HIV-associated gut dysbiosis is independent of sexual practice and correlates with noncommunicable diseases.

Loss of gut mucosal integrity and an aberrant gut microbiota are proposed mechanisms contributing to chronic inflammation and increased morbidity and mortality during antiretroviral-treated HIV disease. Sexual practice has recently been uncovered as a major source of microbiota variation, potentially confounding prior observations of gut microbiota alterations among persons with HIV (PWH). To overcome this and other confounding factors, we examine a well-powered subset of AGEhIV Cohort participants comprising antiretroviral-treated PWH and seronegative controls matched for age, body-mass index, sex, and sexual practice. We report significant gut microbiota differences in PWH regardless of sex and sexual practice including Gammaproteobacteria enrichment, Lachnospiraceae and Ruminococcaceae depletion, and decreased alpha diversity. Men who have sex with men (MSM) exhibit a distinct microbiota signature characterized by Prevotella enrichment and increased alpha diversity, which is linked with receptive anal intercourse in both males and females. Finally, the HIV-associated microbiota signature correlates with inflammatory markers including suPAR, nadir CD4 count, and prevalence of age-associated noncommunicable comorbidities.

Pain and negative mood during rehabilitation after anterior cruciate ligament reconstruction: a daily process analysis.

Daily diary methods were used to examine changes in pain and negative mood over the first 6 weeks of rehabilitation after surgical reconstruction of the anterior cruciate ligament (ACL). Participants (58 men and 33 women) completed measures of personal factors (i.e., age, athletic identity, neuroticism, optimism) before surgery and indices of daily pain, negative mood, and stress for 42 days after surgery. Multilevel modeling revealed that, as would be expected, daily pain ratings decreased significantly over the course of the study and that the rate of decline in pain ratings decreased over time. Age and daily negative mood were positively associated with daily pain ratings. Daily negative mood also decreased significantly over the course of the study and was positively associated with neuroticism, daily pain, and daily stress. Athletic identity and optimism interacted with time since surgery in predicting daily negative mood such that participants with high levels of athletic identity and low levels of optimism reported greater decreases in daily negative mood over time. Overall, the findings reveal a pattern of improved psychological functioning over the early stages of post-operative ACL rehabilitation.

Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors.

Endometrial stromal tumors are divided into three types: benign stromal nodules, endometrial stromal sarcomas, and undifferentiated endometrial sarcomas. A variety of cytogenetic abnormalities involving chromosome 7 have been reported in endometrial stromal sarcomas, including a recurrent t(7;17)(p15;q21). We have identified two zinc finger genes, which we have termed JAZF1 and JJAZ1, at the sites of the 7p15 and 17q21 breakpoints. Analyses of tumor RNA indicate that a JAZF1/JJAZ1 fusion is present in all types of endometrial stromal tumors; however, the fusion appears to be rarer among endometrial stromal sarcomas that would be considered high-grade according to certain classification schemes. These findings suggest that the less malignant endometrial stromal tumors may evolve toward more malignant types, but that some endometrial stromal sarcomas with relatively abundant mitotic activity may compose a biologically distinct group.

The preemptive analgesic effect of intraarticular bupivacaine and morphine after ambulatory arthroscopic knee surgery.

