ß1-Na(+),K(+)-ATPase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with diverse disorders and characterized by disruption of the alveolar-capillary barrier, leakage of edema fluid into the lung, and substantial inflammation leading to acute respiratory failure. Gene therapy is a potentially powerful approach to treat ALI/ARDS through repair of alveolar epithelial function. Herein, we show that delivery of a plasmid expressing ß1-subunit of the Na(+),K(+)-ATPase (ß1-Na(+),K(+)-ATPase) alone or in combination with epithelial sodium channel (ENaC) α1-subunit using electroporation not only protected from subsequent lipopolysaccharide (LPS)-mediated lung injury, but also treated injured lungs. However, transfer of α1-subunit of ENaC (α1-ENaC) alone only provided protection benefit rather than treatment benefit although alveolar fluid clearance had been remarkably enhanced. Gene transfer of ß1-Na(+),K(+)-ATPase, but not α1-ENaC, not only enhanced expression of tight junction protein zona occludins-1 (ZO-1) and occludin both in cultured cells and in mouse lungs, but also reduced pre-existing increase of lung permeability in vivo. These results demonstrate that gene transfer of ß1-Na(+),K(+)-ATPase upregulates tight junction formation and therefore treats lungs with existing injury, whereas delivery of α1-ENaC only maintains pre-existing tight junction but not for generation. This indicates that the restoration of epithelial/endothelial barrier function may provide better treatment of ALI/ARDS.

The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1beta-converting enzyme (caspase-1) activity in human vascular smooth muscle cells.

Interleukin-1beta-converting enzyme (ICE, caspase-1) regulates key steps in inflammation and immunity, by activating the proinflammatory cytokines interleukin (IL-)1beta and IL-18, or mediating apoptotic processes. We recently provided evidence for the regulation of caspase-1 activity via an endogenous inhibitor expressed by human vascular smooth muscle cells (SMCs) (Schönbeck, U., M. Herzberg, A. Petersen, C. Wohlenberg, J. Gerdes, H.-D. Flad, and H. Loppnow. 1997. J. Exp. Med. 185:1287-1294). However, the molecular identity of this endogenous inhibitor remained undefined. We report here that the serine proteinase inhibitor (serpin) PI-9 accounts for the endogenous caspase-1 inhibitory activity in human SMCs and prevents processing of the enzyme's natural substrates, IL-1beta and IL-18 precursor. Treatment of SMC lysates with anti-PI-9 antibody abrogated the caspase-1 inhibitory activity and coprecipitated the enzyme, demonstrating protein-protein interaction. Furthermore, PI-9 antisense oligonucleotides coordinately reduced PI-9 expression and promoted IL-1beta release. Since SMCs comprise the majority of cells in the vascular wall, and because IL-1 is implicated in atherogenesis, we tested the biological validity of our in vitro findings within human atheroma in situ. The unaffected arterial wall contains abundant and homogeneously distributed PI-9. In human atherosclerotic lesions, however, PI-9 expression correlated inversely with immunoreactive IL-1beta, supporting a potential role of the endogenous caspase-1 inhibitor in this chronic inflammatory disease. Thus, our results provide new insights into the regulation of this enzyme involved in immune and inflammatory processes of chronic inflammatory diseases, and point to an endogenous antiinflammatory action of PI-9, dysregulated in a prevalent human disease.

Influence of impaired selective motor control on gait in children with cerebral palsy.

