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DEBRA International is undertaking a long-term initiative to develop clinical practice guidelines (CPGs) for epidermolysis bullosa (EB), to -improve the clinical care of people living with EB. Current neonatal care is based on evidence, clinical expertise and trial and error, with collaboration between the EB specialist team, parent or carer and patient, and is dependent on the neonate's individual presentation and type of EB. Early intervention based on research and clinical practice is needed to establish a foundation of knowledge to guide international practitioners to create and improve standards of care and to be able to work effectively with those newly diagnosed with EB. This CPG was created by an international panel with expertise working with persons with EB. The CPG focuses on neonatal care using a systematic review methodology covering four key areas: (i) diagnosis and parental psychosocial support; (ii) hospital management: medical monitoring, wound care and pain; (iii) feeding and nutrition; and (iv) discharge planning and EB education. These four areas highlight the importance of a multidisciplinary team approach, to provide a patient-specific holistic care model that incorporates the needs and wishes of the parents and carers. The Hospital Implementation Tool included promotes transfer of theory to clinical practice., Competing Interests: Conflicts of interest With one exception (K.M.-C.), this panel of researchers claims no financial conflicts of interest. K.M.-C. declared a potential conflict from her professional work coordinating guideline development for DEBRA International between 1 February 2016 and 6 February 2023. This author was therefore not involved in the final editions of the recommendations after review panel feedback. All other panel members and reviewers self-declared no potential conflicts of interest with respect to the publication of this guideline., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Understanding how mutations affect survivability is a key component to knowing how organisms and complex traits evolve. However, most mutations have a minor effect on fitness and these effects are difficult to resolve using traditional molecular techniques. Therefore, there is a dire need for more accurate and precise fitness measurements methods. Here, we measured the fitness effects in Burkholderia cenocepacia HI2424 mutation accumulation (MA) lines using droplet-digital polymerase chain reaction (ddPCR). Overall, the fitness measurements from ddPCR-MA are correlated positively with fitness measurements derived from traditional phenotypic marker assays (r = 0.297, P = 0.05), but showed some differences. First, ddPCR had significantly lower measurement variance in fitness (F = 3.78, P < 2.6 × 10-13) in control experiments. Second, the mean fitness from ddPCR-MA measurements were significantly lower than phenotypic marker assays (-0.0041 vs -0.0071, P = 0.006). Consistent with phenotypic marker assays, ddPCR-MA measurements observed multiple (27/43) lineages that significantly deviated from mean fitness, suggesting that a majority of the mutations are neutral or slightly deleterious and intermixed with a few mutations that have extremely large effects. Of these mutations, we found a significant excess of mutations within DNA excinuclease and Lys R transcriptional regulators that have extreme deleterious and beneficial effects, indicating that modifications to transcription and replication may have a strong effect on organismal fitness. This study demonstrates the power of ddPCR as a ubiquitous method for high-throughput fitness measurements in both DNA- and RNA-based organisms regardless of cell type or physiology., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: To compare flecainide and dronedarone for sinus rhythm (SR) maintenance following electrocardioversion of persistent atrial fibrillation (AF), in patients with minimal or no structural heart disease., Methods and Results: A systematic search of publications using EMBASE, CENTRAL, CINAHL, and MEDLINE (1989-2019), identified a total of 595 articles. No limitations were applied. Nine articles met the inclusion criteria [five randomized controlled trials (RCTs) and four cohort studies], encompassing 1349 persistent AF candidates. Two retrospective studies compared flecainide with dronedarone, indicating a 6% reduced risk of AF recurrence with flecainide; however, results were not statistically significant [risk ratio (RR) 0.94, 95% confidence interval (CI) 0.71-1.24; P = 0.66]. One RCT compared dronedarone to placebo, demonstrating a 28% reduced risk of AF recurrence at 6 months (RR 0.72, 95% CI 0.58-0.90; P = 0.004). Two RCTs compare flecainide to placebo, when a 16% decreased risk of AF recurrence at 6-12 months was indicated; however, these results were not statistically significant (RR 0.84, 95% CI 0.66-1.07; P = 0.16). Within a 6- to 12-month follow-up period, a combined recurrence rate of AF was examined, in which flecainide and dronedarone maintained SR in 50% and 42%, respectively. Four articles satisfied quality appraisal, one of which focused on flecainide data., Conclusion: Dronedarone and flecainide displayed similar efficacy in maintaining SR in patients following electrocardioversion for persistent AF. The SR maintenance was numerically but not statistically significant in the flecainide group. Side effects uncovered similar pro-arrhythmic activity. However, in light of the deficiency of volume and quality of available evidence, the writer acknowledges the requirement for future research., (Published on behalf of the European Society of Cardiology. © The Author(s) 2020.)

