Delayed Surgery, Low Presurgery Activity, and Poor Preoperative IKDC Scores Are Correlated with Lower Rates of Return to Sports after ACL Reconstruction in an Asian Population.

There is limited literature regarding the early postsurgical outcomes of anterior cruciate ligament (ACL) reconstruction in Asian populations, particularly in the rates of return to sports. We aimed to quantify early clinical outcomes for ACL reconstruction, determine the predictive value of surgeon- and patient-reported outcomes on the rate of return to sports in the early postoperative period, and identify factors predictive of return to sports. We analyzed the data of 55 patients who underwent ACL reconstruction at our tertiary medical center from 2015 to 2016. All patients underwent transportal ACL reconstruction and a standardized post-ACL reconstruction rehabilitation protocol. Patients with concurrent meniscal injury and repair were included. Patients were evaluated at the 3-month, 6-month, 1-year, and 2-year postoperative periods. Surgeon- and patient-reported outcome scores were collected at each follow-up through a systematic questionnaire designed to determine the patient's level of return to sport and reasons for not returning. Surgeon- and patient-reported outcome measures improved significantly over the 2-year postoperative period (p < 0.001). Overall rate of return to sports was 58.2%. The International Knee Documentation Committee (IKDC) subjective (p = 0.02), symptomatic (p = 0.001), composite (p = 0.005), Tegner (p < 0.001) and Lysholm (p = 0.049) scores at 2-year follow-up were significantly worse in patients who failed to return to sports. Earliest difference in scores manifested at 3-month postsurgery (p = 0.011). IKDC grade-D patients were 18.1 times less likely to return to sports (p = 0.035). Delayed surgery (p = 0.01) and presurgery inactivity (p = 0.023) were negatively predictive of return to sports. The rate of return to sport is consistent with the literature analyzing other ethnic populations. Both surgeon- and patient-reported outcome scores at 2-year postsurgery exhibited significant differences between those who did and did not return to sports. Patients should be advised to seek surgical treatment as soon as possible and stay active preoperatively to maximize return to sports.

A multi-institutional retrospective analysis on impact of RV acute mechanical support timing after LVAD implantation on 1-year mortality and predictors of RV acute mechanical support weaning.

BACKGROUND: There is little insight into which patients can be weaned off right ventricular (RV) acute mechanical circulatory support (AMCS) after left ventricular assist device (LVAD) implantation. We hypothesize that concomitant RV AMCS insertion instead of postoperative implantation will improve 1-year survival and increase the likelihood of RV AMCS weaning. METHODS: A multicenter retrospective database of 826 consecutive patients who received a HeartMate II or HVAD between January 2007 and December 2016 was analyzed. We identified 91 patients who had early RV AMCS on index admission. Cox proportional-hazards model was constructed to identify predictors of 1-year mortality post-RV AMCS implantation and competing risk modeling identified RV AMCS weaning predictors. RESULTS: There were 91 of 826 patients (11%) who required RV AMCS after CF-LVAD implantation with 51 (56%) receiving a concomitant RV AMCS and 40 (44%) implanted with a postoperative RV AMCS during their ICU stay; 48 (53%) patients were weaned from RV AMCS support. Concomitant RV AMCS with CF-LVAD insertion was associated with lower mortality (HR 0.45 [95% CI 0.26-0.80], p = 0.01) in multivariable model (which included age, BMI, angiotensin-converting enzyme inhibitor use, and heart transplantation as a time-varying covariate). In the multivariate competing risk analysis, a TPG < 12 (SHR 2.19 [95% CI 1.02-4.70], p = 0.04) and concomitant RV AMCS insertion (SHR 3.35 [95% CI 1.73-6.48], p < 0.001) were associated with a successful wean. CONCLUSIONS: In patients with RVF after LVAD implantation, concomitant RV AMCS insertion at the time of LVAD was associated with improved 1-year survival and increased chances of RV support weaning compared to postoperative insertion.

Prediction of right heart failure after left ventricular assist implantation: external validation of the EUROMACS right-sided heart failure risk score.

