Development of clinical tools to estimate the breathing effort during high-flow oxygen therapy: A multicenter cohort study.

INTRODUCTION AND OBJECTIVES: Quantifying breathing effort in non-intubated patients is important but difficult. We aimed to develop two models to estimate it in patients treated with high-flow oxygen therapy. PATIENTS AND METHODS: We analyzed the data of 260 patients from previous studies who received high-flow oxygen therapy. Their breathing effort was measured as the maximal deflection of esophageal pressure (ΔPes). We developed a multivariable linear regression model to estimate ΔPes (in cmH2O) and a multivariable logistic regression model to predict the risk of ΔPes being >10 cmH2O. Candidate predictors included age, sex, diagnosis of the coronavirus disease 2019 (COVID-19), respiratory rate, heart rate, mean arterial pressure, the results of arterial blood gas analysis, including base excess concentration (BEa) and the ratio of arterial tension to the inspiratory fraction of oxygen (PaO2:FiO2), and the product term between COVID-19 and PaO2:FiO2. RESULTS: We found that ΔPes can be estimated from the presence or absence of COVID-19, BEa, respiratory rate, PaO2:FiO2, and the product term between COVID-19 and PaO2:FiO2. The adjusted R2 was 0.39. The risk of ΔPes being >10 cmH2O can be predicted from BEa, respiratory rate, and PaO2:FiO2. The area under the receiver operating characteristic curve was 0.79 (0.73-0.85). We called these two models BREF, where BREF stands for BReathing EFfort and the three common predictors: BEa (B), respiratory rate (RE), and PaO2:FiO2 (F). CONCLUSIONS: We developed two models to estimate the breathing effort of patients on high-flow oxygen therapy. Our initial findings are promising and suggest that these models merit further evaluation.

Awake prone positioning in nonintubated spontaneous breathing ICU patients with acute hypoxemic respiratory failure (PRONELIFE)-protocol for a randomized clinical trial.

BACKGROUND: It is uncertain whether awake prone positioning can prevent intubation for invasive ventilation in spontaneous breathing critically ill patients with acute hypoxemic respiratory failure. Awake prone positioning could benefit these patients for various reasons, including a reduction in direct harm to lung tissue, and prevention of tracheal intubation-related complications. DESIGN AND METHODS: The PRONELIFE study is an investigator-initiated, international, multicenter, randomized clinical trial in patients who may need invasive ventilation because of acute hypoxemic respiratory failure. Consecutive patients admitted to participating ICUs are randomly assigned to standard care with awake prone positioning, versus standard care without awake prone positioning. The primary endpoint is a composite of tracheal intubation and all-cause mortality in the first 14 days after enrolment. Secondary endpoints include time to tracheal intubation and effects of awake prone positioning on oxygenation parameters, dyspnea sensation, and complications. Other endpoints are the number of days free from ventilation and alive at 28 days, total duration of use of noninvasive respiratory support, total duration of invasive ventilation, length of stay in ICU and hospital, and mortality in ICU and hospital, and at 28, 60, and 90 days. We will also collect data regarding the tolerance of prone positioning. DISCUSSION: The PRONELIFE study is among the first randomized clinical trials investigating the effect of awake prone positioning on intubation rate in ICU patients with acute hypoxemic failure from any cause. The PRONELIFE study is sufficiently sized to determine the effect of awake prone positioning on intubation for invasive ventilation-patients are eligible in case of acute hypoxemic respiratory failure without restrictions regarding etiology. The PRONELIFE study is a pragmatic trial in which blinding is impossible-however, as around 35 ICUs worldwide will participate in this study, its findings will be highly generalizable. The findings of the PRONELIFE study have the potential to change clinical management of patients who may need invasive ventilation because of acute hypoxemic respiratory failure. TRIAL REGISTRATION: ISRCTN ISRCTN11536318 . Registered on 17 September 2021. The PRONELIFE study is registered at clinicaltrials.gov with reference number NCT04142736 (October, 2019).

Thromboelastography clot strength profiles and effect of systemic anticoagulation in COVID-19 acute respiratory distress syndrome: a prospective, observational study.