Background: The proportion of tumors of various histologies that may respond to drugs targeted to molecular alterations is unknown. NCI-MATCH, a collaboration between ECOG-ACRIN Cancer Research Group and the National Cancer Institute, was initiated to find efficacy signals by matching patients with refractory malignancies to treatment targeted to potential tumor molecular drivers regardless of cancer histology., Methods: Trial development required assumptions about molecular target prevalence, accrual rates, treatment eligibility, and enrollment rates as well as consideration of logistical requirements. Central tumor profiling was performed with an investigational next-generation DNA-targeted sequencing assay of alterations in 143 genes, and protein expression of protein expression of phosphatase and tensin homolog, mutL homolog 1, mutS homolog 2, and RB transcriptional corepressor 1. Treatments were allocated with a validated computational platform (MATCHBOX). A preplanned interim analysis evaluated assumptions and feasibility in this novel trial., Results: At interim analysis, accrual was robust, tumor biopsies were safe (<1% severe events), and profiling success was 87.3%. Actionable molecular alteration frequency met expectations, but assignment and enrollment lagged due to histology exclusions and mismatch of resources to demand. To address this lag, we revised estimates of mutation frequencies, increased screening sample size, added treatments, and improved assay throughput and efficiency (93.9% completion and 14-day turnaround)., Conclusions: The experiences in the design and implementation of the NCI-MATCH trial suggest that profiling from fresh tumor biopsies and assigning treatment can be performed efficiently in a large national network trial. The success of such trials necessitates a broad screening approach and many treatment options easily accessible to patients., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Sexually transmitted infections (STIs) are associated with an increased risk of human immunodeficiency virus (HIV) infection, but their biological effect on HIV susceptibility is not fully understood., Methods: Female pig-tailed macaques inoculated with Chlamydia trachomatis and Trichomonas vaginalis (n = 9) or medium (controls; n = 7) were repeatedly challenged intravaginally with SHIVSF162p3. Virus levels were evaluated by real-time polymerase chain reaction, plasma and genital cytokine levels by Luminex assays, and STI clinical signs by colposcopy., Results: Simian/HIV (SHIV) susceptibility was enhanced in STI-positive macaques (P = .04, by the log-rank test; relative risk, 2.5 [95% confidence interval, 1.1-5.6]). All STI-positive macaques were SHIV infected, whereas 3 controls (43%) remained uninfected. Moreover, relative to STI-negative animals, SHIV infections occurred earlier in the menstrual cycle in STI-positive macaques (P = .01, by the Wilcoxon test). Levels of inflammatory cytokines (interferon γ, interleukin 6, and granulocyte colony-stimulating factor [G-CSF]) were higher in STI-positive macaques during STI inoculation and SHIV exposure periods (P ≤ .05, by the Wilcoxon test)., Conclusions: C. trachomatis and T. vaginalis infection increase the susceptibility to SHIV, likely because of prolonged genital tract inflammation. These novel data demonstrate a biological link between these nonulcerative STIs and the risk of SHIV infection, supporting epidemiological associations of HIV and STIs. This study establishes a macaque model for studies of high-risk HIV transmission and prevention., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: Depot medroxyprogesterone acetate (DMPA) has been linked to human immunodeficiency virus type 1 (HIV-1) acquisition., Methods: Vaginal microbiota of women using DMPA for up to 2 years were cultured. Mucosal immune cell populations were measured by immunohistological staining., Results: Over 12 months, the proportion with H2O2-positive lactobacilli decreased (n = 32; 53% vs 27%; P = .03). Median vaginal CD3(+) cells also decreased (n = 15; 355 vs 237 cells/mm(2); P = .03), as did CD3(+)CCR5(+) cells (195 vs 128 cells/mm(2); P = .04), HLA-DR(+) cells (130 vs 96 cells/mm(2); P = .27), and HLA-DR(+)CCR5(+) cells (18 vs 10 cells/mm(2); P = .33)., Conclusions: DMPA contraception does not increase vaginal mucosal CCR5(+) HIV target cells but does decrease CD3(+) T lymphocytes and vaginal H2O2-producing lactobacilli., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Rey Auditory Verbal Learning Test [RAVLT; Rey, A. (1941). L'examen psychologique dans les cas d'encéphalopathie traumatique. Archives de Psychologie, 28, 21] is a commonly used neuropsychological measure that assesses verbal learning and memory. Normative data have been compiled [Schmidt, M. (1996). Rey Auditory and Verbal Learning Test: A handbook. Los Angeles, CA: Western Psychological Services]. When assessing an individual suspected of neurological dysfunction, useful comparisons include the extent that the patient deviates from healthy peers and also how closely the subject's performance matches those with known brain injury. This study provides the means and S.D.'s of 392 individuals with documented neurological dysfunction [closed head TBI (n=68), neoplasms (n=57), stroke (n=47), Dementia of the Alzheimer's type (n=158), and presurgical epilepsy left seizure focus (n=28), presurgical epilepsy right seizure focus (n=34)] and 122 patients with no known neurological dysfunction and psychiatric complaints. Patients were stratified into three age groups, 16-35, 36-59, and 60-88. Data were provided for trials I-V, List B, immediate recall, 30-min delayed recall, and recognition. Classification characteristics of the RAVLT using [Schmidt, M. (1996). Rey Auditory and Verbal Learning Test: A handbook. Los Angeles, CA: Western Psychological Services] meta-norms found the RAVLT to best distinguish patients suspected of Alzheimer's disease from the psychiatric comparison group.