AIMS: Prediction of right heart failure (RHF) after left ventricular assist device (LVAD) implant remains a challenge. The EUROMACS right-sided heart failure (EUROMACS-RHF) risk score was proposed as a prediction tool for post-LVAD RHF but lacks from large external validation. The aim of our study was to externally validate the score. METHODS AND RESULTS: From January 2007 to December 2017, 878 continuous-flow LVADs were implanted at three tertiary centres. We calculated the EUROMACS-RHF score in 662 patients with complete data. We evaluated its predictive performance for early RHF defined as either (i) need for short- or long-term right-sided circulatory support, (ii) continuous inotropic support for ≥14 days, or (iii) nitric oxide for ≥48 h post-operatively. Right heart failure occurred in 211 patients (32%). When compared with non-RHF patients, pre-operatively they had higher creatinine, bilirubin, right atrial pressure, and lower INTERMACS class (P < 0.05); length of stay and in-hospital mortality were higher. Area under the ROC curve for RHF prediction of the EUROMACS-RHF score was 0.64 [95% confidence interval (CI) 0.60-0.68]. Reclassification of patients with RHF was significantly better when applying the EUROMACS-RHF risk score on top of previous published scores. Patients in the high-risk category had significantly higher in-hospital and 2-year mortality [hazard ratio: 1.64 (95% CI 1.16-2.32) P = 0.005]. CONCLUSION: In an external cohort, the EUROMACS-RHF had limited discrimination predicting RHF. The clinical utility of this score remains to be determined.

Predicting and measuring mortality risk after transcatheter aortic valve replacement.

Introduction: Over the last decade, transcatheter aortic valve replacement (TAVR) has emerged as a treatment option for most patients with severe symptomatic aortic stenosis (AS). With growing indications and exponential increase in the number of TAVR procedures, it is important to be able to accurately predict mortality after TAVR.Areas covered: Herein, we review the surgical and TAVR-specific mortality prediction models (MPMs) and their performance in their original derivation and external validation cohorts. We then discuss the role of other important risk assessment tools such as frailty, echocardiographic parameters, and biomarkers in patients, being considered for TAVR.Expert opinion: Conventional surgical MPMs have suboptimal predictive performance and are mis-calibrated when applied to TAVR populations. Although a number of TAVR-specific MPMs have been developed, their utility is also limited by their modest discriminative ability when applied to populations external to their original derivation cohorts. There is an unmet need for robust TAVR MPMs that accurately predict post TAVR mortality. In the interim, heart teams should utilize the currently available TAVR-specific MPMs in conjunction with other prognostic factors, such as frailty, echocardiographic or computed tomography (CT) imaging parameters, and biomarkers for risk assessment of patients, being considered for TAVR.

Incongruent Teen Pregnancy Attitudes, Coparenting Conflict, and Support Among Mexican-Origin Adolescent Mothers.

The current longitudinal study examined whether differences between Mexican-origin adolescent mothers and their mother figures (N = 204 dyads) in attitudes on the status attained through teen pregnancy were associated with conflict in their coparenting relationship and whether coparenting conflict was associated with adolescent mothers' perceptions of social support. Findings revealed that when adolescents held more positive attitudes than their mother figures about the status gained through teen pregnancy, they tended to report greater coparenting conflict with their mother figures. Furthermore, greater coparenting conflict was significantly associated with decreases in adolescents' perceptions of social support (i.e., emotional, instrumental, companion support) 1 year later. Findings underscore the importance of incongruent attitudes and the quality of coparenting relationships between adolescent mothers and their mother figures in relation to support processes. Findings are discussed with respect to understanding Mexican-origin adolescent mothers' social support in the context of family subsystem attitudes and interactions.

The Immunology Quality Assessment Proficiency Testing Program for CD3⁺4⁺ and CD3⁺8⁺ lymphocyte subsets: a ten year review via longitudinal mixed effects modeling.