UNLABELLED: Intraarticular (IA) morphine provides effective postoperative analgesia after arthroscopic knee surgery. Some investigators have suggested that the preemptive administration of opioids may reduce postoperative analgesic requirements and hypersensitivity. We evaluated the analgesic effect of administering IA morphine either before or after surgical incision in patients undergoing arthroscopic knee surgery under local anesthesia. Forty patients undergoing arthroscopic meniscectomy were randomized into two groups. All patients received IA bupivacaine 0.25% before and after surgery together with IV sedation using midazolam and propofol. The Preemptive IA Morphine group received a single 3-mg dose of morphine with their preoperative bupivacaine. The Post-IA Morphine group received 3 mg of morphine at the completion of surgery with the postoperative bupivacaine. After surgery, pain scores, the time to first opioid use, and 24-h analgesic use were recorded. Analgesic duration, defined as the time from completion of surgery until first opioid use, was significantly longer in those patients receiving preoperative (953 +/- 209 min) versus postoperative (556 +/- 121 min) IA morphine. The 24-h acetaminophen and oxycodone use was less in the Preemptive group (2.2 +/- 1.2 pills) versus the Postoperative group (3.0 +/- 1.2 pills). We conclude that IA morphine provides a longer duration of postoperative analgesia with less 24-h opioid use when administered before surgery. IMPLICATIONS: The administration of intraarticular morphine 3 mg before arthroscopic knee surgery provides a longer duration of analgesia with less 24-h opioid use compared with the administration of the drug at the completion of surgery.

Clindamycin phosphate 1% gel in acne vulgaris.

A 12-week study compared Clindagel, a unique water-based gel formulation of clindamycin phosphate 1%, administered once daily, and Cleocin T, a slightly different gel formulation indicated for twice-daily use, in the treatment of acne vulgaris. Clindagel was safe and effective and equivalent to Cleocin T gel, albeit with a better tolerability profile. Clindagel is a viable alternative to Cleocin T gel.

Intravenous interleukin-6 levels predict need for laparotomy in patients with bowel obstruction.

Interleukin-6 (IL-6) has been identified as a marker of ischemia. However, its association with bowel obstruction has not been studied. Fifty-seven patients diagnosed with bowel obstruction were evaluated in a prospective blinded study and managed either medically (n = 29) or surgically (n = 28) per decision of attending surgeon. Serum IL-6 levels were obtained at the time of diagnosis and serially during hospitalization. Mean IL-6 levels at the time of diagnosis were significantly higher in patients who required operation compared with medically treated patients (63.9 vs 19.6 pg/mL respectively; P = 0.027). Levels returned to those seen in medically treated patients 3 days after operation. There was no difference in temperature, white blood cell count, or lactic acid levels. Five patients required resection for ischemic bowel. Patients with ischemic bowel had significantly higher initial mean IL-6 (146.6 vs 45.9 pg/mL; P = 0.034) and lactic acid (23.6 vs 11.8 mg/dL; P = 0.035) at time of diagnosis compared with surgically treated patients without bowel ischemia. No difference in white blood cell count was seen. IL-6 was a sensitive predictor of patients with bowel obstruction requiring operation and for presence of ischemic bowel. IL-6 screening may allow for earlier and more selective operation potentially decreasing morbidity and mortality.

Molecular analysis of DNA rearrangements in leukemias and non-Hodgkin's lymphomas.

Genetic markers for leukemias and lymphomas include chromosomal translocations and antigen-receptor gene rearrangements. Clonal rearrangements of immunoglobulin or T cell receptor (TCR) genes reflect clonal proliferations of lymphocytes, a characteristic feature of lymphoid neoplasia. These rearrangements can be detected as described in this unit by Southern blot hybridization or, in many instances, the polymerase chain reaction (PCR). Specific chromosomal translocations can also serve as markers for clonality, for malignant transformation, and for various defined subtypes of hematopoietic cancers. PCR protocols are described for detection of the two most commonly assayed translocations, t(9;22) of chronic myelogenous leukemia or acute lymphoblastic leukemia, and t(14;18) of follicular lymphomas.

High dose cyclophosphamide plus recombinant human granulocyte-colony stimulating factor (rhG-CSF) in the treatment of follicular, low grade non-Hodgkin's lymphoma: CALGB 9150.