OBJECTIVE: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) infection may yield a hypercoagulable state with fibrinolysis impairment. We conducted a single-center observational study with the aim of analyzing the coagulation patterns of intensive care unit (ICU) COVID-19 patients with both standard laboratory and viscoelastic tests. The presence of coagulopathy at the onset of the infection and after seven days of systemic anticoagulant therapy was investigated. PATIENTS AND METHODS: Forty consecutive SARS-CoV-2 patients, admitted to the ICU of a University hospital in Italy between 29th February and 30th March 2020 were enrolled in the study, providing they fulfilled the acute respiratory distress syndrome criteria. They received full-dose anticoagulation, including Enoxaparin 0.5 mg·kg-1 subcutaneously twice a day, unfractionated Heparin 7500 units subcutaneously three times daily, or low-intensity Heparin infusion. Thromboelastographic (TEG) and laboratory parameters were measured at admission and after seven days. RESULTS: At baseline, patients showed elevated fibrinogen activity [rTEG-Ang 80.5° (78.7 to 81.5); TEG-ACT 78.5 sec (69.2 to 87.9)] and an increase in the maximum amplitude of clot strength [FF-MA 42.2 mm (30.9 to 49.2)]. No alterations in time of the enzymatic phase of coagulation [CKH-K and CKH-R, 1.1 min (0.85 to 1.3) and 6.6 min (5.2 to 7.5), respectively] were observed. Absent lysis of the clot at 30 minutes (LY30) was observed in all the studied population. Standard coagulation parameters were within the physiological range: [INR 1.09 (1.01 to 1.20), aPTT 34.5 sec (29.7 to 42.2), antithrombin 97.5% (89.5 to 115)]. However, plasma fibrinogen [512.5 mg·dl-1 (303.5 to 605)], and D-dimer levels [1752.5 ng·ml-1 (698.5 to 4434.5)], were persistently increased above the reference range. After seven days of full-dose anticoagulation, average TEG parameters were not different from baseline (rTEG-Ang p = 0.13, TEG-ACT p = 0.58, FF-MA p = 0.24, CK-R p = 0.19, CKH-R p  = 0.35), and a persistent increase in white blood cell count, platelet count and D-dimer was observed (white blood cell count p < 0.01, neutrophil count p = 0.02, lymphocyte count p < 0.01, platelet count p = 0.13 < 0.01, D-dimer levels p= 0.02). CONCLUSIONS: SARS-CoV-2 patients with acute respiratory distress syndrome show elevated fibrinogen activity, high D-dimer levels and maximum amplitude of clot strength. Platelet count, fibrinogen, and standard coagulation tests do not indicate a disseminated intravascular coagulation. At seven days, thromboelastographic abnormalities persist despite full-dose anticoagulation.

Lung volumes, respiratory mechanics and dynamic strain during general anaesthesia.

BACKGROUND: Driving pressure (ΔP) represents tidal volume normalised to respiratory system compliance (CRS) and is a novel parameter to target ventilator settings. We conducted a study to determine whether CRS and ΔP reflect aerated lung volume and dynamic strain during general anaesthesia. METHODS: Twenty non-obese patients undergoing open abdominal surgery received three PEEP levels (2, 7, or 12 cm H2O) in random order with constant tidal volume ventilation. Respiratory mechanics, lung volumes, and alveolar recruitment were measured to assess end-expiratory aerated volume, which was compared with the patient's individual predicted functional residual capacity in supine position (FRCp). RESULTS: CRS was linearly related to aerated volume and ΔP to dynamic strain at PEEP of 2 cm H2O (intraoperative FRC) (r=0.72 and r=0.73, both P<0.001). These relationships were maintained with higher PEEP only when aerated volume did not overcome FRCp (r=0.73, P<0.001; r=0.54, P=0.004), with 100 ml lung volume increases accompanied by 1.8 ml cm H2O-1 (95% confidence interval [1.1-2.5]) increases in CRS. When aerated volume was greater or equal to FRCp (35% of patients at PEEP 2 cm H2O, 55% at PEEP 7 cm H2O, and 75% at PEEP 12 cm H2O), CRS and ΔP were independent from aerated volume and dynamic strain, with CRS weakly but significantly inversely related to alveolar dead space fraction (r=-0.47, P=0.001). PEEP-induced alveolar recruitment yielded higher CRS and reduced ΔP only at aerated volumes below FRCp (P=0.015 and 0.008, respectively). CONCLUSIONS: During general anaesthesia, respiratory system compliance and driving pressure reflect aerated lung volume and dynamic strain, respectively, only if aerated volume does not exceed functional residual capacity in supine position, which is a frequent event when PEEP is used in this setting.