PURPOSE: Spastic cerebral palsy (CP) is characterized by four neuromuscular deficits: weakness, short muscle-tendon unit, muscle spasticity and impaired selective motor control (SMC). We examined the influence of impaired SMC on gait in children with bilateral spastic CP. Delineating the influence of neuromuscular deficits on gait abnormalities can guide surgical and therapeutic interventions to reduce long-term debilitating effects of CP. METHODS: The relationship between impaired SMC and gait was assessed using multivariate linear regression analysis of Selective Control Assessment of the Lower Extremity (SCALE) in relation to stance phase knee flexion and temporal-spatial gait parameters calculated using 3D kinematics for 57 children with bilateral spastic CP, ages seven to 11 years. RESULTS: Mean SCALE values were 5.8 (0 to 10, sd 3.0) and 5.7 (0 to 10, sd 2.9) for right and left legs, respectively. Multivariate linear regression models, including right and left SCALE and height, significantly predicted right and left knee flexion at initial contact (R = 0.479, p = 0.003; R = 0.452, p = 0.007, respectively) and right and left knee flexion in midstance (R = 0.428, p = 0.013; R = 0.407, p = 0.022, respectively). The model significantly predicted right and left step length (R = 0.645, p = 0.000; R = 0.523, p = 0.001, respectively) and predicted gait velocity (R = 0.444, p = 0.008). The model including SCALE did not predict step width. CONCLUSION: Results indicate impaired SMC predicts increased knee flexion at initial contact, and reduces step length and velocity. Understanding the influence of impaired SMC on gait can inform decisions regarding therapy and surgery, such as hamstring lengthening. LEVEL OF EVIDENCE: Level II Retrospective Study.

Sunlight exposure increases vitamin D sufficiency in growing pigs fed a diet formulated to exceed requirements.

Traditional confinement practices limit exposure to sunlight and vitamin D synthesis, and vitamin insufficiency occurs even with dietary supplementation. The aim of this study was to determine the effect of limited sun exposure on serum concentration of vitamin D and the expression of vitamin D synthesizing enzymes in the liver and kidney of pigs on a vitamin D sufficient diet. White-pigmented grower pigs (29.7 ± 2.3 kg) fed 15% CP diet ad libitum providing >1,200 IU vitamin D3/kg of feed were exposed to sunlight for 1 h each day at solar noon for 14 d at the spring equinox (March pigs, n = 10) or summer solstice (June pigs, n = 5) and again before slaughter in June (March pigs) and September (June pigs). Blood for the analysis of 25(OH)D was collected before and after sunlight exposure. Traditionally housed pigs served as controls. After initial sun exposure, blood samples were collected from June pigs daily for 5 d and weekly for 8 wk to determine vitamin D3 and 25(OH)D decay, respectively. Kidney and liver samples were collected from the June pigs at slaughter after sun exposure for analysis of messenger RNA expression of vitamin D binding protein and synthesizing/degrading enzymes. Average daily gain (ADG) was not influenced (P > 0.5) by sunlight exposure. June pigs had fewer days on feed, lower (P = 0.003) ADG and were slaughtered at a lighter (P < 0.001) weight. Exposure to sunlight increased (P < 0.001) 25(OH) vitamin D for all pigs. March pigs, obtained from a Midwest producer, had lower (P < 0.001) concentration of 25(OH)D than June pigs born on-farm. Initial sunlight exposure increased serum concentration of 25(OH)D in March pigs by 200% and June pigs by 67%. Serum concentration of vitamin D3 was decreased (P < 0.05) by 72 h with 25(OH)D decreased (P < 0.05) by wk 4 after exposure. Expression of vitamin D binding protein, vitamin D synthesizing CYP2R1, CYP27A1, CYP2D25, or degrading enzyme CYP24A1 were not influenced (P ≥ 0.19) by sunlight exposure. Expression of CYP27B1 was decreased (P = 0.04) in the kidney but tended to be increased (P = 0.06) in the liver after sun exposure. These results suggest limited sun exposure can efficiently increase serum concentration of vitamin D in growing pigs with varying levels of vitamin sufficiency. The lack of major changes in vitamin synthesizing enzymes suggests the 14-d exposure period did not saturate the capacity of slaughter-weight pigs to synthesize vitamin D.

Cancer patient survival: Surveillance, Epidemiology, and End Results Program, 1973-79.