Since 1999, the National Institute of Allergy and Infectious Diseases Division of AIDS (NIAID DAIDS) has funded the Immunology Quality Assessment (IQA) Program with the goal of assessing proficiency in basic lymphocyte subset immunophenotyping for each North American laboratory supporting the NIAID DAIDS HIV clinical trial networks. Further, the purpose of this program is to facilitate an increase in the consistency of interlaboratory T-cell subset measurement (CD3(+)4(+)/CD3(+)8(+) percentages and absolute counts) and likewise, a decrease in intralaboratory variability. IQA T-cell subset measurement proficiency testing was performed over a ten-year period (January 2003-July 2012), and the results were analyzed via longitudinal analysis using mixed effects models. The goal of this analysis was to describe how a typical laboratory (a statistical modeling construct) participating in the IQA Program performed over time. Specifically, these models were utilized to examine trends in interlaboratory agreement, as well as successful passing of proficiency testing. Intralaboratory variability (i.e., precision) was determined by the repeated measures variance, while fixed and random effects were taken into account for changes in interlaboratory agreement (i.e., accuracy) over time. A flow cytometer (single-platform technology, SPT) or a flow cytometer/hematology analyzer (dual-platform technology, DPT) was also examined as a factor for accuracy and precision. The principal finding of this analysis was a significant (p<0.001) increase in accuracy of T-cell subset measurements over time, regardless of technology type (SPT or DPT). Greater precision was found in SPT measurements of all T-cell subset measurements (p<0.001), as well as greater accuracy of SPT on CD3(+)4(+)% and CD3(+)8(+)% assessments (p<0.05 and p<0.001, respectively). However, the interlaboratory random effects variance in DPT results indicates that for some cases DPT can have increased accuracy compared to SPT. Overall, these findings demonstrate that proficiency in and among IQA laboratories have, in general, improved over time and that platform type differences in performance do exist.

Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

A biomechanical study of the meniscofemoral ligaments and their contribution to contact pressure reduction in the knee.

The aim of this study was to test the hypothesis that the meniscofemoral ligaments (MFLs) of the human knee assist the lateral meniscal function in reducing tibiofemoral contact pressure. Five human cadaveric knee joints were loaded in axial compression in extension using a 4-degree of freedom rig in a universal materials testing machine. Contact pressures pre- and post-sectioning of the MFLs were measured using pressure sensitive film. Sectioning the MFLs increased the contact pressure significantly in the joints for two of the four measures. In addition to their known function in assisting the posterior cruciate ligament (PCL) to resist tibiofemoral posterior drawer, the MFLs also have a significant role in reducing contact stresses in the lateral compartment. Their retention in PCL and meniscal surgery is therefore to be advised.

The effects of lateral meniscal allograft transplantation techniques on tibio-femoral contact pressures.

This paper reports a series of comparative tests in vitro, that examined how meniscectomy and meniscal allografting affected tibio-femoral joint contact pressure. Knees were loaded in axial compression and pressure maps obtained from the lateral compartment using Fuji Prescale film inserted below the meniscus. This was repeated after meniscectomy, and then after meniscal allografting with fixation by a bone plug for the insertional ligaments, plus sutures. Finally, the pressure, when the allograft was secured by sutures alone, was measured. The peak pressure rose significantly after meniscectomy, and then was reduced significantly by both allograft methods so that it was not significantly different to normal. Allografts fixed by sutures only allowed slightly higher contact pressure than when they had bone fixation. This study suggests that meniscal allografting should have a chondroprotective effect and that there is a small advantage from adding bony fixation to suture fixation.

Reversal of suprascapular neuropathy following arthroscopic repair of massive supraspinatus and infraspinatus rotator cuff tears.

PURPOSE: The purpose of this study was to study the prevalence of suprascapular neuropathy (SSN) in the setting of massive rotator cuff tears and to determine if arthroscopic rotator cuff repair, even if partial, was associated with reversal of SSN and clinical improvement in pain and function. METHODS: Over a 13-month period, 26 of 216 patients with rotator cuff tears treated operatively were identified to have massive tears associated with retraction and moderate to severe fatty infiltration of the supraspinatus and infraspinatus muscles. All patients had pain and marked weakness in abduction and external rotation which did not improve with conservative treatment. Electrodiagnostic electromyographic/nerve conduction velocity (EMG/NCV) evaluation, as well as pre- and postoperative questionnaire and physical examination, were performed. An arthroscopic repair, either partial or complete, was performed on patients identified to have a massive rotator cuff tear in association with SSN. RESULTS: Fourteen of 26 patients with massive rotator cuff tears (54%) were identified to have a peripheral nerve injury. Seven of these 26 (38%) had isolated suprascapular nerve injury, 4 had axillary nerve injury, 2 had an associated upper trunk brachial plexus injury, and 1 had a cervical radiculopathy. All 7 patients with isolated suprascapular injury underwent arthroscopic treatment, 1 of which was not technically reparable at the time of surgery. In the 6 patients who underwent either partial or complete arthroscopic repair, follow-up EMG/NCV after 6 months demonstrated partial or full recovery of the suprascapular nerve palsy that correlated with complete pain relief and marked improvement in function. CONCLUSIONS: SSN is found in a significant proportion of patients with massive rotator cuff tears, and is associated with pain and dysfunction. Arthroscopic rotator cuff repair can result in reversal of SSN, which may correlate with substantial improvement in pain and function. LEVEL OF EVIDENCE: Level IV, case series.