Long-term pancreatic exocrine and endometabolic functionality after pancreaticoduodenectomy. Comparison between pancreaticojejunostomy and pancreatic duct occlusion with fibrin glue.

OBJECTIVE: Even if pancreatic pathologies, residual fibrosis, residual amount of parenchyma, and anastomotic patency are recognized as main causes of exocrine and glycemic impairment after pancreaticoduodenectomy (PD), few data are reported concerning the role of the different pancreatic remnant treatment techniques. The objective of the study is to assess and compare exocrine functionality, glycemic pattern, nutritional status, and quality of life (QoL) after PD between pancreaticojejunostomy (PJ) and pancreatic duct occlusion (PDO), both in an objective and a subjective manner. PATIENTS AND METHODS: Thirty-two patients (16 PJ and 16 PDO) were evaluated after a mean follow-up of 21 months after surgery. Exocrine insufficiency was objectively evaluated through the 13C-labelled mixed triglyceride breath test. Fasting glucose, fasting insulin, HbA1c and HOMA-IR values were used to assess glucose metabolism. For these two outcomes, anamnestic data were also collected. QoL was assessed with GIQLI, SF-36, EORTC-QLQ-C30, and EORTC-PAN-26 questionnaires. RESULTS: The 13C-labelled mixed triglyceride breath test detected a lipid digestive insufficiency in 56% of patients after PJ and 100% after PDO respectively (p = 0.007). However, no difference was observed between the two groups regarding postoperative necessity of substitutive pancreatic enzymes. Nutritional status, fasting plasma glucose, fasting insulin, HbA1c levels, HOMA-IR values and postoperative necessity of insulin or oral antidiabetic agents were comparable between the two groups. QoL measurements showed similar results. However, in the subdomains analysis, better outcomes were reported regarding digestive symptoms and physical functioning for PJ and PDO respectively. CONCLUSIONS: Even if an objective exocrine major impairment was evidenced after PDO, this result did not impact the need for a higher rate of postoperative substitutive enzymes. In terms of glycemic pattern, nutritional status, and QoL, the two techniques turn out to be comparable.

Taking Care of Relationships in the Intensive Care Unit: Positive Impact on Family Consent for Organ Donation.

BACKGROUND: Organ donation refusal from relatives of potential donors with brain death significantly reduces organ availability. The need for organ donation has increased over time, but the shortage of available donors is the major limiting factor in transplantation. We analyzed the impact of a new systematic communication approach between medical staff and patients' relatives on the rate of consent to organ donation. METHODS: The study was conducted as a single-center, non-randomized, controlled, before-and-after study at an 18-bed intensive care unit (ICU) of a university hospital. We compared the rate of consent for organ donation before and after the introduction of the new communication approach. RESULTS: A total of 291 brain-dead patients were studied. The consent rate increased from 71% in the pre-intervention period (2007-2012) to 78.4% in the post-intervention period (2013-2015), with an 82.75% increase in the 2014 to 2015 period. During these periods, no significant variation of consent to organ donation was recorded at the national and regional levels. CONCLUSIONS: The introduction of a new communication approach between medical staff and relatives of brain-dead patients was associated with a significant increase in the rate of consent to donation. Our results highlight the importance of empathy with relatives in the ICU.

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs.

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.

Successful ablation of ventricular tachycardia after correction of tetralogy of Fallot.

We present a case of a patient who, after correction of tetralogy of Fallot (TOF), experienced runs of ventricular tachycardia (VT). Mapping of the aortic root showed that the critical component of the reentry was located within the noncoronary cusp. The potential explanations of such an unusual isthmus location may be the presence of myocardial extensions in the aortic root or the close vicinity of the right ventricle (RV) to the noncoronary cusp, since in TOF the aorta overrides the RV. Our case highlights the advantage of using electroanatomic mapping systems together with intracardiac echocardiography in such complex cases.

