Semi-automated segmentation to assess the lateral meniscus in normal and osteoarthritic knees.

OBJECTIVE: The goal of this study was to develop an algorithm to semi-automatically segment the meniscus in a series of magnetic resonance (MR) images to use for normal knees and those with moderate osteoarthritis (OA). METHOD: The segmentation method was developed then evaluated on 10 baseline MR images obtained from subjects with no evidence, symptoms, or risk factors of knee (OA), and 14 from subjects with established knee OA enrolled in the Osteoarthritis Initiative (OAI). After manually choosing a seed point within the meniscus, a threshold level was calculated through a Gaussian fit model. Under anatomical, intensity, and range constraints, a threshold operation was completed followed by conditional dilation and post-processing. The post-processing operation reevaluates the pixels included and excluded in the area surrounding the meniscus to improve accuracy. The developed method was evaluated for both normal and degenerative menisci by comparing the segmentation algorithm results with manual segmentations from five human readers. RESULTS: The semi-automated segmentation method produces results similar to those of trained observers, with an average similarity index over 0.80 for normal participants and 0.75, 0.67, and 0.64 for participants with established knee OA with Osteoarthritis Research Society International (OARSI) joint space narrowing (JSN) scores of 0, one, and two respectively. CONCLUSION: The semi-automatic segmentation method produced accurate and consistent segmentations of the meniscus when compared to manual segmentations in the assessment of normal menisci in mild to moderate OA. Future studies will examine the change in volume, thickness, and intensity characteristics at different stages of OA.

Developmental toxicity and genotoxicity studies of 1,1,1,3,3,3-hexachloropropane (HCC-230fa) in rats.

The potential developmental toxicity and the in vitro and in vivo genotoxicity of HCC-230fa were assessed. In the developmental toxicity study, groups of 25 mated Crl:CD(R)(SD)BR rats were exposed (whole body) by inhalation to HCC-230fa over days 7-21 of gestation; the day of confirmed mating was designated as gestation day 1 (GD1). Exposures were 6 h per day at concentrations of 0, 0.5, 2.5, or 25 ppm. Body weight, food consumption, and clinical observation data were collected during the study. On day 22 of gestation, the dams were euthanized and examined grossly. The fetuses were removed and subsequently weighed, sexed, and examined for external, visceral, head, and skeletal alterations. Evidence of maternal and developmental toxicity was observed at 25 ppm and was noted as significant, compound-related reductions in mean maternal body weight, weight change, and food consumption. Significant fetal effects also were observed at 25 ppm as compound-related reductions in mean fetal weight and increased fetal malformations (filamentous tail, situs inversus, absent vertebrae) and variations (rudimentary cervical ribs, delayed sternebral ossification). There was no evidence of either maternal or developmental toxicity at 0.5 or 2.5 ppm. The genotoxicity of HCC-230fa was examined in a bacterial reversion assay and in erythrocyte micronucleus studies in two species by different routes of administration. No increases in the number of revertants were observed in the bacterial reversion assay. In one micronucleus study, HCC-230fa was administered by inhalation to rats as part of a 90-day study at doses indicated above. For the second study, ICR mice were given a single ip dose at 0, 166, 330, or 660 mg/kg. In both micronucleus studies, a significant increase in micronucleated erythrocytes was observed. The results of these studies suggest that HCC-230fa affects rapidly dividing cells and may have long-term consequences for occupational exposures.

Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2.

The phenotypes in myotonic dystrophy types 1 and 2 (DM1 and DM2) are similar, suggesting a shared pathophysiologic mechanism. DM1 is caused by expansion of a CTG repeat in the DMPK gene. Pathogenic effects of this mutation are likely to be mediated, at least in part, by the expanded CUG repeat in mutant mRNA. The mutant transcripts are retained in the nucleus in multiple discrete foci. We investigated the possibility that DM2 is also caused by expansion of a CTG repeat or related sequence. Analysis of DNA by repeat expansion detection methods, and RNA by ribonuclease protection, did not show an expanded CTG or CUG repeat in DM2. However, hybridization of muscle sections with fluorescence-labeled CAG-repeat oligonucleotides showed nuclear foci in DM2 similar to those seen in DM1. Nuclear foci were present in all patients with symptomatic DM1 (n = 9) or DM2 (n = 9) but not in any disease controls or healthy subjects (n = 23). The foci were not seen with CUG- or GUC-repeat probes. Foci in DM2 were distinguished from DM1 by lower stability of the probe-target duplex, suggesting that a sequence related to the DM1 CUG expansion accumulates in the DM2 nucleus. Muscleblind proteins, which interact with expanded CUG repeats in vitro, localized to the nuclear foci in both DM1 and DM2. These results support the idea that nuclear accumulation of mutant RNA is pathogenic in DM1, suggest that a similar disease process occurs in DM2, and point to a role for muscleblind in the pathogenesis of both disorders.