The Oklahoma Premorbid Intelligence Estimate-3 (OPIE-3) combines Wechsler Adult Intelligence Scale-3rd edition (WAIS-III) subtest raw scores (vocabulary, information, matrix reasoning, and picture completion) and demographic data (i.e., age, education, gender, ethnicity, and region) to predict FSIQ scores. Differences between OPIE-3 estimated FSIQ scores and actual FSIQ scores were compared across 13 age groups in a random sample (N=1201) of the WAIS-III standardization sample. There were mean differences in estimated FSIQ between age groups (P<.01). There was a trend that the OPIE-3 algorithms underestimated FSIQ for individuals 16-17 (2.7 points) and 80-89 years old (3.5 points). However, the differences in estimation errors were small and the percentage of individuals misclassified by more than 10 FSIQ points by age group was similar across groups. The OPIE-3(2ST), OPIE-3MR, and OPIE-3VOC yielded robust estimates of FSIQ across age groups in a neurologically intact sample. Limitations, particularly with individuals aged 16-17 and 85-89 years, are discussed.

Repeated neuropsychological assessments are common with older adults, and the determination of clinically significant change across time is an important issue. Regression-based prediction formulas have been utilized with other patient and healthy control samples to predict follow-up test performance based on initial performance and demographic variables. Comparisons between predicted and observed follow-up performances can assist clinicians in making the determination of change in the individual patient. The current study developed regression-based prediction equations for the twelve subtests of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) in a sample of 223 community dwelling older adults. All algorithms included both initial test performances and demographic variables. These algorithms were then validated on a separate elderly sample (n = 222). Minimal differences were present between Observed and Predicted follow-up scores in the Validation sample, suggesting that the prediction formulas would be useful for practitioners who assess older adults. A case example is presented that utilizes the formulas.

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.

Defining the mode(s) of action by which chemicals induce tumors in laboratory animals has become a key to judgments about the relevance of such tumor data for human risk assessment. Frameworks for analyzing mode of action information appear in recent U.S. EPA and IPCS publications relating to cancer risk assessment. This FORUM paper emphasizes that mode of action analytical frameworks depend on both qualitative and quantitative evaluations of relevant data and information: (1) presenting key events in the animal mode of action, (2) developing a "concordance" table for side-by-side comparison of key events as defined in animal studies with comparable information from human systems, and (3) using data and information from mode of action analyses, as well as information on relative sensitivity and exposure, to make weight-of-evidence judgments about the relevance of animal tumors for human cancer assessments. The paper features a systematic analysis for using mode of action information from animal and human studies, based in part on case examples involving environmental chemicals and pharmaceuticals.

The SeedGenes database (http://www.seedgenes.org) presents molecular and phenotypic information on essential, non-redundant genes of Arabidopsis that give a seed phenotype when disrupted by mutation. Experimental details are synthesized for efficient use by the community and organized into two major sections in the database, one dealing with genes and the other with mutant alleles. The database can be queried for detailed information on a single gene to create a SeedGenes Profile. Queries can also generate lists of genes or mutants that fit specified criteria. The long-term goal is to establish a complete collection of Arabidopsis genes that give a knockout phenotype. This information is needed to focus attention on genes with important cellular functions in a model plant and to assess from a genetic perspective the extent of functional redundancy in the Arabidopsis genome.

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway previously identified. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior.