Weight gain in antipsychotic-naive patients: a review and meta-analysis.

BACKGROUND: Weight gain is a long-recognized side-effect of antipsychotic (AP) drugs and a major health concern in the treatment of psychosis. The strength of the causal relationship between AP drug exposure and weight gain can only be gauged by a drugs trial conducted on AP-naive patients. METHOD: We conducted a review of the literature regarding the amount of weight gain induced by APs in AP-naive patients and carried out a meta-analysis of mean weight gains. RESULTS: We found 11 primary studies reporting the effects of APs on body weight or body mass index (BMI) in AP-naive patients. The mean body weight and BMI gains in AP-naive patients were highly significant from the first weeks of treatment. When we limited the analysis to studies conducted on patients hospitalized and without any adjunctive treatment potentially affecting weight, the resultant sample showed less heterogeneity and confirmed the final picture of weight gain at around 3.8 kg and 1.2 points BMI. CONCLUSIONS: Weight gain associated with AP therapy in AP-naive patients occurs rapidly in the first few weeks and continues during the following months. Clinicians should be aware of the high probability of causing weight gain in AP-naive patients and should strictly monitor such patients.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis.

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2d), C57BL6 (H-2b), DBA/2J (H-2d) and CBA/CaH (H-2k) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4+ and CD8+ T-cell subsets, CD14+ macrophages and CD19+ B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8+ T cells and CD19+ B cells were found in any of the lesions. The percentages of CD4+ cells, CD14+ cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14+ cells in sham-immunized mice. The percentage of CD14+ cells was higher than that of CD4+ cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4+ and CD14+ cells predominated in immunized CBA/CaH mice and CD4+ cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14+ cells and CD4+ cells in sham-immunized mice. IgG1+ plasma cells were more dominant than IgG2a+ cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a+ plasma cells were more obvious in sham-immunized mice. IgG2a+ plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

P. gingivalis-specific T-cell lines produce Th1 and Th2 cytokines.

Cytokines produced by T-cells in periodontal lesions may determine the nature of the adaptive immune response. Since different antigen-presenting cells (APC) may direct the Th1/Th2 response, P. gingivalis-specific T-cell lines were established by different APC subpopulations, and their cytokine profiles were determined. Peripheral blood mononuclear cells induced similar percentages of IL-4+ and IFN-gamma+ T-cells and lower percentages of IL-10+ T-cells. Epstein-Barr virus-transformed B-cells (LCL) induced higher percentages of IL-4+ cells than IFN-gamma+ cells, with lower percentages of IL-10+ cells. Peripheral blood mononuclear cells induced a higher percent of IFN-gamma+ CD8 cells than LCL (p = 0.004). Purified B-cells, monocytes, and dendritic cells induced similar percentages of IL-4+ and IFN-gamma+ cells, although again, the percentage of IL-10+ cells was lower. The results of the present study have demonstrated that, as measured by FACS analysis of intracytoplasmic cytokines, P. gingivalis-specific T-cells produce both Th1 and Th2 cytokines, regardless of the APC population.

Role for cyclin-dependent kinase 2 in mitosis exit.

Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity [1]. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B [2]. Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable [5]. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 [6]. Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit.

Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication.

Mre11 complex promotes repair of DNA double-strand breaks (DSBs). Xenopus Mre11 (X-Mre11) has been cloned, and its role in DNA replication and DNA damage checkpoint studied in cell-free extracts. DSBs stimulate the phosphorylation and 3'-5' exonuclease activity of X-Mre11 complex. This induced phosphorylation is ATM independent. Phosphorylated X-Mre11 is found associated with replicating nuclei. X-Mre11 complex is required to yield normal DNA replication products. Genomic DNA replicated in extracts immunodepleted of X-Mre11 complex accumulates DSBs as demonstrated by TUNEL assay and reactivity to phosphorylated histone H2AX antibodies. In contrast, the ATM-dependent DNA damage checkpoint that blocks DNA replication initiation is X-Mre11 independent. These results strongly suggest that the function of X-Mre11 complex is to repair DSBs that arise during normal DNA replication, thus unraveling a critical link between recombination-dependent repair and DNA replication.