Identification of an autoimmune serum containing antibodies against the Barr body.

Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence in situ hybridization with a probe against XIST RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with XIST RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation.

RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase.

Legionella pneumophila replicates within amoebae and macrophages and causes the severe pneumonia Legionnaires' disease. When broth cultures enter the post-exponential growth (PE) phase or experience amino acid limitation, L. pneumophila accumulates the stringent response signal (p)ppGpp and expresses traits likely to promote transmission to a new phagocyte. The hypothesis that a stringent response mechanism regulates L. pneumophila virulence was bolstered by our finding that the avirulent mutant Lp120 contains an internal deletion in the gene encoding the stationary phase sigma factor RpoS. To test directly whether RpoS co-ordinates virulence with stationary phase, isogenic wild-type, rpoS-120 and rpoS null mutant strains were constructed and analysed. PE phase L. pneumophila became cytotoxic by an RpoS-independent pathway, but their sodium sensitivity and maximal expression of flagellin required RpoS. Likewise, full induction of sodium sensitivity by experimentally induced (p)ppGpp synthesis required RpoS. To replicate efficiently in macrophages, L. pneumophila used both RpoS-dependent and -independent pathways. Like those containing the dotA type IV secretory apparatus mutant, phagosomes harbouring either rpoS or dotA rpoS mutants rapidly acquired the late endosomal protein LAMP-1, but not the lysosomal marker Texas red-ovalbumin. Together, the data support a model in which RpoS co-operates with other regulators to induce L. pneumophila virulence in the PE phase.

Subchronic inhalation toxicity of the chlorinated propane 1,1,1,3,3,3-hexachloropropane (HCC-230fa).

Male and female rats were exposed by inhalation (whole body) to HCC-230fa (1,1,1,3,3,3-hexachloropropane) for 6 h/day, 5 days/week over a 15-week period. Concentrations of 0, 0.50, 2.5, and 25 ppm were studied. A total of eight groups/sex were exposed. Four groups of male and four groups of female rats were used to measure clinical signs and growth, clinical pathology, and tissue pathology. The remaining four groups of male rats were used for immunotoxicological and sperm assessment evaluations, and the remaining four groups of female rats were used for immunotoxicological evaluation. Following the exposure period, surviving male rats were kept for a 1- or 3-month recovery period. Male and female rats exposed to 25 ppm had lower mean body weights, mean body weight gains, and food consumption during the exposure period. Male and female rats exposed to 25 ppm and sacrificed immediately after the exposure period had minimally decreased total leukocyte and lymphocyte counts. These changes were considered to be marginally adverse. Pathologic examination revealed hepatocellular hypertrophy in 0-day recovery males and an increased incidence and/or severity in chronic progressive nephropathy in 0-day, 1-month recovery, and 3-month recovery males at 25 ppm. No other pathological changes, including the testis, epididymis, and other accessory sex organs, were noted in rats during the study. Evaluation of sperm parameters at the end of the exposure period showed statistically significant decreases in epididymal sperm number per cauda epididymis, percent motile sperm, and percent normal sperm morphology at 25 ppm. The biological significance of the slight changes observed in the sperm parameters in the absence of histopathological changes is unclear. After a 1-month recovery period, no biologically significant differences in sperm parameters were noted at 25 ppm compared with controls. Exposure to HCC-230fa did not significantly alter the primary humoral immune response to sheep red blood cell (SRBC). Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 2.5 ppm.

Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia.

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice approximately 100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-gamma and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-gamma and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-gamma drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.

Evidence that Dot-dependent and -independent factors isolate the Legionella pneumophila phagosome from the endocytic network in mouse macrophages.

