A new model of short acceleration-based training improves exercise performance in old mice.

In order to identify a more appealing exercise strategy for the elderly, we studied a mouse model to determine whether a less time-consuming training program would improve exercise performance, enzyme activities, mitochondrial respiration, and metabolomic parameters. We compared the effects of short-session (acceleration-based) training with those of long-session endurance training in 23-month-old mice. The short-session training consisted of five acceleration-based treadmill running sessions over 2 weeks (the acceleration group), whereas the endurance training consisted of five-one-hour treadmill sessions per week for 4 weeks (the endurance group). A control group of mice was also studied. In the acceleration group, the post-training maximum running speed and time to exhaustion were significantly improved, relative to pretraining values (+8% for speed, P<.05; +10% for time to exhaustion, P<.01). The post-training maximum running speed was higher in the acceleration group than in the endurance group (by 23%; P<.001) and in the control group (by 15%; P<.05). In skeletal muscle samples, the enzymatic activities of citrate synthase, lactate dehydrogenase, and creatine kinase were significantly higher in the acceleration group than in the endurance group. Furthermore, mitochondrial respiratory activity in the gastrocnemius was higher in the acceleration group than in the control group. A metabolomic urine analysis revealed a higher mean taurine concentration and a lower mean branched amino acid concentration in the acceleration group. In old mice, acceleration-based training appears to be an efficient way of increasing performance by improving both aerobic and anaerobic metabolism, and possibly by enhancing antioxidant defenses and maintaining muscle protein balance.

Molecular evidence of field cancerization in a patient with 7 tumors of the aerodigestive tract.

Exposure of the mucosa of the upper aerodigestive tract to carcinogens can induce genetic changes resulting in various independent clones of neoplastic growth, a concept defined as "field cancerization." The risk of developing multiple tumors in this compartment of the body is well established. We studied 6 distinct tumors of the upper aerodigestive tract of a single patient for loss of heterozygosity (LOH), microsatellite instability (MSI), p53 mutations, and K-ras codon 12 point mutations. We detected a unique pattern of LOH and p53 mutations in all 6 tumors. No tumor showed a K-ras mutation or MSI. The results support the mechanism of "field cancerization" and illustrate the potential power of molecular techniques to elucidate pathogenesis.

Alterations of the p53 tumor-suppressor gene and K-ras oncogene in perihilar cholangiocarcinomas from a high-incidence area.

We observed a clustering of cholangiocarcinoma in a part of West Virginia. We analyzed the frequency and type of alterations in the p53 tumor-suppressor gene and the K-ras oncogene to determine whether cholangiocarcinomas from this high-incidence area differ from other cholangiocarcinomas at the molecular level. We studied 12 carcinomas of patients from the high-incidence area (West Virginia group), and 15 carcinomas of patients from nearby states (non-West Virginia group). Over-expression of the p53 gene product, accompanying most mutations in the p53 gene, was determined by immunohistochemistry. p53 sequence analysis of exons 5, 6, 7, and 8 of the p53-immunohistochemical-positive carcinomas was also performed. K-ras codon 12 mutations were detected by the polymerase chain reaction and allele-specific oligonucleotide hybridization. Significantly more cholangiocarcinomas from the West Virginia group were p53-immunohistochemical-positive than from the non-West Virginia group (67% vs. 20%; p < 0.05). p53 mutations did not differ in the 2 groups in respect to site or specific type. No differences were found between the 2 groups regarding K-ras mutations (17% vs. 27%). Although the higher frequency of p53-immunohistochemical positivity in the West Virginia group may reflect a different etiology of these cholangiocarcinomas, explaining the high incidence in this area, results of p53 sequence analysis were not different in the West Virginia group. The high incidence may be explained by difference in carcinogenic dose or a different etiology not reflected in p53 or K-ras alterations.

p53 alterations in atypical alveolar hyperplasia of the human lung.

Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. We have previously shown that AAHs are neoplastic epithelial proliferations that often harbor activating mutations of the K-ras oncogene. In the current study, we examined a spectrum of AAHs to determine the frequency and timing of p53 alterations in lung tumorigenesis. We analyzed 37 AAHs and their paired overt lung neoplasms for p53 protein accumulation using the monoclonal antibody DO7. DNA sequence analysis of the p53 gene was performed on those cases demonstrating p53 protein accumulation. AAHs were classified as low-grade, high-grade, or AAH-like carcinoma based on cytoarchitectural features. Accumulation of the p53 protein was found in none (0%) of 20 low-grade AAHs, in 1 (9%) of 11 high-grade AAHs, and in three (50%) of six AAH-like carcinomas. There was a statistically significant trend toward p53 accumulation with increasing grade of the AAHs. A missense mutation in exon 7 of the p53 gene was found in 1 AAH-like carcinoma, whereas mutations in exons 5 through 8 could not be detected in the other three AAHs with p53 protein accumulation. Three of the paired overt carcinomas harbored p53 mutations that were not present in the AAHs. Alterations of p53 do not appear to be common events in AAHs, especially when these lesions exhibit low-grade cytoarchitectural features. Alterations of p53, however, are more frequent at the level of AAH-like carcinoma and may be associated with the transition from a benign to a malignant proliferation of pneumocytes.

Mutational analysis of the P16-binding domain of cyclin-dependent kinase 4 in tumors in the head region of the pancreas.

Alterations in genes involved in cell cycle regulation are common in many tumor types. In pancreatic adenocarcinomas, inactivating mutations in the CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, are frequently observed. CDKN2 mutations have also been identified in the germline of 50% of patients with hereditary melanoma. Interestingly, such patients also have an increased risk for pancreatic cancers. In melanoma-prone kindreds with CDKN2 wild-type status, a mutation in one of the targets of p16, cyclin-dependent kinase 4 (CDK4) was reported, which abolishes p16 inhibition. To test the possible involvement of CDK4 mutations in pancreatic carcinoma, we analyzed sequence alterations in the p16-binding domain of CDK4 in DNA isolated from 32 tumors in the head region of the pancreas. Alterations in the CDK4 region between codon 1 and codon 56 were not observed in any of the tumors. Our results do not support disruption of the p16 pathway through CDK4 mutation as an oncogenic mechanism in pancreatic head tumorigenesis.

Multiple hyperplastic polyps in the stomach: evidence for clonality and neoplastic potential.

The origin and neoplastic potential of gastric epithelial polyps remains an area of great interest, and treatment choices are a topic of controversy. This report describes a patient diagnosed with three concurrent hyperplastic gastric polyps that were studied for genetic alterations. The polyps were investigated for alterations in the K-ras oncogene and the p53 tumor suppressor gene and for p21WAF1/Cip1 and MDM2 protein overexpression. In addition, loss of heterozygosity at several loci that are frequently involved in human cancer was analyzed, microsatellite instability, a hallmark of the "mutator" phenotype, was determined, and Epstein-Barr virus infection was investigated. All separate areas from the three independent polyps harbored the same activating point mutation in codon 12 of the K-ras oncogene, indicating a clonal origin. DNA sequence alterations in p53 were not found, although high p53 protein levels could be shown by immunohistochemistry in areas of carcinoma within the largest polyp. No alterations in any of the other molecular markers were observed. The results strongly favor a clonal origin of the three independent gastric polyps and support the notion that these hyperplastic polyps may carry a risk for malignancy.

Potential false-positive results with antigen enhancement for immunohistochemistry of the p53 gene product in colorectal neoplasms.

Immunohistochemistry (IHC) for intranuclear p53 gene product is a simple, routine alternative to molecular genetic analysis of the p53 gene. Several methods for antigen enhancement are currently in use for IHC. This study evaluates the effect of extreme antigen enhancement for p53, using a monoclonal antibody (DO7) and a polyclonal antibody (CM1). The cases studied were five colorectal carcinomas, two specimens of normal colorectal mucosa, and four colorectal carcinomas with genetic alterations which are expected to preclude p53 gene product expression, namely mutation to a STOP codon in the p53 gene detected by denaturing gradient gel electrophoresis with subsequent sequencing and allelic loss of 17p in the region where p53 is located, detected by restriction fragment length polymorphism analysis. The findings suggest that extreme antigen enhancement may cause false-positive results with a distinct nuclear staining pattern when MAb DO7 is used as a primary antibody. It is concluded that all antigen enhancement methods should be thoroughly tested to evaluate their validity and that there may be a limit to the extent to which antigen enhancement can be applied in IHC for p53 protein.

Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pancreatic carcinoma.