Genetic variation in the recognition of Porphyromonas gingivalis antigens in mice.

T-cell cytokine profiles, anti-Porphyromonas gingivalis antibodies and Western blot analysis of antibody responses were examined in BALB/c, CBA/CaH, C57BL6 and DBA/2J mice immunized intraperitoneally with different doses of P. gingivalis outer membrane antigens. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by FACS analysis and levels of anti-P. gingivalis antibodies in the serum samples determined by enzyme-linked immunosorbent assay. Western blot analysis was performed on the sera from mice immunized with 100 microg of P. gingivalis antigens. The four strains of mice demonstrated varying degrees of T-cell immunity, although the T-cell cytokine profiles exhibited by each strain were not affected by different immunizing doses. While BALB/c and DBA/2J mice exhibited responses that peaked at immunizing doses of 100-200 microg of P. gingivalis antigens, CBA/CaH and C57BL6 demonstrated weak T-cell responsiveness compared with control mice. Like the T-cell responses, serum antibody levels were not dose dependent. DBA/2J exhibited the lowest levels of anti-P. gingivalis antibodies followed by BALB/c with CBA/CaH and C57BL6 mice demonstrating the highest levels. Western blot analysis showed that there were differences in reactivity between the strains to a group of 13 antigens ranging in molecular weight from 15 to 43 kDa. Antibody responses to a number of these bands in BALB/c mice were of low density, whereas CBA/CaH and C57BL6 mice demonstrated high-density bands and DBA/2J mice showed medium to high responses. In conclusion, different immunizing doses of P. gingivalis outer membrane antigens had little effect on the T-cell cytokine responses and serum anti-P. gingivalis antibody levels. Western blot analysis, however, indicated that the four strains of mice exhibited different reactivity to some lower-molecular-weight antigens. Future studies are required to determine the significance of these differences, which may affect the outcome of P. gingivalis infection.

Costimulatory molecules in human periodontal disease tissues.

An immunoperoxidase technique was used to examine CD28, CD152, CD80 and CD86 positive cells in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups (small, intermediate, large) according to the size of the infiltrate. The percent CD28+ T cells in the connective tissue infiltrates was highly variable with no differences between the healthy/gingivitis and periodontitis groups. While there was an increase in positive cells in intermediate infiltrates from both healthy/gingivitis (28.5%) and periodontitis (21.4%) patients compared with small infiltrates (8.6% and 11.8%, respectively), this was not significant, although the percent CD28+ T cells did increase significantly in tissues with increased proportions of B cells relative to T cells (p=0.047). A mean of less than 5% infiltrating T cells were CD152+ which was significantly lower than the mean percent CD28+ T cells in intermediate healthy/gingivitis lesions (p = 0.021). The mean percent CD80+ and CD86+ B cells and macrophages was 1-7% and 8-16%, respectively, the difference being significant in intermediate healthy/gingivitis tissues (p = 0.012). Analysis of these cells in relation to increasing numbers of B cells in proportion to T cells and also to macrophages, suggested that CD80 was expressed predominantly by macrophages while CD86 was expressed by both macrophages and B cells. Few endothelial cells expressed CD80 or CD86. Keratinocytes displayed cytoplasmic staining of CD80 rather than CD86 although the numbers of positive specimens in the healthy/gingivitis and periodontitis groups reduced with increasing inflammation. In conclusion, percentages of CD28, CD152, CD80 and CD86 did not reflect differences in clinical status. However, the percent CD28+ T cells increased with increasing size of infiltrate and with increasing proportions of B cells suggesting increased T/B cell interactions with increasing inflammation. The percent CD152+ cells remained low indicating that CD152 may not be involved in negative regulation of T cells in periodontal disease. CD80 and CD86 have been reported to promote Th1 and Th2 responses, respectively, and the higher percent CD86+ cells suggests a predominance of Th2 responses in both healthy/gingivitis and periodontitis tissues. Nevertheless, other factors including cytokines themselves and chemokines which modulate T cell cytokine profiles must be monitored to determine the nature of Th1/Th2 responses in periodontal disease.

Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage.

Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.

