CD8 expression alters the fine specificity of an alloreactive MHC class I-specific T hybridoma.

The influence of CD8 on the fine specificity of MHC class I-restricted T cell allorecognition was evaluated by comparing the reactivity of CD8- and CD8-transfected forms of an allospecific, H-2Kb-restricted T hybridoma. The CD8- T hybridoma responded to cells expressing H-2Kb, H-2Kbm6, and the individual H-2Kb----bm10 back mutations 165V----M, 173K----E, and 174N----L. Under the same conditions the CD8- T hybridoma responded poorly or not at all to cells expressing H-2Kbm10, H-2Kbm8, the individual H-2Kb----bm10 back mutants 163T----A and 167W----S, and the individual H-2Kb----bm8 back mutations 22Y----F and 24E----S. In contrast, T hybridoma cells expressing high levels of CD8 reacted strongly with antigen presenting cells (APC) expressing H-2Kb and H-2Kbm6 molecules, as well as APC expressing H-2Kbm10 (weakly), H-2Kbm8, and all five individual H-2Kb----bm10 and the two H-2Kb----bm8 back mutants 22Y----F and 24E----S. The mutations which distinguish the T cell recognition of both H-2Kbm10 and H-2Kbm8 from H-2Kb are predicted to control the interaction of these class I molecules with antigenic peptides in the binding site, implying an important role for peptide antigen in T cell allorecognition. Nonetheless, CD8 expression by the H-2Kb-restricted T cells conferred novel or enhanced alloreactivity with cells expressing H-2Kbm10, H-2Kbm8, and each of the individual H-2Kb----bm10 and H-2Kb----bm8 back mutants. These findings reflect an important role for CD8 in influencing the fine specificity of MHC class I recognition by T cells and may indicate a limited structural role for peptide antigen in defining the ligand recognized by these alloreactive T cells.     

Effects of mercury on human polymorphonuclear leukocyte function in vitro.

A variety of heavy metals are recognized as environmental pollutants, and although a significant body of literature exists on the acute toxicity of these metals in various tissues, little is known about the effects of metals such as mercury on host defense. Therefore, the effect of mercuric chloride (HgCl2) on human polymorphonuclear leukocytes (PMN) function in vitro was evaluated. The acute toxicity of HgCl2 for human PMN was calculated initially using vital dye exclusion (trypan blue), and lactate dehydrogenase (LDH) release. Concentrations of HgCl2 less than or equal to 10(-6) M did not induce significant LDH release, or uptake of trypan blue. Additionally, HgCl2 at less than or equal to 10(-7) M produced no ultrastructural alterations in the PMN. The effects of HgCl2 on human PMN functions involved in host defense were evaluated next. HgCl2 consistently suppressed human PMN adherence, polarization, chemotaxis, and erythrophagocytosis at concentrations between 10(-6) and 10(-17) M. Because of the established role of oxygen metabolites in host defense, the effects of HgCl2 on human PMN chemiluminescence and H2O2 production were evaluated next. These studies demonstrated that low concentrations of HgCl2 (ie, 10(-9)-10(-15) M) significantly enhanced chemiluminescence, as well as stimulated H2O2 production by the PMN. These studies clearly demonstrate the ability of extremely low levels of HgCl2 not only to suppress various PMN functions involved in host defense, but also to stimulate oxygen metabolism. In vivo, these HgCl2 effects would not only compromise host defense but also promote tissue injury via the local production of oxygen metabolites.     

Considerations in choosing a primary endpoint that measures durability of virological suppression in an antiretroviral trial

