Linkage of M-CSF signaling to Mitf, TFE3, and the osteoclast defect in Mitf(mi/mi) mice.

Osteoclasts are multinucleated hematopoietic cells essential for bone resorption. Macrophage colony-stimulating factor (M-CSF) is critical for osteoclast development and function, although its nuclear targets in osteoclasts are largely unknown. Mitf and TFE3 are two closely related helix-loop-helix (HLH) transcription factors previously implicated in osteoclast development and function. We demonstrate that cultured Mitf(mi/mi) osteoclasts are immature, mononuclear, express low levels of TRAP, and fail to mature upon M-CSF stimulation. In addition, M-CSF induces phosphorylation of Mitf and TFE3 via a conserved MAPK consensus site, thereby triggering their recruitment of the coactivator p300. Furthermore, an unphosphorylatable mutant at the MAPK consensus serine is specifically deficient in formation of multinucleated osteoclasts, mimicking the defect in Mitf(mi/mi) mice. These results identify a signaling pathway that appears to coordinate cytokine signaling with the expression of genes vital to osteoclast development.

Contingent neutrality in competing viral populations.

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degrees C. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.

Update on screening for type 2 diabetes. The why, who, how, and what of testing and diagnosing.

Type 2 diabetes can cause serious complications even as it remains undiagnosed. Screening is recommended only in people with risk factors for the disease. The ADA recommends FPG as the test of choice, but RPG is also a practical alternative because it is easier and more convenient. Frequency of rescreening if the first screen result is normal depends on the number of risk factors present. Physicians should realize the importance of a confirmatory test and not base a diagnosis of diabetes on a single value unless the value is so high that the diagnosis is unequivocal.

Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus-thymidine kinase in combination with acyclovir.

Adenovirus expressing herpes simplex virus-thymidine kinase (HSV-TK) sensitizes internal rat glioma cells to radiation in combination with acyclovir (ACV). However, relatively high concentrations of ACV (>10 microM) are required to obtain significant radiosensitization. Serum levels rarely reach more than the lower micromolar range, preventing the full use of this genetic approach to radiosensitize cells in vivo. To better use the lower concentrations of ACV available in sera, we constructed an adenovirus expressing a mutant HSV-TK (HSV-TK(75)) isolated for its approximately 20 times greater sensitivity to ACV than wild-type (wt) HSV-TK. We demonstrate that rat RT2 glioma cells infected with adenovirus AdCMV-TK(75) and exposed to either ACV or ganciclovir become more sensitive to lower concentrations (1-3 microM) of the drugs compared with cells infected with AdCMV-TK(wt), which expresses wt HSV-TK. Most importantly, the RT2 cells become more sensitive to low doses (2-4 Gy) of 60Co radiation than cells infected with an adenovirus expressing wt HSV-TK. This sensitization is accompanied by an increased rate of apoptosis. In summary, we show that infection of rat glioma cells with an adenovirus expressing a mutant HSV-TK sensitizes the cells to low doses of radiation after exposure to ACV at lower concentrations than those required for wt HSV-TK. This finding suggests that this mutant adenovirus may improve the in vivo efficacy of HSV-TK-based cancer gene therapy approaches.

Role of the p38 and MEK-1/2/p42/44 MAP kinase pathways in the differential activation of human immunodeficiency virus gene expression by ultraviolet and ionizing radiation.

Ultraviolet (UV) radiation is a potent activator of human immunodeficiency virus (HIV) gene expression in a HeLa cell clone having stably integrated copies of an HIV cat (cat gene under control of the HIV promoter) reporter construct, whereas ionizing radiation is ineffective. UV-activated HIV gene expression is completely blocked by the specific p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 and by expression of a kinase-inactive p38 mutant that interferes with normal p38 function, suggesting that this stress-activated protein kinase plays an important role in UV-mediated transcriptional activation of HIV. In support of these findings, we show here that Western blot analysis demonstrated rapid and significant activation of p38 MAP kinase by UV. On the other hand, gamma-radiation activated p38 MAP kinase very poorly in HeLa cells at both low and high doses at times (5-30 min) when UV radiation was effective. UV radiation also activated HIV gene expression (< or = 9-fold) in 1G5 Jurkat T-cells stably transfected with a luciferase reporter gene under control of the HIV promoter. In these cells, gamma-radiation stimulated HIV gene expression but to a lesser extent (< or = 3-fold) and with different kinetics than after UV radiation, and this response was obliterated by the incubation of cells with the mitogen-activated protein kinase/Erk kinase (MEK)-1/2 inhibitor PD98059. This result suggests that in these cells signaling in response to gamma-radiation is transduced through the MEK-1/2/p42/44 MAP kinase pathway to increase HIV gene expression. All combined, these results suggest that activation of p38 MAP kinase is necessary for efficient HIV gene expression triggered by DNA damaging agents, and, in a cell type-specific manner, activation of the MEK-1/2/p42/44 MAP kinase pathway is important for triggering a response to gamma-radiation. Thus, it appears as if UV signaling leading to HIV gene expression requires the p38 MAP kinase pathway whereas activation by gamma-radiation requires the MEK-1/2/p42/44 MAP kinase pathway.

