurred in the absence of Gram-negative bacteremia. Using a logistic regression model, multiple organ failure occurred 10.3 times more frequently and depression of left ventricular ejection fraction (less than or equal to 45 percent) 4.8 times more frequently in endotoxemic patients. In patients with positive blood cultures, endotoxemia was associated with a high mortality. We conclude that endotoxemia occurs frequently in septic shock and is associated with severe manifestations of this syndrome, including cardiac depression and multiple organ failure. This study suggests that endotoxin is an important mediator of septic shock and supports efforts to develop anti-endotoxin therapies for treating patients with this disease.

Adverse exposures and universal precautions practices among a group of highly exposed health professionals.

An anonymous national survey of a representative population of healthcare workers who were thought likely to have frequent and intensive exposures to blood and other body fluids (certified nurse-midwives [CNMs]), was conducted to assess the type and frequency of self-reported occupational exposures to blood and body fluids experienced, the extent to which barrier precautions and other infection control measures were used, whether or not reported use of barriers was associated with a lower perceived rate of exposures and factors that influenced the use of infection control procedures. Of those responding, 74% had soiled their hands with blood at least one time in the preceding six months, 51% had splashed blood or amniotic fluid in their faces and 24% reported one or more needlestick injuries during that same period. Our study also found evidence of an association between the practice of needle recapping and the occurrence of needlestick injury (p = .003). Despite a high level of training and knowledge, only 55% reported routinely practicing universal precautions (UPs). Several factors that potentially influenced the use of UPs were studied, including healthcare worker perceptions of risk of occupational bloodborne infection, knowledge of routes of transmission of bloodborne pathogens and rationale for not using appropriate barriers. Our data suggest that occupational exposures occur frequently and that healthcare workers' (HCWs') perceptions of risk for occupational infection play an important role in influencing use of UPs. This study emphasizes the importance of developing new strategies for UP training.

Liver transection: a controlled study of four different techniques in pigs.

The optimal method of transecting liver parenchyma has not been established and presently a variety of methods are in use. In a controlled study in pigs standard resections were performed with four different transection techniques: ultrasonic dissection, suction dissection, electrocautery, and sharp dissection. The blood loss, number of vessels identified before their division, need for additional hemostatic measures, and time for each procedure were evaluated. Also, the histologic appearance of the fresh and the healing cut surface of the liver was studied. The blood loss was the lowest when ultrasonic dissection was used (median blood loss of 58 ml per resection). The comparisons with suction dissection (median blood loss of 87 ml) and cautery (median blood loss of 79 ml) were not significant. The ultrasonic and suction dissection techniques were both effective in isolating vessels, but the ultrasonic dissector did this more atraumatically. Cautery and ultrasonic dissection had a hemostatic effect on the parenchyma in that a significantly smaller number of vessels needed to be clipped or tied. On histologic study of the fresh cut liver surface, a smooth surface was seen with ultrasonic dissection, parenchymal hemorrhage after suction dissection, and coagulation necrosis after electrocautery. Ultrasonic dissection was the only technique that combined lowered blood loss because medium- and large-size vessels were dissected free and ligated before transection and a hemostatic effect on small vessels.

Computerized technetium/thallium scans and parathyroid reoperation.

Twenty-three consecutive patients who underwent reoperation for persistent primary hyperparathyroidism were studied with computerized technetium/thallium scans to evaluate it as a localization test. Prospective and blinded retrospective readings of the scans were correlated with findings at surgery. Computerized technetium/thallium scanning detected 52% of the parathyroid adenomas found at surgery. Forty-two percent of adenomas were localized exactly. There were no false positive scans. No normal parathyroid glands were imaged. No mediastinal glands were imaged. The study was not observer dependent. We conclude that computerized technetium/thallium scanning is helpful in patients undergoing parathyroid reoperation. If positive, it can accurately localize adenomas. If negative, we recommend more invasive studies.

A prospective, randomized study of adjuvant parenteral nutrition in the treatment of sarcomas: results of metabolic and survival studies.