The main objectives of this study were to determine the feasibility of administering high doses of cyclophosphamide plus recombinant human granulocyte-colony stimulating factor (rhG-CSF) every 14-21 days to patients with follicular small cleaved cell lymphoma. For each patient, the treatment was not considered feasible if fewer than four cycles of cyclophosphamide chemotherapy could be administered on schedule (i.e. at least every 29 days) or (1) hospitalization of the patient for longer than three days was necessary for neutropenic fever (38 degrees C) or bacteriologically documented infection in > 50% of the cycles, or (2) grade > or = 2 hemorrhage in association with thrombocytopenia of grade > or = 3 severity occurred in > 50% of the cycles or (3) non-hematologic toxicity (excluding nausea/vomiting and alopecia) of grade > or = 3 occurred in > 50% of cycles. The goal was to have a treatment program feasible in 75% or more of the treated patients. The secondary objectives were to determine the toxicities, the complete and partial response rates, and the time to treatment failure (TTF). The trial also attempted to assess the effectiveness of this treatment program in eradicating Bcl-2 rearrangements by PCR, and to assess complete remission duration in relationship to PCR results in patients who respond to this chemotherapy program. Patients were required to have histologically documented non-Hodgkin's lymphoma of the subtypes follicular, predominantly small cleaved cell (IWF-B) or follicular mixed, (IWF-C). Patients were required to have Stage IV disease including histologic evidence of bone marrow involvement. Measurable disease was required and patients were also required to have one of the following risk factors: > or = 2 extranodal sites, node or nodal group > or = 5 cm. Submission of fresh bone marrow for molecular genetic studies for the presence of Bcl-2-Ig fusion DNA was mandatory in previously untreated patients. Patients had to be between 18 and physiologic age 55 years (carefully selected patients over age 55 years were also eligible), expected survival > 2 years, performance status 0-1, and have adequate renal, hepatic and bone marrow function, and a cardiac ejection fraction > or = 50%. Cyclophosphamide 4.5 g/m2 i.v. was given with mesna every 14 days with rhG-CSF support. Twenty-nine patients were accrued to this trial. The median follow-up time is 5.0 years, with a range of 2.5-6.7 years. The overall response rate was 75% (9 CRs 37.5%, 9PRs 37.5%). The median duration of survival is 5.53 years. The 1-year estimated probability of freedom from treatment failure was 50% and of survival at 1 year was 92%. No strong association was observed between TTF and age, symptomatic stage, histology performance status, number of extranodal sites or baseline Bcl-2 status. At 3 years the survival of all patients was 78% and failure free survival was 17%. 15 (62%) of the 24 eligible previously untreated patients met the criteria for feasibility specified in the protocol. The 95% CI for the feasibility rate is (44 and 82%). Twenty-two of the 24 (92%) previously untreated patients had specimens submitted for testing for Bcl-2 rearrangements. Thirteen of the 22 (59%) were found to have rearrangements at baseline. Post-treatment specimens were submitted for seven of the 13 patients. Four of the seven converted to Bcl-2 negative following treatment. Eight of 13 Bcl-2 positive patients (62%) had a clinical response to treatment. The 95% exact binomial CI for the total response rate in this subgroup is (28 and 88%). This study demonstrates that repetitive doses of cyclophosphamide at 4.5 g/m2 every two weeks with rhG-CSF support can be administered to selected younger patients with advanced follicular lymphoma with morphologic involvement of the bone marrow with acceptable non-hematologic toxicity.

Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations.

We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.

Postoperative analgesia for outpatient arthroscopic knee surgery with intraarticular clonidine and/or morphine.

Both clonidine, an alpha(2) agonist, and morphine, an opioid agonist, provide enhanced patient analgesia after arthroscopic knee surgery when administered via the intraarticular (IA) route. Clonidine potentiates morphine analgesia in the animal model. We designed this study to determine whether clonidine or morphine results in better analgesia and whether their combination would provide superior analgesia to either drug alone. We evaluated 60 patients undergoing arthroscopic knee meniscus repair under local anesthesia with sedation. After surgery, patients were randomized into four IA groups: Group B received 30 mL 0.25% bupivacaine; Group BC received 30 mL 0.25% bupivacaine and clonidine 1 microg/kg; Group BM received 30 mL 0.25% bupivacaine and morphine 3 mg; and Group BCM received 30 mL 0.25% bupivacaine, clonidine 1 microg/kg, and morphine 3 mg. This study revealed a significant benefit from the individual IA administration of both clonidine and morphine. The combination of these drugs resulted in decreased postoperative pain and analgesic use, as well as an increased analgesic duration compared with either drug alone. We conclude that IA clonidine and morphine improved comfort compared with either drug alone in patients undergoing knee arthroscopy.