Effects of a single episode of intercourse on vaginal flora and epithelium were examined in subjects randomly assigned to groups that used no condom or lubricated nonspermicide condoms. Subjects were evaluated at visits before (1 month and 1-2 days) and after (8-12 h, 2-3 days, and 6-8 days) an index episode of sexual intercourse. The 22 subjects who used no condoms had significantly more Escherichia coli and a high concentration (> or =10(5) cfu/mL) of E. coli in the vagina (both, P<.001) and urine (all <10(5) cfu/mL; P=.004) at visit 3 than at visits 1 and 2. The 20 subjects who used condoms had a trend toward more vaginal E. coli (P=.06) and a significant increase in other enteric gram-negative rods (P=.001) after intercourse. Intercourse was not associated with gross, colposcopic, or histologic vaginal epithelial abnormalities.

The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.

The objective of this study was to examine genital tissue, vaginal fluid, and vaginal microbial flora at 3 phases of the menstrual cycle in asymptomatic women. Vaginal examinations were performed 3 times in 74 women: at the menstrual phase (days 1-5), the preovulatory phase (days 7-12), and the postovulatory phase (days 19-24). Flora of 50 women without bacterial vaginosis (BV) was analyzed separately from flora of 24 women with BV. The volume of vaginal discharge increased and the amount of cervical mucus decreased over the menstrual cycle. Among subjects without BV, the rate of recovery of any Lactobacillus changed little (range, 82% to 98%; P = .2); however, a small increase occurred in the rate of recovery of heavy (3+ to 4+ semiquantitative) growth of Lactobacillus over the menstrual cycle (P = .04). A linear decrease occurred in the rate of recovery of heavy growth of any non-Lactobacillus species, from 72% at days 1-5 to 40% at days 19-24 (P = .002). A linear decrease also occurred in the rate of recovery of Prevotella species, from 56% on days 1-5 to 28% on days 19-24 (P =. 007), while a small linear increase occurred in the rate of recovery of Bacteroides fragilis (P=.05). Among subjects with BV, the only significant change was an increase in the rate of recovery of Lactobacillus, from 33% at days 1-5 to 54% at days 19-24 (P = .008). Among all subjects, the rate of recovery of heavy growth of Lactobacillus increased over the menstrual cycle and, in contrast, the concentration of non-Lactobacillus species tended to be higher at menses, which is evidence that the vaginal flora becomes less stable at this time.

A primate model of chlamydial pelvic inflammatory disease was used to characterize serum antibody responses to the 60 kDa chlamydial heat shock protein (CHSP60). Forty monkeys were infected in the fallopian tubes with Chlamydia trachomatis and then were treated. Twenty-three (58%) monkeys developed antibodies against CHSP60, of whom 6 (15%) had CHSP60 responses that persisted throughout the study and 17 (42.5%) had a transient response. A persistent CHSP60 antibody response was correlated with being culture- or ligase chain reaction-positive in the fallopian tubes (P=.004), but not in the cervix pretreatment, and with being tubal-positive posttreatment (P=. 02). Compared with tubal-negative monkeys, tubal-positive monkeys had more intense CHSP60 responses (P=.006) that lasted longer (P=. 002). Among CHSP60 responders, an OD>0.5 was correlated with more severe salpingeal pathology before treatment (P=.04). CHSP60 antibody response may be useful as a marker of persistent chlamydial infection in the fallopian tubes.

Biopsy specimens were obtained from the distal end of the Fallopian tubes of 62 women with tubal infertility and examined by light and electron microscopy. Ciliary beat frequency (CBF) measurements were obtained using laser light spectroscopy. Neither demographic nor behavioural characteristics nor serological evidence of past chlamydial infection were associated with CBF measurements. In contrast, CBF were significantly lower (P < 0.05) in tissues with oedema compared to tissues without oedema (6.7 versus 12.9) and in tissues with erythema compared to tissues without erythema (9.2 versus 13.7). Furthermore, CBF measurements did vary by chlamydial serotype pattern, with lower values observed among the tissues of women with antibodies to serotype C or E (without D) as compared to the tissues of women with other serotypes (P < 0.04). However, these data must be interpreted with caution as the numbers of subjects with chlamydial antibodies to serotype C (n = 3) or E without D (n = 5) were few in number and serotyping of IgG antibodies in blood is not as accurate as it is in bacterial isolates. Confirmation of the suggested association between chlamydial serotype and risk of adverse sequelae could indicate potential new avenues for vaccine research.