Legionella pneumophila survives within macrophages by evading phagosome-lysosome fusion. To determine whether L. pneumophila resides in an intermediate endosomal compartment or is isolated from the endosomal pathway and to investigate what bacterial factors contribute to establishment of its vacuole, we applied a series of fluorescence microscopy assays. The majority of vacuoles, aged 2.5 min to 4 h containing post-exponential phase (PE) L. pneumophila, appeared to be separate from the endosomal pathway, as judged by the absence of transferrin receptor, LAMP-1, cathepsin D and each of four fluorescent probes used to label the endocytic pathway either before or after infection. In contrast, more than 70% of phagosomes that contained Escherichia coli, polystyrene beads, or exponential phase (E) L. pneumophila matured to phagolysosomes, as judged by co-localization with LAMP-1, cathepsin D and fluorescent endosomal probes. Surprisingly, neither bacterial viability nor the putative Dot/Icm transport complex was absolutely required for vacuole isolation; although phagosomes containing either formalin-killed PE wild-type or live PE dotA or dotB mutant L. pneumophila rapidly accumulated LAMP-1, less than 20% acquired lysosomal cathepsin D or fluorescent endosomal probes. Therefore, a Dot-dependent factor(s) isolates the L. pneumophila phagosome from a LAMP-1-containing compartment, and a formalin-resistant Dot-independent activity inhibits vacuolar accumulation of endocytosed material and delivery to the degradative lysosomes.

Multicenter trial of sentinel node biopsy for breast cancer using both technetium sulfur colloid and isosulfan blue dye.

OBJECTIVE: To determine the factors associated with false-negative results on sentinel node biopsy and sentinel node localization (identification rate) in patients with breast cancer enrolled in a multicenter trial using a combination technique of isosulfan blue with technetium sulfur colloid (Tc99). SUMMARY BACKGROUND DATA: Sentinel node biopsy is a diagnostic test used to detect breast cancer metastases. To test the reliability of this method, a complete lymph node dissection must be performed to determine the false-negative rate. Single-institution series have reported excellent results, although one multicenter trial reported a false-negative rate as high as 29% using radioisotope alone. A multicenter trial was initiated to test combined use of Tc99 and isosulfan blue. METHODS: Investigators (both private-practice and academic surgeons) were recruited after attending a course on the technique of sentinel node biopsy. No investigator participated in a learning trial before entering patients. Tc99 and isosulfan blue were injected into the peritumoral region. RESULTS: Five hundred twenty-nine patients underwent 535 sentinel node biopsy procedures for an overall identification rate in finding a sentinel node of 87% and a false-negative rate of 13%. The identification rate increased and the false-negative rate decreased to 90% and 4.3%, respectively, after investigators had performed more than 30 cases. Univariate analysis of tumor showed the poorest success rate with older patients and inexperienced surgeons. Multivariate analysis identified both age and experience as independent predictors of failure. However, with older patients, inexperienced surgeons, and patients with five or more metastatic axillary nodes, the false-negative rate was consistently greater. CONCLUSIONS: This multicenter trial, from both private practice and academic institutions, is an excellent indicator of the general utility of sentinel node biopsy. It establishes the factors that play an important role (patient age, surgical experience, tumor location) and those that are irrelevant (prior surgery, tumor size, Tc99 timing). This widens the applicability of the technique and identifies factors that require further investigation.

The effect of reading ability and response formats on patients' abilities to respond to a patient satisfaction scale.

This study investigated the relationship between reading ability and patient satisfaction measured with a 5-point Likert scale, a yes/no/uncertain choice, and a pictorial format. The study sample included 48 patients, 16 patients with third to fifth grade reading levels and 32 patients with reading levels of sixth grade or above. The mean percent agreement among the positively worded items on all three scales exceeded 80% regardless of reading level and response format. All of the patients had difficulty with the negatively worded items in all three formats, with the percent agreement dropping to only 49% between the negative items on the yes/no/uncertain and pictorial formats in the lower reading group.

Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles.

After ingestion by macrophages, Legionella pneumophila inhibits acidification and maturation of its phagosome. After a 6-10-h lag period, the bacteria replicate for 10-14 h until macrophage lysis releases dozens of progeny. To examine whether the growth phase of intracellular L. pneumophila determines the fate of its phagosome, interactions between the endosomal network and pathogen vacuoles were analyzed throughout the primary infection period. Surprisingly, as L. pneumophila replicated exponentially, a significant proportion of the vacuoles acquired lysosomal characteristics. By 18 h, 70% contained lysosomal-associated membrane protein 1 (LAMP-1) and 40% contained cathepsin D; 50% of the vacuoles could be labeled by endocytosis, and the pH of this population of vacuoles averaged 5.6. Moreover, L. pneumophila appeared to survive and replicate within lysosomal compartments: vacuoles harboring more than five bacteria also contained LAMP-1, inhibition of vacuole acidification and maturation by bafilomycin A1 inhibited bacterial replication, bacteria within endosomal vacuoles responded to a metabolic inducer by expressing a gfp reporter gene, and replicating bacteria obtained from macrophages, but not broth, were acid resistant. Understanding how L. pneumophila first evades and then exploits the endosomal pathway to replicate within macrophages may reveal the mechanisms governing phagosome maturation, a process also manipulated by Mycobacteria, Leishmania, and Coxiella.

Legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages.

Legionella pneumophila first commanded attention in 1976, when investigators from the Centers for Disease Control and Prevention identified it as the culprit in a massive outbreak of pneumonia that struck individuals attending an American Legion convention (). It is now clear that this gram-negative bacterium flourishes naturally in fresh water as a parasite of amoebae, but it can also replicate within alveolar macrophages. L. pneumophila pathogenesis is discussed using the following model as a framework. When ingested by phagocytes, stationary-phase L. pneumophila bacteria establish phagosomes which are completely isolated from the endosomal pathway but are surrounded by endoplasmic reticulum. Within this protected vacuole, L. pneumophila converts to a replicative form that is acid tolerant but no longer expresses several virulence traits, including factors that block membrane fusion. As a consequence, the pathogen vacuoles merge with lysosomes, which provide a nutrient-rich replication niche. Once the amino acid supply is depleted, progeny accumulate the second messenger guanosine 3',5'-bispyrophosphate (ppGpp), which coordinates entry into the stationary phase with expression of traits that promote transmission to a new phagocyte. A number of factors contribute to L. pneumophila virulence, including type II and type IV secretion systems, a pore-forming toxin, type IV pili, flagella, and numerous other factors currently under investigation. Because of its resemblance to certain aspects of Mycobacterium, Toxoplasma, Leishmania, and Coxiella pathogenesis, a detailed description of the mechanism used by L. pneumophila to manipulate and exploit phagocyte membrane traffic may suggest novel strategies for treating a variety of infectious diseases. Knowledge of L. pneumophila ecology may also inform efforts to combat the emergence of new opportunistic macrophage pathogens.

Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy.

Myotonic dystrophy (DM1) is an autosomal dominant neuromuscular disorder associated with a (CTG)(n) expansion in the 3'-untranslated region of the DM1 protein kinase (DMPK) gene. To explain disease pathogenesis, the RNA dominance model proposes that the DM1 mutation produces a gain-of-function at the RNA level in which CUG repeats form RNA hairpins that sequester nuclear factors required for proper muscle development and maintenance. Here, we identify the triplet repeat expansion (EXP) RNA-binding proteins as candidate sequestered factors. As predicted by the RNA dominance model, binding of the EXP proteins is specific for dsCUG RNAs and proportional to the size of the triplet repeat expansion. Remarkably, the EXP proteins are homologous to the Drosophila muscleblind proteins required for terminal differentiation of muscle and photoreceptor cells. EXP expression is also activated during mammalian myoblast differentiation, but the EXP proteins accumulate in nuclear foci in DM1 cells. We propose that DM1 disease is caused by aberrant recruitment of the EXP proteins to the DMPK transcript (CUG)(n) expansion.

Two-generation reproduction and developmental neurotoxicity study with sodium chlorite in the rat.

The potential for sodium chlorite to produce reproductive toxicity, developmental neurotoxicity and alterations in hematology and thyroid hormones was evaluated in Sprague-Dawley rats administered sodium chlorite in the drinking water continuously for two generations. The F(0) generation animals (30 of each gender per group) and F(1) generation animals (25 of each gender per group) selected to rear the F(2) generation were allowed free access to drinking water containing 0, 35, 70 or 300 ppm sodium chlorite for a 10-week prebreed period, through mating for males and through mating, gestation and lactation for females. These drinking water concentrations corresponded to sodium chlorite doses of approximately 4, 8 and 30 mg kg(-1) day(-1) for males and 5, 10 and 39 mg kg(-1) day(-1) for females, respectively. Evaluations included standard reproductive and postnatal indices, sperm morphology and motility, estrous cyclicity, a functional observational battery, motor activity, auditory startle, swim maze, hematology, serum thyroid hormone analyses and histopathology of reproductive and nervous system tissues. Sodium chlorite resulted in a decrease in water consumption in all groups and a decrease in food consumption and body weights in the 70 and 300 ppm groups. There was no evidence of reproductive toxicity. Pup body weight was decreased in the 300 ppm group and small delays were observed in the time to preputial separation and vaginal opening. Mild anemia and mild methemoglobinemia were observed for animals in the 300 ppm group. Thyroid hormone levels were not affected by treatment. Changes to the nervous system were limited to small decreases in amplitude of auditory startle response for postnatal day (PND) 25 pups in the 70 and 300 ppm groups and a small decrease in absolute brain weight for PND 11 pups in the 300 ppm group. These effects were considered to be of questionable neurotoxicological significance. Based on the results of this study, the no-observed-effect level (NOEL) for effects on reproduction and thyroid hormones is 300 ppm. The no-observed-adverse-effect levels (NOAEL) for hematological toxicity and neurotoxicity are considered to be 70 and 300 ppm, respectively.