Data from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute were used in the evaluation of cancer patient survival experience for almost 10% of the U.S. population. This first report contains actuarial (life table) survival analysis on 368,263 patients with first primary cancer diagnosed in 1973-79 from nine SEER areas: the entire States of Connecticut, Iowa, New Mexico, Utah, and Hawaii and the metropolitan areas of Atlanta, Detroit, San Francisco, and Seattle. Both observed and relative survival rates were shown for whites and blacks, sex, age, primary site, and time period (each yr, 1973-78). The largest racial difference in survival was for cancer of the corpus uteri: 87% 5-year relative survival rate for white females versus only 54% for black females. Survival rates varied considerably by primary site. For most sites, as age increased the relative and observed survival rates decreased. Analysis of relative survival rates by ech year of diagnosis, 1973-78, and for each year following diagnosis suggested increasing rates for cancers of the colon, lung, and prostate gland, whereas there were no definite increases for cancers of the female breast and corpus uteri. A dramatic increase in survival from acute lymphocytic leukemia was found for children under 15 years of age, with the 4-year relative survival rate increasing from 51% among those diagnosed in 1973-75 to 65% for those diagnosed in 1976-78.

Marital status and incidence of ovarian cancer: the U.S. Third National Cancer Survey, 1969--71.

As determined in the U.S. Third National Cancer Survey, 1969--71, the incidence of ovarian cancer was 60-70% higher in women who had never married than in those who had. This relationship was present in both whites and blacks and in all age groups over 25 years. Among ovarian epithelial tumors, those for which the incidence rates between never-married and ever-married women differed most were endometrioid and clear cell tumors. The incidence of tumors of germ cell and sex cordmesenchyme origin, however, showed no relationship to marital status.

Cancer patient survival among ethnic groups in the United States.

Data from the Surveillance, Epidemiology and End Results Program of the National Cancer Institute were used in the evaluation of cancer patient survival experience for 8 racial-ethnic groups in the U.S. population--Anglos, Hispanics, blacks, American Indians, Chinese, Japanese, Filipinos, and Hawaiians. This report contains an actuarial (life table) survival analysis on 402,752 patients with first primary cancer diagnosed in 1973-79 and followed through December 31, 1981. Relative and observed survival rates are shown for each sex separately by primary site. Survival rates for females exceeded those for males for each primary site except for gallbladder and urinary bladder. The primary site having the highest survival rate among each group studied was the thyroid gland with a 5-year relative rate of 91% for all races combined. Rates were uniformly low among each group for cancers of the esophagus, liver, and pancreas. Survival rates for Hispanics were almost identical to those for Anglos; the largest differences were found for bladder (73% for Anglos vs. 64% for Hispanics), Hodgkin's disease (70% for Anglos vs. 61% for Hispanics), and ovary (35% for Anglos vs. 42% for Hispanics). For many primary sites Japanese experienced the highest survival rates and American Indians the lowest. In comparison to Anglos, Japanese had higher survival for cancers of the stomach, colon, prostate gland, and breast. The higher survival for stomach and breast cancers persisted even when controlling for age of patient and stage of disease at diagnosis. Survival rates for blacks were much lower than those for whites for cancers of the corpus uteri and urinary bladder. Survival rates for Chinese and Hawaiians were roughly comparable to those for Anglos, whereas survival rates for Filipinos tended to be similar to those for blacks.

Risk of leukemia associated with the first course of cancer treatment: an analysis of the Surveillance, Epidemiology, and End Results Program experience.

The risk of leukemia associated with the first course of cancer treatment was evaluated in over 440,000 patients diagnosed during 1973-80 (average follow-up = 1.91 yr) from the National Cancer Institute's Surveillance, Epidemiology, and End Results Program. Although the reporting of the first course of therapy probably was incomplete, 34 acute nonlymphocytic leukemias (ANLL) developed compared with 7.6 expected among 70,674 patients known to receive initial chemotherapy [relative risk (RR) = 4.5, 95% confidence interval (Cl) = 3.1-6.3]. Significant ANLL excesses were observed following chemotherapy for breast cancer (RR = 8.1), ovarian cancer (RR = 22.2), and multiple myeloma (RR = 9.5). Patients initially treated with radiation (with no record of chemotherapy) also had a significantly increased ANLL risk; 45 leukemias occurred versus 17.9 expected (RR = 2.5, 95% Cl = 1.8-3.4). In this group, excess ANLL were found following irradiation for uterine corpus cancer (RR = 4.0). Kidney and renal pelvis cancer patients had a twofold leukemia risk (all types) that was unrelated to treatment (RR = 2.2).