Medical care in the United States continues to consume an increasing amount of the Gross Domestic Product. To control the rising costs of health care many industries have turned to a controlled form of financing and delivery of health care--often referred to as managed care. Many types of managed care exist, including preferred provider organizations (PPO), exclusive provider organization (EPO), and health maintenance organizations (HMO). HMOs involve prepaid premiums, limited panels of providers and assumption of financial risk on the part of the providers. A variety of HMOs are currently operating in the United States. Managed care involves taking risks by those who administer it. Some methods of controlling patient and physician behaviour by taking risks are capitation, risk pools and withholds. With capitation the physician is paid a 'per member per month' fee regardless of whether the patient uses the service. Risk pools are concerned with who shares the risk; for example, the primary physician shares the financial risk with specialists. Withholds involve a fee-for-service with a portion withheld which may be returned to the provider if he/she is parsimonious. A concern expressed about HMOs is the possibility of restricted services. Moreover, hospital expenses make up a large portion of the total health care dollar. In 1995 the average length of stay for a Medicare patient was 6.1 days as opposed to 3.9 days for the non-Medicare patient. Indeed, HMOs were the leaders in the development of same-day surgery and out-patient treatment. Increasingly, in the United States, public and social insurance plans are turning to managed care as a method to control health care expenditure. Some government insurance plans, such as Medicare and Medicaid, also increasingly offer managed health options. The trend, for now, in the United States increases enrollment in managed care plans. Although this is occurring at a rapid pace, managed care will probably not be the final solution to provision of medical care in the United States.

To evaluate the effects of antimicrobial and antiinflammatory drugs on oviductal pathology in chronic chlamydial upper genital tract infection, the fallopian tubes of 40 female Macaca nemestrina were inoculated with Chlamydia trachomatis and randomly assigned to treatment with doxycycline (n = 10), doxycycline plus ibuprofen (n = 10), doxycycline plus triamcinolone (n = 10), or placebo (n = 10). Before therapy, all animals were positive for culture or ligase chain reaction (or both), and laparoscopy demonstrated the presence of upper genital tract pathology. After therapy, cervical cultures remained positive in 5 animals given placebo versus 0 given doxycycline alone (P = .03), 0 given doxycycline plus ibuprofen (P = .03), and 1 given doxycycline plus triamcinolone (P = .14). At hysterectomy, neither gross nor histologic pathology was affected by any of the treatment regimens, but immunocytochemistry and in situ hybridization evidence of persistent tubal infection was significantly more frequent among animals given placebo or doxycycline plus antiinflammatory agents than among those given doxycycline alone.

In Kartagener's syndrome (KS), primary defects of the ciliary axoneme cause dyskinetic ciliary motion. Because ciliary motion is an important factor in normal ovum transport, ciliary dyskinesia may cause infertility. On the other hand, the existence of some ciliary activity, albeit abnormal, may account for fertility in some women with KS. In this case study, an infertile woman diagnosed with KS had normal results in all usual infertility tests. Biopsies of tubal mucosa were obtained at laparoscopy for ovum recovery during an in-vitro fertilization cycle. Ciliary activity, measured by laser light-scattering spectroscopy, was detected in all tubal specimens; however the majority of regions sampled showed no activity. In active regions, beat frequency ranged from 5 to 10 Hz, approximately 30% of normal. Electron microscopy showed similar morphological defects in both tubal and nasal mucosa. The number of cilia per cell was approximately 20% of normal. The major ultrastructural abnormality of cilia was an absence of the central microtubules. The only demonstrable explanation for this patient's infertility was primary ciliary dyskinesia associated with KS.

Chlamydia trachomatis infects the upper genital tract of millions of women, causing infertility and pelvic inflammatory disease, yet the inflammatory response to C. trachomatis infection is poorly understood. The cytokine response and the phenotype of infiltrating lymphocytes during C trachomatis infection of fimbria and ampulla autografts in subcutaneous pockets in Macaca nemestrina were characterized. About two-thirds of the infiltrating lymphocytes were CD8 T cells, with the remainder being CD4 T cells and B cells. Interleukin (IL)-2, IL-6, IL-10, interferon-gamma (IFN-gamma), and perforin mRNA were produced by the infiltrating cells, but IL-4 mRNA was absent. The presence of CD8 T cells and perforin mRNA suggest that activated cytolytic T cells are present. The presence of IL-2 and IFN-gamma mRNA and the absence of IL-4 mRNA suggest that Th1-type cytokines predominate during the acute phase of C. trachomatis infection of the upper genital tract.