Activation of NF-kappa B and p38 MAP kinase is not sufficient for triggering efficient HIV gene expression in response to stress.

Recent studies have established an essential role for p38 MAP kinase in UV activation of human immunodeficiency virus (HIV) gene expression. However, p38 MAP kinase is not involved in activation of NF-kappa B, a key transcriptional activator of HIV gene expression, in response to UV, suggesting that NF-kappa B acts independently of p38 MAP kinase. In this study, we have investigated whether activation of HIV gene expression occurs when p38 MAP kinase and NF-kappa B are activated by separate stress-causing treatments, each relatively specific for activating only one of the factors. Treatment of cells with sorbitol (hyperosmotic shock) strongly activates p38 MAP kinase, whereas the cytokine TNF-alpha is a poor activator of p38 MAP kinase. On the other hand, TNF-alpha is a strong activator of NF-kappa B whereas sorbitol is not. Sorbitol, however, activates AP-1 DNA binding activity in a manner similar to that of UV. Most importantly, both sorbitol and TNF-alpha are poor activators of HIV gene expression in HeLa cells stably transfected with an HIVcat reporter gene, whereas UV elicits a strong response. The combined treatment with UV and hyperosmotic shock produces an additive effect on HIV gene expression, suggesting that these agents activate at least in part by different mechanisms. The combined treatment with sorbitol and TNF-alpha activates p38 and NF-kappa B to levels similar to those with UV, yet only results in 25-30% of the CAT levels elicited by UV. Inhibition of NF-kappa B activation by the protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) prevents UV activation of HIV gene expression, but does not inhibit p38 MAP kinase activation. We conclude that whereas both p38 MAP kinase and NF-kappa B are important for UV activation of HIV gene expression they act independently from each other and activation of both factors is not sufficient for triggering a full HIV gene expression response. Activation of HIV gene expression by UV must therefore involve additional cellular processes, such as those triggered by DNA damage, for generation of a full gene expression response.

Barriers to control of blood glucose in diabetes mellitus.

The purpose of this study was to characterize the barriers to tight control of diabetes mellitus. The study collected data from multiple sources at a primary health care clinic in an academic teaching hospital serving an urban population, including patients' charts, structured interviews with patients, a survey of physicians' general perspectives and beliefs concerning diabetes mellitus, and a physician's structured review of barriers to tight control for individual patients. One hundred thirty-five patients with scheduled appointments were eligible for the study, of whom 94% had had a recent hemoglobin A1C (HbA1C). Seventy-seven of 88 patients (88%) who presented for their appointments consented to the interview, 48 of 50 providers (96%) returned useable surveys, and providers completed individual assessments for 96 patients (71%). Patients had a mean age of 61 years, an average of 7.60 diagnoses, and an average of 8.96 prescriptions. Their diabetes control was less than ideal, with less than 15% having normal or near normal control and almost a quarter having poor control. Correlations of HbA1C with age and show rate were seen. Physicians' assessments showed that motivation and understanding of diabetes and compliance with diet and medications correlated with diabetes control. Neither patient knowledge nor physician knowledge appeared to be a problem, nor did either correlate with diabetes control. The number of barriers to control were many, with over half of the patients having five or more barriers. Tight control of blood glucose is felt to be an important aspect of quality care for diabetic patients. In this study, a representative sample of diabetic patients had less than ideal diabetes control. This population was receiving their care in an urban setting and had many comorbid illnesses and many barriers to control. Age, motivation, understanding of the disease, show rate, and compliance with diet and medications had statistical correlations with diabetes control. In order to improve the quality of care for diabetic patients, barriers to care must be addressed.

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells.

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.

The elderly psychiatric patient with positive syphilis serology: the problem of neurosyphilis.

OBJECTIVE: Older patients have a high prevalence of neurological and psychiatric disorders. They also have a baseline prevalence of late latent syphilis or positive syphilis serology. Thus the clinical question arises as to whether a neuropsychiatric disorder in a geriatric patient is neurosyphilis or if the positive serology is incidental. METHOD: An illustrative case example is used to illustrate this dilemma. The relevant literature is reviewed. RESULTS: The cerebrospinal fluid (CSF) protein is an important indicator of inflammatory activity in the central nervous system and is used as a clinical guide in the diagnosis. Elderly patients have higher values of normal CSF protein than younger patients. CONCLUSIONS: Given the importance of CSF protein in the diagnosis of neurosyphilis, physicians must include this knowledge, that elderly patients have higher CSF protein values, in their clinical decision making in the differentiation between neurosyphilis and late latent syphilis in the elderly patient.

Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3.

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.

Delivery of vaccines to adults: correlations with physician knowledge and patient variables.

Our objective was to explore reasons why vaccines are not provided to adult patients receiving primary health care. The study setting was a primary health care clinic in a urban hospital staffed primarily by residents and teaching faculty. The patients were identified as all continuity care patients with a scheduled visit to the clinic during the 4-week study period in April 1995. The providers were all primary care providers for the patients during the study period. The providers were given two surveys before the study: one to assess their knowledge of published preventive health care guidelines and one to assess their perspective of the guidelines. During the study period, the charts of the patients were reviewed for the services they had received. An assessment was placed in each patient's chart for the provider's completion during the visit. The assessment enabled the provider to explain why services had not been provided. The results showed that influenza, pneumococcal, and diphtheria-tetanus vaccines were provided at varying rates. Each vaccine had a different profile as to noncompliance with guidelines. Lack of provider knowledge of the guideline was most apparent with pneumococcal vaccine. Providers' ambivalence regarding the scientific basis and/or clinical importance was most apparent with influenza vaccine. Patient refusal was a prominent cause with influenza vaccine in the elderly. Patient appointment behavior (opportunity for care and compliance) also seemed to play an important role. We conclude that explanations for nondelivery of vaccines to adults seem to be multiple. Lack of physician knowledge and physician perception of the guidelines provide some explanations. Patient-related factors including refusal, decreased opportunity for care, and noncompliance also play important roles in why vaccinations are not provided. Improvement in the rates at which immunizations to adults are provided will require interventions in multiple areas.

Assessment of preventive health care: design considerations.

OBJECTIVE: To design an instrument to assess the performance of a clinic in the delivery of preventive health services to a general population. METHODS: The study utilized a chart review of services delivered, abstraction of data from electronic databases, and a standardized provider assessment of each eligible patient. The study was conducted in a primary care clinic staffed primarily by internal medicine residents in an urban academic medical center. Patients who were receiving continuity care in the clinic and who were scheduled for an appointment during the 2-week study period were eligible for inclusion. Patients were identified prospectively from the appointment schedule. Charts were reviewed for the delivery of preventive health services prior to the patient's visit. Assessment forms were provided to the primary providers for review and completion. Demographic and appointment information was electronically abstracted from current databases. RESULTS: The rate at which services were provided varied considerably by service and over time. The reasons for nondelivery included disagreement with guidelines, patient resistance/refusal, and lack of priority. CONCLUSIONS: It should be possible to assess a clinic's performance over a range of services over its entire population over time. There may be legitimate reasons for services not being provided to a sizable proportion of the population. These issues are complex and require sensitive detailed investigation.

Assessment of delivery of preventive health services.

Our objective was to design and evaluate an instrument to assess the performance of a clinic in the delivery of preventive health services to a general medical clinic population. The patients were identified prospectively; data were obtained retrospectively with review of the charts. The study was conducted in a primary care clinic staffed primarily by internal medicine residents in an urban academic medical center. Patients who were receiving continuity care in the clinic and who were scheduled for an appointment during the 4-week study period were eligible for inclusion. Patients were identified by the appointment schedule. Charts were reviewed for the delivery of preventive health services. Data were abstracted utilizing a standard instrument. We found that the rate at which services were provided varied considerably by service and over time. The techniques used provided some insight into methods for the evaluation of the delivery of preventive services. It should be possible to assess a clinic's performance over a range of services over its entire population over time. An understanding of this more global performance may provide a better tool for managers and researchers addressing these issues. There may be legitimate reasons for services not being provided. These issues are complex and require sensitive, detailed investigation.

Experience with community academic practice: strategies for the ambulatory education of residents.

The expectation that training programs will place more of their physician graduates into primary care has caused internal medicine programs to explore different educational experiences for their residents. One proposal is to transfer the ambulatory training site for residents from the hospital clinic to a community academic practice (CAP) site. We discuss the theoretical aspects, the practical considerations, and our experience with one CAP site, including the problems that have been solved and the problems remaining.

Enhancing physician adoption of practice guidelines. Dissemination of influenza vaccination guideline using a small-group consensus process.