Cancer incidence and mortality trends among whites in the United States, 1947-84.

Cancer incidence trends from the late 1940s to 1983-84 were assessed among white residents of five geographic areas (Atlanta, Connecticut, Detroit, Iowa, San Francisco-Oakland) by means of data derived from several National Cancer Institute surveys, the Connecticut Tumor Registry, and the Surveillance, Epidemiology, and End Results Program. Incidence trends were compared with mortality trends for the entire United States and for the same five study areas. This study documented rising incidence and mortality rates for four cancers: lung cancer, melanoma of the skin, multiple myeloma, and non-Hodgkin's lymphomas. Increases in lung cancer continued through the early 1980s, but the rate of increase has been moderating during recent years, particularly among males and at younger ages for whom recent declines are evident. Overall, lung cancer incidence rates increased more than 220 and 400% among males and females, respectively. Although much rarer than lung cancer, melanoma of the skin and multiple myeloma increased greatly until the early 1980s among both males and females. The overall rate of increase in melanoma incidence among males was greater than that for lung cancer, and the rate of increase in multiple myeloma mortality among females was exceeded only by that for lung cancer. Increases of 70-120% were observed for non-Hodgkin's lymphomas. Increases in incidence and mortality rates for pancreatic cancer were apparent during the early years but less conspicuous in recent years. Laryngeal and kidney cancer rates generally increased substantially, although the changes were not remarkable for laryngeal cancer mortality among males and kidney cancer mortality among females. The rates for cancers of the mouth and pharynx increased among females but not males. Prostate, colon, and bladder cancer incidence rates increased more than 65% among males, whereas mortality rates changed only moderately. The incidence of thyroid cancer increased more than 75% among both sexes until the late 1970s, but mortality rates have declined during the period of study. Breast cancer incidence increased 30%, whereas mortality rates remained remarkably constant. The incidence of corpus uteri cancer increased dramatically during the mid-1970s and decreased substantially thereafter; these changes were not reflected in the mortality rates, which continually declined during the entire time period. The incidence of testicular cancer increased more than 90% and that of Hodgkin's disease did not change greatly; however, mortality rates for both cancers declined more than 50% since the late 1960s and early 1970s.(ABSTRACT TRUNCATED AT 400 WORDS)

Incidence of cancer in United States blacks.

Incidence rates for the black population of six Standard Metropolitan Statistical Areas in the United States are examined using data collected in the Third National Cancer Survey, 1969 to 1971. For all sites combined, black males had the highest rates among the four major race-sex groups; black females had the lowest rates. For fourteen common sites accounting for 80% of the cancers among blacks, black/white ratios, survival data, trends between 1935 and 1969, and geographic variation are presented. United States black data adjusted to an African Standard are compared with similar data from Nigeria, Rhodesia, and South Africa.

Memory and the lifespan of human T lymphocytes.

The properties of human CD45RA and CD45R0 T cells are described. CD45R0 cells respond to recall antigens and provide help for B lymphocytes. They produce a wide variety of cytokines including IL-2, IL-4 and IFN-gamma. CD45RA T cells respond poorly to recall antigens and produce mainly IL-2. The phenotype of CD45R0 cells suggests that they may be in cycle and in vivo data shows that they have a short lifespan while CD45RA cells are long lived. The lineage relationship of the two subsets is not clear but in vivo and in vitro evidence suggests bidirectional conversion between CD45RA and CD45R0 phenotypes.

Psychological benefits of Spiritual Baptist "mourning," II: An empirical assessment.

Members of a West Indian Christian sect called the Spiritual Baptists participate in a "mourning" ritual that is characterized by praying, fasting, and the experiencing of dreams and visions while in isolation. Sixteen individuals who underwent mourning were evaluated with the SCL-90-R before and after the ritual. In general, the mourners showed significant improvement on the self-report scales.

In vitro proliferative responses of human peripheral blood mononuclear cells to non-recall antigens.