To further investigate a proposed relationship between Chlamydia pneumoniae and coronary heart disease, coronary atheromas were collected from patients undergoing percutaneous atherectomy. Fifty-eight atheroma specimens were examined by culture and polymerase chain reaction (PCR) and 22 by electron microscopy. All were negative for C. pneumoniae, except a single specimen that was PCR-positive. These results differ from studies in other populations, in which this organism was identified by nonculture methods within coronary atheromas obtained at autopsy. Anti-C. pneumoniae antibody titers from 65 of the patients were compared with those of 28 asymptomatic controls. IgG titers were higher in controls than in patients. There is no evidence that C. pneumoniae exists within atheromas in this study population, nor does seroprevalence correlate with the presence of coronary disease in these patients.

The effects of azithromycin on the infectivity and growth of Chlamydia trachomatis were investigated in primary human amniotic epithelial cells. Infection was prevented when cultures were exposed to the drug 6 h after inoculation and growth was completely inhibited when the drug was added to cultures 24, 48, 72 h or 7 days after infection. The same inhibition was observed at 0.5, 1.0 and 5.0 mg/L. Ultrastructural observations depicted interruption in the growth cycle of the chlamydia and ghost-like envelopes were present in the near empty inclusions. Azithromycin is effective in inhibiting chlamydial growth no matter when treatment is initiated after infection.

Chlamydia pneumoniae TWAR has been associated with coronary heart disease by seroepidemiologic studies and direct detection of the organism in atheromatous lesions of coronary arteries and aorta. In this study, 38 fresh tissue specimens from patients with coronary artery lesions that were treated by directional coronary atherectomy were tested for C. pneumoniae. Twenty-three specimens were from patients with primary lesions and 15 were from patients with restenoses. C. pneumoniae was detected by polymerase chain reaction (PCR), immunocytochemical stain (ICC), or both in 20 of 38 specimens. Using cell identity markers, the organism was localized to macrophages. Ultrastructural evidence of the organism was found in the 2 specimens examined by transmission electron microscopy, which were also positive by both ICC and PCR. C. pneumoniae was found more frequently in tissues from restenoses than in primary lesions (P = .17). There was no relation between the frequency of detection of the organism and C. pneumoniae-specific antibody titers.

The ultrastructural lung pathology of Swiss Webster mice on days 2, 4, 7, 11, 15, and 21 after intranasal inoculation of Chlamydia pneumoniae AR-39 is described. The inflammatory infiltrate was predominantly polymorphonuclear leukocytes on day 2. By day 7, mononuclear cells were most prominent in the infiltrate. On day 2, chlamydial inclusions were found frequently in the bronchial ciliated epithelial cells and less frequently in the interstitial cells that appeared to be macrophages. Free particles of all developmental forms of the chlamydial microorganism were found in the bronchial lumen and alveolar space. These particles were likely organisms released from infected cells. Inclusions as well as free particles were difficult to find after day 4. These ultrastructural observations suggest an immunopathologic basis for the acute phase of the disease process.

The diagnosis of Epstein-Barr virus (EBV)-associated carcinomas is often heralded by high antibody titers to the viral replicative antigens, suggesting EBV reactivation may be a factor in tumor evolution. EBV DNA and nuclear antigen was detected in a newly diagnosed thymic carcinoma. Polymerase chain reaction analysis revealed the presence of a rearranged EBV DNA fragment, BamHI WZhet. This rearrangement is found in a defective EBV that up-regulates the BZLF1 (BamHI Z leftward reading frame) gene product in vitro and induces the EBV lytic cycle. Molecular analysis of the EBV termini demonstrated low levels of the lytic (linear) genomic configuration among a predominantly latent (episomal) population at diagnosis. The episomal populations were of uniform molecular weight at diagnosis and relapse, indicating clonal tumor expansion from a single EBV-infected progenitor. The presence within malignant epithelium of defective virus that can disrupt EBV latency, and perhaps cellular gene regulation, suggests a potential mechanism for EBV reactivation and concurrent malignant progression.

The role of delayed hypersensitivity in the pathogenesis of Chlamydia t trachomatis salpingitis was studied in the monkey "pocket" model. Pigtailed monkeys (Macaca nemestrina) were sensitized by inoculation of live C. trachomatis organisms (E/UW-5/Cx) into subcutaneous pockets containing salpingeal autotransplants. At 21 days, affinity-purified recombinant C. trachomatis heat-shock protein (rhsp60) was injected into pockets either previously sensitized with C. trachomatis or not sensitized in the same monkey. Delayed-type hypersensitivity reaction was observed, characterized by mononuclear cell infiltration with peak reaction at 48 h. Injection of rhsp60 into the pockets of a naive animal did not induce inflammation. This study showed that C. trachomatis infection in monkeys induced delayed hypersensitivity, which is mediated by hsp60. Histologic findings of the salpinx were consistent with delayed hypersensitivity reaction observed in ocular C. trachomatis infection, further suggesting a similar pathogenesis for both salpingitis and trachoma.