BACKGROUND: A dissemination intervention to facilitate adoption of a preventive practice guideline (influenza vaccination for older adults) in group practices was developed and evaluated. The intervention, small-group consensus process, started with a physician expert presenting the guideline and followed with the group participating in a structured discussion of ways to implement the guideline that culminated in a public commitment (ie, "buy in") to adopt the guideline. METHODS: Thirteen group practices and their primary care physicians (mean size, 5) were assigned randomly to intervention or control arms. In each group practice, physicians in the intervention arm met for 1 hour. Control physicians participated in an unrelated discussion (non-steroidal drug use). Guideline adoption was determined by changes in physicians' vaccination rates that were obtained through prechart and postchart reviews of 51 physicians. Prequestionnaires and postquestionnaires measured influenza knowledge and prevention attitudes. RESULTS: Using analysis of covariance, the small-group consensus process was found to increase physician vaccination rates by 34% compared with the control arm (F (1,48) = 19.49). All intervention arm physicians increased vaccination rates from before to after compared with 54% of control arm physicians. Attitudes and knowledge did not change and were unrelated to increased vaccination rates. CONCLUSIONS: A case is made for the small-group consensus process as an effective utilization-focused dissemination method. Interventions based on group dynamics and sensitive to local practice contexts can be useful in facilitating adoption of guidelines by physicians in group practices.

Measuring physician mental workload: reliability and validity assessment of a brief instrument.

This study examined the reliability and validity of a brief six-item instrument to measure the mental workload experienced by residents for specific patient visits to an ambulatory care clinic. Participating in the study were twenty-two residents in postgraduate years 1 through 3 who were working in the general outpatient clinic of an inner city, private, nonprofit community hospital. Cronbach's alpha coefficient for the instrument was 0.80. Findings supported several theory-based hypotheses on determinants and performance consequences of mental workload. Mental workload was positively correlated with fatigue (r = 0.42, P = 0.03) and inversely correlated with residents' self-rated experience with patients' problems (r = -0.65, P less than 0.001). Residents' performance was measured through self-ratings and faculty physicians' ratings. Mental workload was inversely correlated with self-rated performance (r = -0.67, P less than 0.001). The correlation of mental workload with faculty physician ratings that reflected the technical dimension of patient care (physician examination, medications, and procedures) was r = -0.38 (P = 0.04). With mental workload squared, the correlation was r = -0.45 (P = 0.02) and the form of the relationship, consistent with the hypothesis, was a slightly downward sloping curve. Limitations of this research are discussed as well as suggestions for further research.

Postmortem management of patient affairs: the resident's perspective.

The role of residents continues to evolve, and they are finding themselves increasingly being asked to request an autopsy from a grieving family. Although much has been written previously concerning the need for residents to be more aggressive in obtaining autopsy, this article suggests that structural problems must be addressed if the current low autopsy rate is to be improved.

The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site-directed mutagenesis.

The analysis of the antigenic surface of staphylococcal nuclease was begun by generating and characterizing a panel of mAb. Twelve mAb were selected from a large number of anti-nuclease mAb and characterized for affinity and isotype, by their ability to block enzyme activity, and by complementation and competitive inhibition assays for the relative location of epitopes. The mAb were placed in complementation groups based on their distinct binding patterns. These groups define a series of eight overlapping epitopes that are estimated to cover a large portion of the nuclease surface. Four mAb blocked the enzyme activity of nuclease. The epitopes defined by two of these four mAb were localized on the surface of nuclease using single amino acid variant Ag generated by site-directed mutagenesis of the cloned nuclease coding sequence. mAb-25 maps to residue 46 which is located at the edge of the enzyme active site consistent with its ability to inhibit enzyme activity. mAb-19, which also blocks enzyme activity and belongs to the same complementation group as mAb-25, was unaffected by the substitution at position 46. This suggests that mAb-19 and mAb-25, if they do react with the same epitope, have differences in fine specificity. mAb-22 blocks enzyme activity and belongs to an overlapping complementation group. The fourth mAb, mAb-1, which belongs to a distinct, nonoverlapping, complementation group, does not blocks enzyme activity, and is directed to a region of nuclease that includes the amino acid at position 133. This residue is located a short distance from the active site in a region that has been suggested to participate in binding of DNA, a substrate for nuclease. Therefore, the four epitopes defined by these mAb are localized at or near the enzyme active site.

A measure of physician mental work load in internal medicine ambulatory care clinics.

Physician mental work load is an important variable intervening between work demands imposed on physicians and physician performance. A brief instrument was developed to measure the mental work load experienced during a clinic session in internal medicine ambulatory care hospital clinics. The instrument covered six dimensions of mental work load: performance, time load, mental effort, physical effort, psychologic stress, and difficulty. Cronbach's alpha reliability coefficient for the instrument was 0.83. The instrument exhibited construct validity. As hypothesized, mental work load was found to be positively associated with number of patients seen and with fatigue, and mental work load was inversely associated with physician satisfaction with the patient care they provided and with their self-rating of the quality of care they provided. The importance of measuring physician mental work load is discussed.