Primary in vitro proliferative responses of naive T cells to antigens other than superantigens and alloantigens have been little studied. Two tissue culture techniques have been reported which support in vitro antigen priming of T cells. These methods require various degrees of cellular manipulation and culture vessels other than standard microtitre plates. We report here that primary proliferative responses to non-recall antigens can be readily obtained using unselected human PBMC prepared from either adult or cord blood. Cells proliferate whether cultured in 2 ml volumes, 200 microliters microcultures or 20 microliters hanging drops. The variation in the proliferative responses increases as the culture volume is decreased such that considerable errors are apparent when Terasaki culture plates are used. The lowest stimulation indices are also observed in the 20 microliters microcultures. Nevertheless, similar response patterns are noted for the differing culture vessels; generally, proliferative responses reach peak magnitude only after 7 days of culture. The initial concentration of PBMC in culture influences the magnitude of the reactions such that halving the cell numbers frequently leads to greater than 50% reduction in the measured responses. The results of this study indicate that neither a specialised culture vessel nor complex cellular manipulation are required for in vitro priming of T cell immunity. Consequently, this area of immunology should be readily amenable to further study.

Treadmill walking protocol for the diagnostic evaluation and exercise programming of cardiac patients.

This study (1) describes a treadmill walking protocol which has been used safely in over 40,000 tests since 1960, (2) presents normative data on oxygen consumption (VO2) so that it may be determined from work load without direct measurement, and (3) demonstrates simple techniques for early activity prescription or later therapeutic walking programs for patients after myocardial infarction (MI) based on this treadmill test. Normative data are presented on 131 subjects. There were no significant differences in VO2 values at the various work loads dependent on age, gender, fitness level, familiarity with the test procedure, clinical status, or the presence or absence of beta blockade. The protocol was then applied in cardiac rehabilitation of 25 patients. As a low level test, in 2 to 9 minutes patients attained 56 to 83% of age-predicted maximal heart rate. The translation of these data into early activity guidelines is shown. For a walking program, the maximal speed attained at 10% grade on the treadmill when walked on level ground put patients in the target heart rate zone for cardiovascular conditioning.

Effect of polymorphism of the HLA-DPA1 chain on presentation of antigenic peptides.

Human T-cell clones that recognize a peptide from mycobacterial heat shock protein 60 in the context of HLA-DP were found to be sensitive to changes in the DPA1 chain of the restricting element, optimal responses being seen with the combination HLA-DPA1*0201 and HLA-DPB*0301. HLA-DP dimers containing HLA-DPA1*01 were only able to present antigenic peptides to T-cell clones when peptides were present throughout the period of coculture of T cells with antigen presenting cells. In contrast the optimal HLA-DP dimer could also stimulate T-cell clones maximally when incubated with peptides for 1 h and then thoroughly washed. This suggests that the DPA1 polymorphism influenced the strength of binding of antigenic peptides to the HLA-DP dimer. Modeling studies identified amino acid 31 of DPA1 as the polymorphic residue most likely to account for this effect. This is the first demonstration that the relatively limited polymorphism displayed by DPA1 has functional consequences.

Use of a competitive protein-binding assay for adenosine 3':5'-phosphate for the study of bovine corpus luteum adenylate cyclase.

The use of a competitive protein-binding assay for cyclic AMP, utilizing the binding protein purified from bovine adrenal cortex, for the study of adenylate cyclase activity of the washed 600 g sediment of bovine corpus luteum is validated. A specific assay for cyclic AMP could only be achieved by removal of the degradation products of ATP on a precipitate of nascent BaSO4. Simple dilution of the sample before assay was not sufficient to eliminate interference from degradation products of ATP. An observed variability in optimal ATP substrate and tissue concentrations is though to reflect variability in the enzymic profile of the cyclic corpus luteum. Optima with respect to F-, Mg2+ and pH are more clearly defined and are similar to those reported for adenylate cyclase systems for other tissues.

Gel filtration of human urinary immunoreactive luteinizing hormone.