Chlamydia pneumoniae is a human respiratory pathogen that causes acute respiratory disease and approximately 10% of community-acquired pneumonia. The infections are geographically widespread. Antibody prevalence studies have shown that virtually everyone is infected with the C. pneumoniae organisms at some time and that reinfection is common. In addition to respiratory disease, seroepidemiologic studies have shown an association of this organism with coronary artery disease. C. pneumoniae was detected in coronary artery atheromas by immunocytochemistry (15/36) and by polymerase chain reaction (PCR) (13/30) in 20 of 36 autopsy cases from Johannesburg, South Africa. Sequence analysis of the C. pneumoniae rRNA genes amplified by PCR confirmed that the amplified gene products were C. pneumoniae. Electron microscopy revealed typical pear-shaped C. pneumoniae elementary bodies in 6 of 21 atheromatous plaques. These findings support the seroepidemiologic studies and offer further evidence that C. pneumoniae may be involved in the atherosclerotic process.

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.

This study was conducted to determine the efficacy of the male mouse as a model for epididymitis caused by human genital serovar E, Chlamydia trachomatis. C. trachomatis was reisolated from all tissues removed on Days 3, 5, and 7 post inoculation (pi). Although some infected epididymides removed on Days 10, 14, and 21 pi were positive, control tissues remained negative. Histopathology of tissues showed a heavy, mixed inflammatory infiltrate consisting of polymorphonuclear cells and lymphocytes. Serum antibody to C. trachomatis was detected in the infected mice only (titer greater than or equal to 1:32). Chlamydial inclusions and individual elementary bodies were confirmed by immunofluorescent and immunoperoxidase staining up to Day 7 pi. These data show that the male mouse is susceptible to C. trachomatis infection and is appropriate for studies dealing with the effect of C. trachomatis on male fertility.

Thirteen Campylobacter-like organisms (CLOs) isolated from rectal cultures from homosexual men were studied. Like catalase-positive Campylobacter species, CLOs were curved gram-negative rods that did not grow aerobically, were motile, were oxidase- and catalase-positive, and did not utilize glucose. However, CLOs could not be classified within any of the Campylobacter species because they grew slowly and had unusual colony morphology; did not grow at 25 C, hydrolyze hippurate, produce H2S in triple sugar-iron agar, or tolerate 2% NaCl; were inhibited by 30-micrograms disks of nalidixic acid; and tolerated 1% glycine and 0.04% triphenyltetrazolium chloride. Three groups of CLOs were identified based on differences in nitrate reduction, growth at 42 C, and sensitivity to cephalothin. By the colony hybridization technique, whole-cell DNA isolated from a strain in each CLO group hybridized with DNA from other strains in the same group, but not with strains in other groups or with reference strains of catalase-positive Campylobacter species.

The effects of setting exercise quotas were examined using 26 subjects with chronic pain. The subjects were participants in a residential treatment program. Each subject was assigned a group of exercises to perform twice daily. A single subject multiple baseline design across responses was used to evaluate treatment effects. The data analysis revealed that the subjects responded to quotas by showing steady and gradual increases in exercise activity. These effects, however, occurred only when quotas were set and were not generalized from "quota" to "nonquota" exercises. These data were discussed in terms of the usefulness of exercise quotas, the applicability of multiple baseline designs, and the need for more research in the area of generalization.

Acute infection with Chlamydia trachomatis serotype E was established in monkey fallopian tube fimbriae by subcutaneous implantation. Depending upon monkey species, from eight to 20 implants could be established in each animal. Animals were given estrogen before percutaneous inoculation of the autografts with Chlamydia. Acute inflammatory changes were found in homografts examined in the first week after infection, with chronic inflammatory changes noted thereafter. Chlamydial inclusions were detected within fimbrial epithelial cells up to seven days postinoculation by fluorescent-antibody staining and immunoperoxidase staining with C. trachomatis-specific monoclonal antibody. Organisms were recovered from autografts up to five days after infection. Analysis of serum antibody by microimmunofluorescence revealed that serotype E-specific antibody of both IgM and IgG classes was produced after infection. We conclude that subcutaneously implanted fallopian tube autografts may provide a useful primate model for kinetic studies of chlamydial infection and immunity.