Mutations in the K-ras oncogene and in the p53 tumor suppressor gene are commonly identified in sporadic cases of pancreatic adenocarcinoma. Although these genes might serve as useful markers for early diagnosis of pancreatic carcinoma in patients at risk for the development of this disease, familial pancreatic carcinomas have not been studied for these mutations. We recently had the opportunity to examine a pancreas prophylactically removed from a patient with a strong family history of pancreatic carcinoma. This gave us the unique opportunity to study the early events in the development of familial adenocarcinoma of the pancreas. Histopathological examination of the pancreas revealed multifocal papillary and nonpapillary mucinous duct hyperplasia. Seven of these foci were microdissected and analyzed for K-ras and p53 mutations. The K-ras mutations were detected by combined mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. Five of the seven duct lesions harbored activating point mutations in codon 12 of K-ras; a G to A transition was found in four and a G to C transversion in one. In contrast, these lesions did not harbor detectable p53 mutations as determined by denaturing gradient gel electrophoresis of exons 5 to 8, nor was there overexpression of the p53 protein as determined by immunohistochemistry. These findings suggest that mutations in K-ras represent an early event in the pathogenesis of pancreatic carcinoma. In addition, monitoring of patients with a strong family history of pancreatic carcinoma for K-ras mutations may identify patients at risk for the development of invasive carcinoma.

Extrapulmonary pneumocystosis: clinical features in human immunodeficiency virus infection.

Pneumocystis carinii infection is reported with increasing frequency as a cause of disease outside of the respiratory tract in patients with human immunodeficiency virus (HIV) infection. Extrapulmonary pneumocystosis is not limited to patients in any discrete risk group for HIV infection. Patients with HIV infection who develop extrapulmonary pneumocystosis frequently do not have concurrent P. carinii pneumonia. Signs and symptoms of extrapulmonary pneumocystosis are nonspecific but when present are frequently referable to the tissues or organs involved. Extrapulmonary pneumocystosis can be diagnosed by examination of tissue biopsies from affected sites using standard histologic techniques. Therapy with antimicrobial agents used to treat P. carinii pneumonia has been effective in some patients. An association between use of aerosolized pentamidine for prevention of P. carinii pneumonia and development of extrapulmonary pneumocystosis has been suggested but remains unconfirmed. Other factors such as the use of zidovudine and duration of immunodeficiency may also be important to the pathogenesis of extrapulmonary pneumocystosis. Further studies are needed to better identify risk factors that may predispose patients to the development of extrapulmonary pneumocystosis.

Sensitivity of treponemal tests for detecting prior treated syphilis during human immunodeficiency virus infection.

To evaluate the sensitivity of treponemal tests as a marker of prior syphilis in individuals with human immunodeficiency virus (HIV) infection, the syphilis serology of 109 homosexual men with a documented history of treated syphilis was compared with records of prior results and confirmed on stored serum samples. None of the HIV-seronegative individuals lost reactivity to a treponemal test, whereas 7% of the seropositive asymptomatic individuals and 38% of those with symptomatic HIV infection had loss of reactivity. Symptomatic HIV infection was associated with loss of reactivity, as a T4 lymphocyte count less than 200 X 10(6)/1, a T4-to-T8 ratio less than 0.6, a single prior episode of syphilis, and a low VDRL titer at the time of the last documented episode of syphilis. Although no conclusions can be drawn about the sensitivity of treponemal tests in patients with active syphilis and HIV infection, these data suggest that treponemal tests may not identify those previously infected with Treponema pallidum.

A survey of adolescents with epilepsy.

Thirty-four adolescents with epilepsy, controls matched for age and sex (A) and controls matched for age, sex and general ability (B), were studied. The adolescents with epilepsy were more likely to arrive at school by car or taxi and to have more difficult behaviour in class. Competitive sports were less popular with them and significantly fewer anticipated ever driving a car. Illness and parental marital problems were not a feature of their families. Their comprehension of reading material was significantly poorer than that of control group A. Within the group, the lowest over-all reading scores were found in children with myoclonic seizures, partial seizures with secondary generalisation, or generalised tonic-clonic seizures; and in those whose EEG findings included two-per-second spike and wave, photosensitivity, generalised slow waves, or generalised spike and wave of non-specific frequency. Right focal slow waves, sharp waves and spikes on EEG were associated with problems of comprehension, even when the over-all reading score was acceptable.

Sequential serological responses to Aspergillus fumigatus in patients with cystic fibrosis. Use of antigen 'stretching' to delineate IgG and IgE activity.