Calcium depletion dissociates and activates heterodimeric notch receptors.

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.

Intracellular forms of human NOTCH1 interact at distinctly different levels with RBP-jkappa in human B and T cells.

The cellular transcriptional repressor RBP-Jkappa associates with the Epstein-Barr virus nuclear antigens (EBNAs) determined to be essential for transformation of human primary B lymphocytes. It was demonstrated through genetic analysis that interaction between the viral transactivator EBNA2 and RBP-Jkappa is essential for EBV immortalization of primary B lymphocytes. We have shown that the association of RBP-Jkappa with intracellular NOTCH1 differs significantly in B and T cells. Immunoprecipitation analyses with antibodies to both the intracellular forms of NOTCH1 and to RBP-Jkappa demonstrated that little or no RBP-Jkappa is associated with NOTCH1 in B cell lines compared to the RBP-Jkappa associated with NOTCH1 in T cell lines and was further demonstrated in human primary lymphocytes. Additionally, EBNA2 can compete with intracellular NOTCH1 for binding to GST-RBP-Jkappa in vitro. Northern blot for the cellular gene hairy enhancer of split (HES1) demonstrated that HES1 is upregulated in the EBV transformed lymphoblastoid cells expressing high levels of EBNA2 and in a T cell line SupT1 overexpressing intracellular activated NOTCH1. Hence, EBNA2 may be able to compete for the available pool of RBP-Jkappa more effectively in human B cells than in T cells and provides a possible explanation for the ability of EBV to potently and efficiently infect and immortalize human B cells. Leukemia (2000) 14, 84-92.

Requirement for presenilin 1 in facilitating lagged 2-mediated endoproteolysis and signaling of notch 1.

Presenilin 1 (PS1), a polytopic membrane protein, is required for endoproteolytic processing at gamma-secretase site within the transmembrane domain of amyloid precursor proteins (APP). In addition, PS1 and its orthologues facilitate signaling of Notch family members, cell-surface receptors that specify cell fates during development. To clarify the mechanism(s) by which PS facilitates Notch signaling, we examined human Jagged-2-dependent metabolism and activity of a chimeric full-length Notchl-GFP molecule expressed in fibroblasts with heterozygous, or homozygous deletions of PS1. We demonstrate that PS1 is required for facilitating Jagged 2-mediated proteolysis and that translocation and accumulation of NICD in the nucleus correlates with signaling activity. Moreover, in a ligand-independent, Ca2+-depletion paradigm, we demonstrate that PS1 facilitates endoproteolysis of a plasma-membrane-associated, Notch1-GFP derivative. Finally, we report that NICD production is inhibited by L-685,458, a potent and selective inhibitor that blocks solubilized gamma-secretase activity and Abeta production in cultured cells. These findings strongly suggest that intramembranous processing of APP and Notch 1 are mediated by similar, if not identical, proteases that require PS1 for their activation.

Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics.