The murine biovar (mouse pneumonitis) of Chlamydia trachomatis was inoculated into the left oviduct of female Swiss Webster mice to establish acute salpingitis. Chlamydial inclusions were observed in secretory epithelial cells by both transmission electron microscopy and light microscopy using immunoperoxidase staining of deparaffinized sections. By days 5-8 after infection, a mixed polymorphonuclear and mononuclear cellular infiltrate was observed in the submucosa and mucosa. Epithelial cell deterioration occurred in the endosalpinx in areas of heavy mononuclear cellular infiltration. During the acute phase of the disease a cellular infiltrate consisting mainly of T cells was identified by staining frozen tissue sections with monoclonal antibodies to mouse lymphocyte antigens. Occasionally B lymphocytes were observed. Widespread deciliation of the mucosa was observed by scanning electron microscopy. No histopathologic or immunopathologic responses were observed in the control oviducts. These observations suggest an immunologic basis for the structural abnormalities seen in the infected oviducts.

Medullary carcinoma (MC) of the thyroid, in contrast to papillary-follicular carcinoma, fails to concentrate iodine and thus has not been treated with radioactive iodine. We have successfully treated a 16-yr-old Mexican-American girl with residual MC after maximal thyroidectomy (Tx), utilizing radioiodine (131I) to deliver radiation to residual follicular cells in the tumor bed. Immediately after Tx, plasma thyrocalcitonin levels before and during calcium infusion were all elevated (640--1200 pg/ml). 131I (150 mCi) was administered 12 days after Tx after four daily im injections of bovine TSH. Three months after 131I therapy, thyrocalcitonin levels before and during calcium infusion were all normal (less than 50 pg/ml). Ten months after 131I therapy, thyrocalcitonin levels before and after iv pentagastrin were all normal (less than 60 pg/ml). These results suggest that parafollicular cells are radiosensitive, and that therapeutic levels of radiation can be delivered to these cells after Tx if iodine trapping by the remaining follicular cells is enhanced by high levels of circulating TSH. 131I may be the therapy of choice for MC after Tx, if disease has not spread beyond the area proximate to the thyroid gland.

The effects of repeated infections with Chlamydia trachomatis, serovars F, D, and J, were examined in pig-tailed macaques. The fallopian tubes of three experimental monkeys were inoculated at the middle of the menstrual cycle on three consecutive months. Monkey 1 received homologous F; monkey 2 received heterologous F, D, and J; and monkey 3 received homologous inoculations in the right and heterologous inoculations in the left fallopian tubes. One control monkey (4) received repeated inoculations of HeLa cell materials only and remained normal throughout the experiment. Infection was confirmed by isolating the microorganism from both the endosalpinx and endocervix. Antibody to the infecting strains was demonstrated in sera, tears, and cervical secretions by using microimmunofluorescence. Mild chronic salpingitis developed in monkeys 1 and 3, and chronic follicular salpingitis developed in monkey 2. Peritubal and periadnexal scarring and endosalpingeal adhesion formation were produced after reinfection. The right fallopian tube of monkey 3 was distally obstructed (confirmed by hysterosalpingography). Systemic complications, including perihepatitis and conjunctivitis, were also documented in these monkeys. We conclude that repeated infections produced extensive tubal scarring, chronic salpingitis, and distal tubal obstruction, findings not apparent in primary infection.

Experimental acute conjunctivitis was produced in cynomolgus monkeys by ocular inoculation with serovar B of Chlamydia trachomatis. The cellular responses to chlamydial conjunctivitis infection were examined by light, transmission, and scanning electron microscopy. A self-limited, acute conjunctivitis resulted from a single primary inoculation. A moderate lymphocytic infiltrate was present in the conjunctiva. After repeated inoculation, a chronic conjunctivitis (trachoma) developed. Prominent lymphoid follicles with distinct germinal centers were present in these tissues. Scanning electron microscopy revealed patchy areas of cellular alteration and loss of microvilli. Intracellular injury, documented by transmission electron microscopy, included disruption of the plasmalemmal membranes and rupture of the cytoplasmic organelles. The inflammatory infiltrate consisted of plasma cells, polymorphonuclear cells, lymphocytes, eosinophils, and degranulating mast cells. The immune response following single and repeated chlamydial infections was characterized by immunoperoxidase staining with monoclonal antibodies to pan-leukocytes, macrophages, and B cells. The center of the follicle was comprised of B cells, with T cells in the cap region. Large macrophages were also found in the germinal center. Further study is required to determine which cellular mechanisms are involved in the histopathologic and immunologic alterations induced in the conjunctiva after chlamydial infection.