Persistence of the mini pivot shift after anatomically placed anterior cruciate ligament reconstruction.

We hypothesized that traces of pivot-shift instability (a minipivot) would persist after anterior cruciate ligament reconstruction in some knees despite objective restoration of anteroposterior laxity to normal. We tested intact cadaver knees after anterior cruciate ligament transection and then after anatomically placed single-bundle anterior cruciate ligament reconstructions. We measured the rotational laxities and pivot-shift kinematics over a range of graft tensions. Increasing graft tension decreased anteroposterior laxity; anteroposterior laxity was greater than normal at 0 and 10 N tensions but not different than normal at 20 to 60 N tensions. Anterior cruciate ligament deficiency had little effect on rotation laxity, and reconstruction had little effect on rotational laxity, which was not reduced by increasing graft tension. During the pivot-shift test, increasing graft tension reduced the anteroposterior subluxation-reduction events, but tibial rotational was not restored to normal. More sophisticated reconstruction methods may be required to control rotation. Objective restoration of anteroposterior laxity to normal does not necessarily return knee kinematics, especially rotational behavior, to normal.

Phosphorylation of Bad is not essential for PKB-mediated survival signaling in hemopoietic cells.

This study was designed to investigate Bad phosphorylation at several of its key regulatory Ser residues in cytokine-dependent hemopoietic cells. These studies were initiated in light of numerous studies that have reported a key role for phosphorylated Bad in preventing apoptosis. One key question is whether the survival signaling effect of the PI 3-kinase pathway is mediated by PKB phosphorylation of Bad. We confirm previous reports that if Bad is overexpressed or if active PKB is overexpressed, then the increased phosphorylation of Bad at Ser136 is apparent. However, we were unable to detect phosphorylation of endogenous Bad at Ser136 in the MC/9 mast cell line or in murine bone marrow-derived macrophages. On the other hand, phosphorylation of Bad at Ser112 and Ser155 was observed in response to IL-3 or GM-CSF, which activate the MEK/erk pathway, but not with IL-4, which activates the PI 3-kinase, but not the MEK/erk pathway, and also promotes cell survival. In contrast to previous reports, we found that ceramide had no effect on the phosphorylation status of Bad. In summary, our results suggest that Bad phosphorylation at any of the three major sites is not a required event for cytokine-dependent cell survival, and in particular, the activation of PI 3-kinase/PKB pathway can be dissociated from phosphorylation of Bad at Ser136.

The remains of anterior cruciate ligament graft tension after cyclic knee motion.

BACKGROUND: There is sometimes a return of excess knee laxity after anterior cruciate ligament reconstruction. One of the contributing factors might be a loss in graft tension. It is unknown whether the tension imposed on an anterior cruciate ligament graft degrades with time and, if so, the effect of that loss of tension on knee laxity. HYPOTHESES: The pretension in the anterior cruciate ligament graft reduces significantly within the first 500 motion cycles, and this decrease in graft tension causes an increase in knee laxity. STUDY DESIGN: Controlled laboratory study. METHODS: This study measured the remains of bone-patellar tendon-bone graft pretension after cyclical flexion-extension and the effect of any tension loss on knee laxity, using 8 cadaveric knees. A tension transducer was inserted into the graft and calibrated in situ. The reconstruction tension was 40 N at 20 degrees of flexion. In test 1, the graft tension was measured under cyclical flexion-extension in a motorized rig up to 1500 cycles. Test 2, with a new graft, also included anteroposterior and internal-external rotational knee laxity measurements at 0, 500, and 1500 cycles. RESULTS: The graft tension at 0 degrees of flexion dropped from 208 N, by 25% after 50 cycles, 41% by 500, and 46% by 1500 cycles. Anterior laxity increased from +1.4 to +2.8 mm by 500 cycles, and performing these laxity tests also caused significant tension losses. CLINICAL RELEVANCE: These results provide one possible explanation for early slackening of anterior cruciate ligament reconstructions.