Hematopoiesis is a balance between proliferation and differentiation that may be modulated by environmental signals. Notch receptors and their ligands are highly conserved during evolution and have been shown to regulate cell fate decisions in multiple developmental systems. To assess whether Notch1 signaling may regulate human hematopoiesis to maintain cells in an immature state, we transduced a vesicular stomatitis virus G-protein (VSV-G) pseudo-typed bicistronic murine stem cell virus (MSCV)-based retroviral vector expressing a constitutively active form of Notch1 (ICN) and green fluorescence protein into the differentiation competent HL-60 cell line and primary cord blood-derived CD34(+) cells. In addition, we observed endogenous Notch1 expression on the surface of both HL-60 cells and primary CD34(+) cells, and therefore exposed cells to Notch ligand Jagged2, expressed on NIH3T3 cells. Both ligand-independent and ligand-dependent activation of Notch resulted in delayed acquisition of differentiation markers by HL-60 cells and cord blood CD34(+) cells. In addition, primary CD34(+) cells retained their ability to form immature colonies, colony-forming unit-mix (CFU-mix), whereas control cells lost this capacity. Activation of Notch1 correlated with a decrease in the fraction of HL-60 cells that were in G0/G1 phase before acquisition of a mature cell phenotype. This enhanced progression through G1 was noted despite preservation of the proliferative rate of the cells and the overall length of the cell cycle. These findings show that Notch1 activation delays human hematopoietic differentiation and suggest a link of Notch differentiation effects with altered cell cycle kinetics.

Breakpoints of the t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma lie within or near the previously undescribed gene MALT1 in chromosome 18.

Lymphomas arising in mucosa-associated lymphoid tissue (MALT) are indolent B-cell tumors that have a predilection for epithelial sites and often develop in a setting of chronic inflammation or autoimmunity. As many as 50% of low-grade MALT lymphomas contain an (11;18)(q21; q21) chromosomal translocation. Using fluorescence in situ hybridization, we have analyzed the position of recombination within chromosome 18 DNA in three examples of MALT lymphoma bearing this translocation. In all three cases, the breakpoint maps to DNA in BAC b357H2, covering about 150 kb of sequence. A previously undescribed, ubiquitously expressed gene, which we refer to as MALT1, was identified within this sequence and was found to be broken in one case for which we have definitively located the position of recombination between chromosomes 18 and 11. The sequence of this gene indicates the presence of two immunoglobulin-like C2 domains and a region of partial homology to caspases, suggesting a possible role for MALT1 in the regulation of apoptosis.

Effect of atenolol or metoprolol on arbutamine stress echocardiography in patients suspected of having coronary artery disease.

Arbutamine stress echocardiography was performed in 81 patients with suspected coronary artery disease. Arbutamine infusion, using a dedicated closed-loop delivery device, provided comparable myocardial stress in patients receiving beta-1 blockers versus those who were not.

Safety and efficacy of closed-loop arbutamine stress echocardiography for detection of coronary artery disease. International Arbutamine Study Group.

Closed-loop arbutamine stress echocardiography has been shown to be safe and effective for detecting coronary artery disease (CAD) using a standardized infusion protocol and centralized core laboratory analyses. However, the accuracy of arbutamine stress echocardiography using local test interpretation has not been previously tested in a large population. The present study reports the safety, sensitivity, and specificity of arbutamine stress echocardiography in a multicenter trial allowing user-defined, nonstandardized protocols and local test interpretation. In all, 1,070 patients underwent arbutamine stress testing at 81 sites. Heart rate increased from 73 +/- 13 to 124 +/- 15 beats/min, systolic blood pressure from 144 +/- 24 to 174 +/- 25 mm Hg, and pressure rate product x 10(3) from 10.5 +/- 2.8 to 19.6 +/- 3.9. Among 1,070 patients, there were only 2 (0.2%) significant adverse events related to arbutamine, both of which resolved completely with appropriate therapy. There were no incidents of ventricular fibrillation, sustained ventricular tachycardia, or death related to testing. Among 242 patients who underwent arbutamine stress echocardiography and diagnostic coronary angiography within 12 weeks, sensitivity and specificity for detection of CAD were 71% (95% confidence interval 64% to 77%) and 67% (95% confidence interval 52% to 80%), respectively. Closed-loop arbutamine stress echocardiography is a safe and effective method for evaluating CAD in clinical practice.

Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.