Immunogens from Aspergillus fumigatus were fractionated on the basis of molecular weight. Nine fractions ranging from 900 to 10 kDa were used in ELISA and in a radioallergosorbent test (RAST) with sera from cases of allergic bronchopulmonary aspergillosis (ABPA) and from cystic fibrosis (CF) patients with ABPA or other Aspergillus involvement and compared with control subjects. The profile of IgG reactivity to the nine fractions did not vary substantially for all Aspergillus-involved groups producing peaks at greater than 900 kD and 170 kD whereas the profile for control subjects had a peak at greater than 900 kD only. The IgE profile for CF patients with ABPA did not differ from the profile of the RAST-positive CF patients without ABPA and provided only one peak of activity at 24 kD. Recovery from an episode of ABPA in CF patients was accompanied by a fall in both IgG and IgE antibody levels to all nine fractions, whereas increases in IgG and IgE to all fractions were seen during an episode of ABPA. Although there was an exaggerated IgG increase to antigens in the 43-170 kD range during ABPA, a meaningful increase was also observed to unfractionated A. fumigatus antigen preparations. With IgE in one detailed study the 24-kD fraction provided a better indication of Aspergillus involvement than the unfractionated A. fumigatus antigens. Sequential studies of IgG and IgE levels were not able to predict an episode of ABPA but were useful in conjunction with clinical assessment in following the course of the illness.

Pulmonary diagnostic evaluation in patients suspected of having an HIV-related disease.

The wide range of pulmonary infections associated with human immunodeficiency virus (HIV)-seropositivity, coupled with the possible presence of HIV-related neoplasms and non-HIV-related pulmonary processes, requires that most patients who are known to be or suspected of being HIV-seropositive should undergo a thorough diagnostic evaluation. This usually should include a screening evaluation that includes history, physical examination, screening laboratory tests, chest radiography, and arterial blood gas measurements. Pulmonary function testing and lung scanning also may be sensitive in screening, although these and the other procedures are nonspecific. Specific infectious organisms, particularly Pneumocystis carinii, may be identified through examination of induced sputum and by bronchoscopic procedures, including bronchoalveolar lavage and transbronchial biopsy. Open lung biopsy is not a routine part of the diagnostic evaluation. Although detection of recurrent episodes of pneumonia due to P carinii is difficult, empiric diagnosis and treatment of this disorder should be employed primarily when specific diagnostic techniques are unavailable or are refused.

Diagnosis of pulmonary diseases.

A broad spectrum of lung disease occurs in association with HIV infection. Included are both infectious and neoplastic processes and idiopathic disorders. To insure prompt, accurate, and efficient diagnosis, a logical, staged sequence of tests should be applied. Chest films and, in some instances, pulmonary function tests and gallium-67 citrate lung scans serve to provide objective indications of lung disease. Each of these tests is sensitive but nonspecific. Specific infecting organisms, particularly P. carinii, can be identified by examining sputum induced by inhalation of 3 per cent saline. Bronchoscopic procedures, including BAL and TBB, are highly sensitive and should be performed in patients having nondiagnostic sputum examinations. Tests involving antigen and antibody detection are of little use in the evaluation of individual patients. Detection of recurrent episodes of PCP is difficult because abnormalities in the usual screening tests may be residual from previous episodes. Finding P. carinii in sputum or bronchoscopic specimens soon (within 2 to 3 months) after a confirmed episode of PCP likely represents residual organisms rather than recrudescence of the infection. Empiric diagnosis of P. carinii should be employed only in limited circumstances when specific diagnostic studies are not available, are contraindicated, or are refused.

Juvenile myoclonic epilepsy.

The clinical and electroencephalographic features of 10 adolescents with juvenile myoclonic epilepsy are presented. The mean age on onset was 12.3 years. Myoclonic jerks, predominantly on awakening, occurred in all 10 and were associated with infrequent generalised tonic-clonic seizures in nine. Five had first degree relatives with seizures. The neurodevelopmental status was normal in eight and social integration good in seven. Waking interictal electroencephalograms showed normal background activity in nine, polyspike and wave in six, and single spike and wave in eight. Four were photosensitive. Failure to respond to other antiepileptic drugs was usual, but valproate monotherapy resulted in good or complete seizure control. Juvenile myoclonic epilepsy is a well defined clinical entity that responds well to valproate and is usually associated with a good outlook.