The influence of genetic variation on the splenic T cell cytokine and specific serum antibody responses to Porphyromonas gingivalis in mice.

BACKGROUND: T cell cytokine profiles in the spleens and anti-Porphyromonas gingivalis antibodies in the sera of P. gingivalis-immunized BALB/c (H-2d), CBA/CaH (H-2k), C57BL6 (H-2b), and DBA/2J (H-2d, C5 deficient) mice were examined. METHODS: Mice were immunized either by intraperitoneal injections of P. gingivalis outer membrane antigens and Freund's incomplete adjuvant weekly for 3 weeks or sham-immunized with PBS and adjuvant, followed by subcutaneous challenge with live organisms 1 week after the final immunization. Spleens were excised and blood samples collected by heart puncture at 0 and 7 days after challenge. Splenic CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IF)-gamma, and IL-10 and levels of anti-P. gingivalis antibodies in the serum samples determined by ELISA. RESULTS: Lesion sizes in immunized BALB/c mice remained stable for the 7-day experimental period. Immunized CBA/CaH and C57BL6 mice exhibited large lesions at day 1 reducing by day 7 particularly in the latter strain. Lesions in immunized DBA/2J mice were still larger than the other strains at day 7. With the exception of DBA/2J mice, sham-immunized mice demonstrated lesions which did not show signs of healing by day 7. T cell cytokine responses in sham-immunized mice at day 0 were low, increasing to a variable degree by day 7 after challenge in the 4 strains. Immunized BALB/c mice demonstrated intermediate T cell responses while generally exhibiting a stronger IFN-gamma response than IL-4 or IL-10. Immunized CBA/CaH and C57BL6 mice showed weak T cell cytokine responses while immunized DBA/2J displayed the strongest T cell responses particularly in regard to IL-4 positive cells. Sham-immunized mice had low levels of serum anti-P. gingivalis antibody levels at day 0 with levels increasing significantly by day 7 after challenge. Antibody levels in immunized mice seemed to correlate with lesion sizes. Immunized C57BL6 mice had the highest antibody levels followed by CBA/CaH, BALB/c with DBA/2J exhibiting low levels. The T cell and B cell antibody responses in each strain appeared to exhibit an inverse relationship. CONCLUSIONS: This study has shown that genetic differences at the level of H-2 haplotype induce variations in the local and T and B cell responses to P. gingivalis antigens. The responses of DBA/2J mice which have the same haplotype as BALB/c mice suggest that factors other than H-2 haplotype such as the C5 deficiency may influence this immune response. The significance of the specific antibody and T cell responses and of their inverse relationship to susceptibility to periodontal disease remains to be determined.

Chemokine expression in Porphyromonas gingivalis-specific T-cell lines.

Autologous non-T cells (monocytes and B cells) were added to Porphyromonas gingivalis-specific T cell lines established from 9 healthy adults together with P. gingivalis outer membrane antigens for 4-6, 16-18, 24 and 48 h. Flow cytometry was employed to analyze the CD4 and CD8 cells, monocytes and B cells for intracytoplasmic IP-10 (interferon-gamma inducible protein 10), MCP-1 (monocyte chemoattractant protein 1), MIP-1 alpha (macrophage inflammatory protein 1 alpha) and RANTES (regulated on activation normal T cell expressed and secreted) at the four time periods. All cell types were positive for each chemokine throughout the 48-h time period. There were significantly fewer MCP-1-positive cells compared with the other 3 chemokines. However, the percentages of MCP-1, MIP-1 alpha- and RANTES-positive CD8 cells were significantly higher than the percentages of positive CD4 cells in all cultures. IP-10-positive CD4, CD14-positive monocytes and CD19-positive B cells were predominant compared with MIP-1 alpha- and RANTES-positive cells at 24 h. In conclusion, the present study has shown that P. gingivalis-specific T cells, monocytes and B cells produce chemokines in response to P. gingivalis outer membrane antigens, IP-10 being predominant, with MCP-1 being significantly reduced in comparison with IP-10, MIP-1 alpha and RANTES. Increased percentages of CD8 cells were induced to produce chemokines in comparison with CD4 cells, indicating a more preferential action on CD8 rather than CD4 cells.