IL-10 levels in Dengue patients: some findings from the exceptional epidemiological conditions in Cuba

The pathogenesis associated with Dengue haemorrhagic fever, has yet to be fully elucidated, with no definitive in vivo evidence. The exceptional epidemiological circumstances in Cuba allow the evaluation of different mediators in a well-defined situation. In the present study, we describe the determination of levels of IL-12, IL-10 and RANTES in the sera of Cuban patients hospitalised with Dengue fever or Dengue haemorrhagic fever. The results showed that levels of serum IL-10 were higher in patients than controls, and those patients with secondary infections had consistently higher levels. All the Dengue haemorrhagic fever patients had increased levels of IL-10. In contrast, levels of IL-12 did not differ between patients and controls. Finally, RANTES serum levels detected in patients were lower than those observed in the controls. The association of increased levels of IL-10 in Dengue patients with a sequential infection suggests a possible role of this cytokine in the pathogenesis of Dengue disease (AU)

The dynamics of bone marrow stromal cells in the proliferation of multipotent hematopoietic progenitors by substance P: an understanding of the effects of a neurotransmitter on the differentiating hematopoietic stem cell.

Communication within the hematopoietic-neuroendocrine-immune axis is partly mediated by neurotransmitters (e.g. substance P, SP) and cytokines. SP mediates neuromodulation partly through the stimulation of bone marrow (BM) progenitors. This study shows that SP, through the neurokinin-1 receptor, stimulates the proliferation of primitive hematopoietic progenitors: cobblestone-forming cells (CAFC, CD34+). This effect is optimal when macrophage is included within the fibroblast support. Indirect induction of IL-1 could be important in the proliferation of CAFC colonies by SP. Phenotypic and functional studies suggest that SP might directly interact with the CD34+/CD45(dim) population. These studies indicate that SP can initiate a cascade of biological responses in the BM stroma and stem cells to stimulate hematopoiesis.

Twitching motility of Ralstonia solanacearum requires a type IV pilus system.

Twitching motility is a form of bacterial translocation over firm surfaces that requires retractile type IV pili. Microscopic colonies of Ralstonia solanacearum strains AW1, K60 and GMI1000 growing on the surface of a rich medium solidified with 1.6% agar appeared to exhibit twitching motility, because early on they divided into motile 'rafts' of cells and later developed protruding 'spearheads' at their margins. Individual motile bacteria were observed only when they were embedded within masses of other cells. Varying degrees of motility were observed for 33 of 35 strains of R. solanacearum in a selected, diverse collection. Timing was more important than culture conditions for observing motility, because by the time wild-type colonies were easily visible by eye (about 48 h) this activity ceased and the spearheads were obscured by continued bacterial multiplication. In contrast, inactivation of PhcA, a transcriptional regulator that is essential for R. solanacearum to cause plant disease, resulted in colonies that continued to expand for at least several additional days. Multiple strains with mutations in regulatory genes important for virulence were tested, but all exhibited wild-type motility. Many of the genes required for production of functional type IV pili, and hence for twitching motility, are conserved among unrelated bacteria, and pilD, pilQ and pilT orthologues were identified in R. solanacearum. Colonies of R. solanacearum pilQ and pilT mutants did not develop spearheads or rafts, confirming that the movement of cells that had been observed was due to twitching motility. Compared to the wild-type parents, both pilQ and pilT mutants caused slower and less severe wilting on susceptible tomato plants. This is the first report of twitching motility by a phytopathogenic bacterium, and the first example where type IV pili appear to contribute significantly to plant pathogenesis.

Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors.

Peripheral blood mononuclear cells from patients with human immunodeficiency virus (HIV) infection exhibit a progressively marked decrease in the production of virus-induced interferon (IFN)-alpha, a finding that correlates with and is highly predictive of disease progression and opportunistic infections. The major IFN-alpha producing population has recently been defined as the precursor to type 2 dendritic cells (pDC2) or plasmacytoid DC (pDC). Using four-color flow cytometry, we have enumerated the pDC2 vs non-IFN-alpha producing myeloid DC1 in peripheral blood from HIV-infected patients and healthy controls and related these values to CD4 cell numbers, viral load, and functional activity. The patients had reductions in the numbers of both pDC2 (lin-/HLA-DR+/CD123(bright)) and DC1 (lin1-/HLA-DR+/CD123(dim)/CD11c+), both at an absolute level and as a percentage of cells. The decreases were most evident in patients with decreased CD4 levels. Viral load correlated with the functional frequency of the IFN producing cells but not with absolute pDC2 levels. Using intracellular flow cytometric analysis for IFN-alpha, the patients were demonstrated to have fewer pDC2, as well as a lower percentage of responding cells among those remaining. We conclude that deficient production of IFN-alpha by pDC2 from HIV-infected patients results from both selective loss of these cells and their qualitative dysfunction. Given the central role of DC, and in particular, DC2, in linking innate and adaptive immune responses, these qualitative and quantitative changes in pDC2 are likely to be key contributors to HIV pathogenesis.