A prospective, randomized study was performed to evaluate the use of total parenteral nutrition (TPN) in a group of young patients receiving aggressive chemotherapy for metastatic or locally recurrent sarcomas. Fourteen patients were randomly selected to receive TPN and 18 to receive conventional oral nutritional support (CN). During the study period (from first dose of chemotherapy to recovery from myelosuppression), the TPN patients received between 1020 and 2100 calories/m2/day (median 1650) and between 5.3 and 12.4 gmN/m2/day (median 8.9), while the CN patients received between 380 and 880 calories/m2/day (median 685) and between 0.0 and 3.7 gmN/m2/day (median 1.5). The mean daily nitrogen balance during the study period for the TPN group (-3.0 to + 1.3 gmN/m2/day, median -0.7) was significantly higher (p = 0.005) than that of the CN group (-6.2 to -0.7 gmN/m2/day, median -2.6). Serum protein levels (albumin, total protein, and transferrin) did not differ between the two treatment groups. The proportion of patients responding to therapy and the long-term survival rates were similar in the treatment groups. Thus despite established improvement in nitrogen balance, no survival or therapeutic advantage was demonstrated for the adjuvant parenteral nutrition group. Further studies of the role of parenteral nutrition as an adjuvant to cancer chemotherapy are needed to determine which populations of patients will benefit from its use.

Prospective evaluation of aspiration needle, cutting needle, transbronchial, and open lung biopsy in patients with pulmonary infiltrates.

Twenty consecutive patients with pulmonary infiltrates undiagnosed by routine, noninvasive methods were entered into a prospective study designed to evaluate the diagnostic yield of four methods of lung biopsy. Percutaneous aspiration needle, cutting needle, transbronchial, and open (anterior thoracotomy) biopsy were performed synchronously on all patients. Specimens were evaluated by microbiological, virological, and pathological methods. The diagnostic yields of the four methods were as follows: aspiration needle, 29%; cutting needle, 53%; transbronchial, 59%; and open lung biopsy, 94%. Open lung biopsy was significantly better in yielding a diagnosis than aspiration needle (p less than 0.001), cutting needle (p less than 0.001), and transbronchial biopsy (p less than 0.04).

Effects of restricting levothyroxine dosage strength availability.

We conducted a prospective, randomized, controlled trial to assess whether hospital formulary restrictions involving limiting dosage strengths of levothyroxine affect physicians' ability to manage patients effectively and provide pharmacy cost savings in a tertiary care federal government research hospital. Thirty-three endocrinologists were randomly assigned to prescribe levothyroxine from a restrictive (dosage strengths of 25, 50, 100, 125, and 150 micrograms) or a nonrestrictive (dosage strengths of 25, 50, 75, 100, 112, 125, 150, 175, 200, and 300 micrograms) formulary through a central computer system. Their 241 respective outpatients' laboratory results and drug compliance were outcome measures. Achievement of treatment objectives was measured by thyroid function tests (free and total thyroxine, total triiodothyronine, thyrotropin), number of clinic visits, and compliance (survey method). Additional measures were drug distribution patterns, drug costs, and pharmacy inventory costs. Restriction of levothyroxine's dosage strength did not significantly alter therapeutic outcomes. However, the restricted formulary was associated with more complex dosing regimens, and resulted in no significant cost savings. It is not known whether such restriction would adversely affect the care of patients of nonspecialists. Prospective studies are required to verify presumed cost-containment measures before such measures are adopted for widespread application.

Rhabdomyosarcoma. A new classification scheme related to prognosis.

We classified 159 cases of rhabdomyosarcoma (RMS) according to the conventional scheme adopted by the World Health Organization and a modified conventional scheme established at the National Cancer Institute (NCI), Bethesda, Md. The major modification in the NCI scheme was the inclusion of compact round-cell RMS with scant myogenesis in the group of alveolar RMS despite lack of an alveolar architecture. These tumors were previously considered to be embryonal RMS, but their cytologic features are quite different from those seen in embryonal RMS and are indistinguishable from those encountered in alveolar RMS. These tumors are referred to as "solid alveolar RMS." Survival curves were constructed with the method of Kaplan-Meier and compared with the unstratified and stratified methods of Mantel-Haenszel (with stratification factors being stage, site, and age) and with the Cox regression analysis. Both histologic schemes showed a statistically significant prognostic value in unstratified analyses, but the NCI scheme demonstrated prognostic value even in stratified analyses and in the Cox regression analysis in our series of cases. The data indicate that the NCI scheme can serve as a highly predictive, independent prognostic factor in RMS and that the alveolar category should be expanded to include the solid round-cell RMS, even in the absence of a classic alveolar architecture.

Levels and chemical composition of cotton gin dust.