Anterior thoracotomy wound complications in minimally invasive direct coronary artery bypass.

BACKGROUND: The minimally invasive anterior thoracotomy for beating heart coronary bypass offers a modest 10-cm incision and avoids the morbidity of extracorporeal circulation. This study examines minimally invasive direct coronary artery bypass (MIDCAB) wound complications and contributing comorbid factors. METHODS: A retrospective, single-institution review of 165 consecutive MIDCAB cases performed between March 1996 and August 1999 examined all wound abnormalities. Two surgeons performed all cases. RESULTS: Wound complications occurred in 15 patients (9.1%), including three (1.8%) incisional hernias, four (2.4%) superficial dehiscences, three (1.8%) wound infections, three (1.8%) chronic pain syndromes, and two (1.2%) seromas. Two patients with incisional hernias required operative repair. The remaining wound abnormalities responded to conservative therapy. Two chronic pain syndrome cases resolved spontaneously, but the third required advanced pain management. In contrast to MIDCAB, the sternotomy wound complications proved significantly less prevalent (n = 5259, 1.1% vs 9.1%, p < 0.005). CONCLUSIONS: Although MIDCAB offers several advantages over standard approaches, these data suggest that anterior thoracotomy wound complications are not insignificant and may be underestimated by those exploring minimally invasive options.

HuR binding to cytoplasmic mRNA is perturbed by heat shock.

AU-rich elements (AREs) located in the 3' untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)(+) RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12-15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)(+) RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.

Effect of Morphine and incision length on bowel function after colectomy.

PURPOSE: Return of bowel function remains the rate-limiting factor in shortening postoperative hospitalization of patients with colectomies. Narcotics are most commonly used in the management of postoperative pain, even though they are known to affect gut motility. Narcotic use has been felt to be proportional to the length of the abdominal incision. The aim of this study was to determine whether return of bowel function after colectomy is directly related to narcotic use and to evaluate the effect of incision length on postoperative ileus. METHODS: A prospective evaluation of 40 patients who underwent uncomplicated, predominantly left colon and rectal resections was performed. Morphine administered by patient controlled analgesia was the sole postoperative analgesic. The amount of morphine used before the first audible bowel sounds, first passage of flatus and bowel movement, and incision length were recorded. Spearman correlation coefficients were calculated between all variables. RESULTS: The strongest correlation was between time to return of bowel sounds and amount of morphine administered (r = 0.74; P = 0.001). There were also significant correlations between morphine use and time to report of first flatus (r = 0.47; P = 0.003) and time to bowel movement (r = 0.48; P = 0.002). There was no relationship between incision length and morphine use or incision length and return of bowel function in the total group. CONCLUSIONS: Return of bowel sounds, reflecting small-intestine motility after colectomy, correlated strongly with the amount of morphine used. Similarly, total morphine use adversely affects colonic motility. Because no relationship with incision length was found, efforts to optimize the care of patients with colectomies should be directed less toward minimizing abdominal incisions and more toward diminishing use of postoperative narcotics.

Sustained CD4+ T cell response after virologic failure of protease inhibitor-based regimens in patients with human immunodeficiency virus infection.

The relationship between plasma human immunodeficiency virus (HIV) RNA levels and peripheral CD4+ T cell counts was examined in 380 HIV-infected adults receiving long-term protease inhibitor therapy. Patients experiencing virologic failure (persistent HIV RNA >500 copies RNA/mL) generally had CD4+ T cell counts that remained greater than pretherapy baseline levels, at least through 96 weeks of follow-up. The CD4+ T cell response was directly and independently related to degree of viral suppression below the pretreatment baseline. For any given HIV RNA level measured 12 weeks after virologic failure, subsequent CD4+ T cell decline was slower in patients receiving a protease inhibitor-based regimen than in a historical control group of untreated patients. These observations suggest that transient or partial declines in plasma HIV RNA levels can have sustained effects on CD4+ T cell levels.

A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II.

Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.