In human urinary concentrates and also in a urinary gonadotrophin standard (2nd IRP-HMG), gel-filtration analysis revealed three main peaks of immunoassayable luteinizing hormone (LH). A similar analysis of LH extracted from human pituitaries showed most of the activity in a peak of larger molecular weight, and only minor fractions in the positions of the urinary peaks. In an extract of normal human serum, analyss showed only one similar peak of large molecular weight, which also emerged before the urinary peaks. During an i.v. infusion of pituitary LH into normal men, the urinary LH activity increased but was still found only in the same three peaks on gel filtration, and all were of a molecular weight smaller than that of the infused material; but a higher proportion of the urinary LH was found in the earliest of these peaks compared with that found before infusion. Conversely, 20-35 h after the i.v. infusion, there was a slightly higher proportion of LH activity in the third peak of smallest molecular weight. These findings suggest that the urinary immunoassayable LH, which is found in three peaks of different molecular weights, is derived from the pituitary or serum LH of higher molecular weight. The changes in the proportions of larger or smaller molecular weight fractions in the urine during and after LH infusion suggest that the earliest peak may be disaggregated serum LH, while the last or smallest molecular weight peak may comprise metabolites of LH.

Thigh injuries in athletes.

The thigh, which consists of the heavily muscled region around the femur, is vulnerable to many types of athletic injury. This review addresses the assessment, prevention, and rehabilitation of both the common thigh injuries associated with participation in sports, such as contusions and myositis ossificans traumatica, and a few relatively uncommon but diagnostically important entities that sports physicians must recognize when an athlete has pain or dysfunction attributable to the thigh. Dividing the thigh into anatomic compartments aids in both differential diagnosis and understanding of the pathomechanics that lead to injury. Rehabilitation is especially emphasized because improper selection of modalities or misuse of exercise can seriously impede or worsen recovery. Conversely, the use of appropriate rehabilitation principles and new concepts in exercise prescription can decrease morbidity and lead to rapid resumption of sports.

Plasma lactate and ventilation thresholds in trained and untrained cyclists.

Six trained male cyclists and six untrained sedentary men were studied to determine whether the plasma lactate threshold (PLT) and ventilation threshold (VT) occur at the same work rate in both fit and unfit populations. The PLT was determined from a marked increase in plasma lactate concentration ([La]) and VT from a nonlinear increase in expired minute ventilation (VE) during incremental leg-cycling tests; work rate was increased 30 W every 2 min until volitional exhaustion. The trained subjects' mean VO2 max (63.8 ml O2 X kg-1 X min-1) and VT (65.8% VO2 max) were significantly higher (P less than 0.05) than the untrained subjects' mean VO2max (35.5 ml O2 X kg-1 X min-1) and VT (51.4% VO2 max). The trained subjects' mean PLT (68.8% VO2 max) and VT did not differ significantly, but the untrained subjects' mean PLT (61.6% VO2 max) was significantly higher than their VT. The trained subjects' mean peak [La] (10.5 mmol X l-1) did not differ significantly from the untrained subjects' mean peak [La] (11.5 mmol X l-1). However, the time of appearance of the peak [La] during passive recovery was inversely related to VO2 max. These results suggest that variance in lactate diffusion and/or removal processes between the trained and untrained subjects may account in part for the different relationships between the VT and PLT in each population.

Neural regulation of glucose 6-phosphate dehydrogenase in rat muscle: effect of denervation and reinnervation.

We tested the hypothesis that glucose 6-phosphate dehydrogenase (G6PD) in rat extensor digitorum longus (EDL) muscle is under neural control by studying changes in G6PD activity in EDL muscles following nerve crush-induced denervation and reinnervation. Changes in G6PD were correlated with choline acetyltransferase activity, as well as with neurological function, muscle weights, and muscle isometric twitch tension. The data show a dramatic increase in G6PD following denervation. The gradual recovery of enzyme activity toward normal levels correlates with the return of functional synaptogenesis manifested by the return of neurological function, choline acetyltransferase, and muscle twitch tension. We conclude, therefore, that muscle G6PD is under neural control. G6PD activity provides a facial biochemical indicator of muscle reinnervation.