Dust levels were determined in the three principal work areas of five high-capacity, saw-type cotton gins processing spindle-picked cotton. Dust levels measured by the vertical elutriator, OSHA personal and stationary personal samplers averaged 0.66, 0.96 and 0.87 mg/m3, respectively. Gross chemical analyses of dust samples collected indicated that the composit0n of the dust was highly variable and different for the principal work areas within each gin -- 15 to 53% ash, 2 to 5% moisture, 8 to 18% protein, 19 to 55% cellulose and 8 to 16% water-extractable constituents. Major elements were silicon, potassium, aluminum, calcium and magnesium.

Plasma polymerized N-isopropylacrylamide: synthesis and characterization of a smart thermally responsive coating.

A lower critical solution temperature (LCST) in an aqueous environment has been observed with poly(N-isopropylacrylamide) (pNIPAM) deposited onto solid surfaces from a plasma glow discharge of NIPAM vapor. The synthesis and spectroscopic data (ESCA, FTIR) for the plasma polymerized NIPAM (ppNIPAM) shows a remarkable retention of the monomer structure. The phase transition at 29 degrees C was measured by a novel AFM method. The phase transition was surprising because of the expectation that the plasma environment would destroy the specific NIPAM structure associated with the thermal responsiveness. The phase change of ppNIPAM is also responsible for the changes in the level of the meniscus when coated capillaries are placed in warm and cold water. Plasma polymerization of NIPAM represents a one-step method to fabricate thermally responsive coatings on real-world biomaterials without the need for specially prepared substrates and functionalized polymers.

Nitric oxide increases tumor necrosis factor production in differentiated U937 cells by decreasing cyclic AMP.

Nitric oxide (NO) increases tumor necrosis factor (TNF) synthesis in human peripheral blood mononuclear cells by a cGMP-independent mechanism. NO has been shown to inhibit adenylate cyclase in cell membranes. Since cAMP down-regulates TNF transcription, we examined the possibility that NO enhances TNF synthesis by decreasing cAMP. U937 cells were induced to differentiate using phorbol myristate acetate (100 nM for 48 h) and then were incubated for 24 h with sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP). These NO donors increased TNF production (7.0- and 15.6-fold, respectively, at 500 microM) in a dose-dependent manner (p = 0.002). However, SNP and SNAP did not elevate cGMP levels in U937 cell cultures, and the cGMP analog, 8-bromo-cGMP, had no effect on TNF production. In contrast, SNP (p = 0.001) and SNAP (p = 0.009) decreased intracellular cAMP levels by up to 51.5% over 24 h and, in the presence of a phosphodiesterase inhibitor, blunted isoproterenol-stimulated increases in cAMP by 21.8% (p = 0.004) and 27.6% (p = 0.008), respectively. H89, an inhibitor of cAMP-dependent protein kinase, dose dependently increased TNF production in phorbol myristate acetate-differentiated U937 cells in the absence (6.5-fold at 30 microM; p = 0.035), but not in the presence (p = 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt2cAMP) blocked SNAP-induced TNF production (p = 0.001). SNP and SNAP (500 microM) increased relative TNF mRNA levels by 57.5% (p = 0.045) and 66.2% (p = 0.001), respectively. This effect was prevented by Bt2cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP.

A Sp1 binding site of the tumor necrosis factor alpha promoter functions as a nitric oxide response element.

Regulation of gene transcription is an incompletely understood function of nitric oxide (NO). Human leukocytes produce increased amounts of tumor necrosis factor alpha (TNF-alpha) in response to NO. This effect is associated with decreases in intracellular cAMP, suggesting that NO might regulate gene transcription through promoter sequences sensitive to cAMP such as cAMP response elements (CRE) and Sp1 binding sites. Here we report that a Sp1 binding site in the TNF-alpha promoter conveys NO responsiveness. Human U937 cells were differentiated for TNF-alpha production with phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of cAMP-dependent protein kinase increased, while dibutyryl cAMP (Bt(2)cAMP) decreased TNF-alpha promoter activity. Deletion or mutation of the proximal Sp1 site, but not the CRE site, abolished the activating effects of NO donors and H89. Further, NO- and H89-mediated increases in TNF-alpha promoter activity were associated with decreased Sp1 binding. The insertion of Sp1 sites into a minimal cytomegalovirus promoter conferred NO responsiveness, an effect blocked by Bt(2)cAMP. Mutation of these inserted Sp1 sites prevented this heterologous promoter from responding to NO, H89 and Bt(2)cAMP. These results identify the Sp1 binding site as a promoter motif that allows NO to control gene transcription.