Relationship between oxidative burst activity and CD11b expression in neutrophils and monocytes from healthy individuals: effects of race and gender.

Oxidative burst activity and the expression of adhesion molecules have been used as indicators of leukocyte activation status. The aim of the study was to delineate the relationship of oxidative burst activity and the expression of adhesion molecules in neutrophils and monocytes from a pool of healthy volunteers (n = 96). We also tested the potential role of gender and a racial background in the individual response differences. Basal and phorbol myristate acetate (PMA)-stimulated oxidative burst and CD11b expression were determined using dihydrorhodamine 123 and phycoerythrin (PE)-conjugated anti-CD11b monoclonal antibodies. PMA markedly increased CD11b expression and cellular oxidant content in neutrophils and monocytes in all samples. However, the responses showed considerable variability among individuals. A positive correlation was observed between the responsiveness of neutrophils and monocytes in their basal or PMA-stimulated CD11b expressions and PMA-stimulated oxidative burst activities. In contrast, no correlation was found between the level of adhesion molecule expression and cellular oxidant content in monocytes or neutrophils either under basal or under PMA-stimulated conditions. The reactivity of oxidative burst (i.e., PMA-stimulated over basal) was significantly lower in neutrophils from African American males compared with cells from African American females, white females, or white males. In contrast, reactivity of monocytes was significantly elevated in white males compared with all other groups. These findings indicate that leukocytes with a relatively high degree of adhesion molecule expression may display an average or decreased oxidative burst activity, and vice versa. Our findings also indicate that ethnic background may influence the oxidative burst activity in neutrophils and monocytes. This needs consideration in clinical studies utilizing healthy volunteers with mixed gender and ethnic backgrounds.

Augmented TNF-alpha and IL-10 production by primed human monocytes following interaction with oxidatively modified autologous erythrocytes.

The presence of dysfunctional/damaged red blood cells (RBCs) has been associated with adverse clinical effects during the inflammatory response. The aim of this study was to elucidate whether oxidatively modified, autologous RBCs modulate monocyte cytokine responses in humans. Monocyte tumor necrosis factor alpha (TNF-alpha) and IL-10 production was measured in whole blood from healthy volunteers using ELISA and flow cytometry. Oxidatively modified RBCs (15 mM phenylhydrazine, 1 h, OX-RBC) or vehicle-treated RBCs (VT-RBC) opsonized by autologous serum were administered alone or in combination with one of three priming agents: E. coli lipopolysaccharide (LPS, 0.2 ng/ml), zymosan A (1 mg/ml), or phorbol 12-myristate 13-acetate (PMA, 50 ng/ml). OX-RBC or VT-RBC alone did not result in the release of TNF-alpha or IL-10. LPS, zymosan, and PMA caused marked and dose-dependent increases in TNF-alpha and IL-10 production. Addition of OX-RBC augmented the LPS-, zymosan-, and PMA-induced TNF-alpha release by approximately 100%. OX-RBC augmented LPS- and zymosan-induced IL-10 release by 400-600%. Flow cytometry analyses showed that monocytes were responsible for TNF-alpha and IL-10 production in whole blood. The presence of OX-RBC alone increased the complexity of CD14+ monocytes but caused no cytokine production. LPS alone induced cytokine production without altering cell complexity. After the combined (OX-RBC+LPS) treatment, monocytes of high complexity were responsible for TNF-alpha production. The presence of mannose or galactose (at 10-50 mM) did not alter the observed augmentation of cytokine production by OX-RBC, suggesting that lectin receptors are not involved in the response. These studies indicate that the interaction between damaged autologous erythrocytes and monocytes has a major impact on the cytokine responses in humans. An augmented cytokine production by the mononuclear phagocyte system may adversely affect the clinical course of injury and infections especially in genetic or acquired RBC diseases or after transfusions.

Inhibition of human immunodeficiency virus type 1 transcription by chemical cyclin-dependent kinase inhibitors.

Cyclin-dependent kinases (cdk's) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH(2), 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdk's. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G(1)/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdk's 9 and 7 was determined to be approximately 0.6 microM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdk's are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.