OBJECTIVES: At present, many clinical trials of anti-HIV-1 therapies compare treatments by a primary endpoint that measures the durability of suppression of HIV-1 replication. Several durability endpoints are compared. DESIGN: Endpoints are compared by their implicit assumptions regarding surrogacy for clinical outcomes, sample size requirements, and accommodations for inter-patient differences in baseline plasma HIV-1-RNA levels and in initial treatment response. METHODS: Virological failure is defined by the non-suppression of virus levels at a prespecified follow-up time T(early virological failure), or by relapse. A binary virological failure endpoint is compared with three time-to-virological failure endpoints: time from (i) randomization that assigns early failures a failure time of T weeks; (ii) randomization that extends the early failure time T for slowly responding subjects; and (iii) virological response that assigns non-responders a failure time of 0 weeks. Endpoint differences are illustrated with Agouron's trial 511. RESULTS: In comparing high with low-dose nelfinavir (NFV) regimens in Agouron 511, the difference in Kaplan-Meier estimates of the proportion not failing by 24 weeks is 16.7% (P = 0.048), 6.5% (P = 0.29) and 22.9% (P = 0.0030) for endpoints (i), (ii) and (iii), respectively. The results differ because NFV suppresses virus more quickly at the higher dose, and the endpoints weigh this treatment difference differently. This illustrates that careful consideration needs to be given to choosing a primary endpoint that will detect treatment differences of interest. CONCLUSION: A time from randomization endpoint is usually recommended because of its advantages in flexibility and sample size, especially at interim analyses, and for its interpretation for patient management.     

Glucose homeostasis in acromegaly: effects of long-acting somatostatin analogues treatment

OBJECTIVE: Acromegaly is a syndrome with a high risk of impaired glucose tolerance (IGT) and diabetes mellitus (DM). Somatostatin analogues, which are used for medical treatment of acromegaly, may exert different hormonal effects on glucose homeostasis. Twenty-four active acromegalic patients were studied in order to determine the long-term effects of octreotide-LAR and SR-lanreotide on insulin sensitivity and carbohydrate metabolism. DESIGN: Prospective study. PATIENTS: We studied 24 patients with active acromegaly, 11 males and 13 females, aged 50.7 +/- 12.7 years, body mass index (BMI) 30.1 +/- 4.8 (kg/m2). MEASUREMENTS: All patients underwent an oral glucose tolerance test (OGTT) and 12 also had an euglycaemic hyperinsulinaemic clamp. All patients were evaluated at baseline and after 6 months of somatostatin analogues therapy. RESULTS: Acromegalic patients showed low M-values in respect to the control group at baseline (P<0.05), followed by a significant improvement after 6 months of therapy (P<0.005 vs. baseline). Serum glucose levels at 120 min during OGTT worsened (P<0.05) during somatostatin analogs therapy in patients with normal glucose tolerance, but not in those with impaired glucose tolerance or diabetes mellitus. This was associated with a reduced (P<0.05) and 30 min delayed insulin secretion during OGTT. Also, HbA1c significantly deteriorated in all subjects after treatment (4.7 +/- 0.6% and 5.1 +/- 0.5%, basal and after six months, respectively, P<0.005). CONCLUSION: In acromegalic patients, somatostatin analogues treatment reduces insulin resistance, and also impairs insulin secretion. This may suggest that the use of oral secretagogue hypoglycaemic agents and/or insulin therapy should be considered rather than insulin sensitizers, as the treatment of choice in acromegalic patients who develop frank hyperglycaemia during somatostatin analogues therapy.     

Investigation of the molecular reactivity of bioactive oxiranylmethyloxy anthraquinones

The design of a multitarget and multifunctional small molecule containing two functional groups reacting through different mechanisms represents an attractive goal for the medicinal chemist. The preparation of two bifunctional oxiranylmethyloxy anthraquinones, previously investigated as anticancer agents, is described here. These compounds combine a planar, DNA‐intercalating and pro‐oxidant anthraquinone scaffold and the alkylating epoxide functions which can covalently react with the nucleic acid. Their multilevel molecular reactivity was studied through a combination of analytical techniques: The DNA‐binding properties were investigated using a mass spectrometry‐based binding assay and by nuclear magnetic resonance, highlighting the formation of a covalent adduct with a nucleobase. Moreover, the contribution of the pro‐oxidant redox cycling was evaluated.     

Differential association of beta2-microglobulin mutants with MHC class I heavy chains and structural analysis demonstrate allele-specific interactions.

Dynamic interactions between major histocompatibility complex (MHC) class I heavy chains and beta2-microglobulin (beta2m) play a critical role in their stability on the cell surface, and their ability to present peptide antigens to CD8+ T-cells. A cursory review of protein sequence homologies and three-dimensional crystal structures of MHC complexes might indicate very similar modes of interaction between the heavy and light chains. In this report, a panel of human beta2m mutants was screened to probe the interactions of beta2m with the murine MHC molecules H-2Kb, -Db, -Kd, -Ld, and -Dd. Binding experiments coupled with analyses of existing three-dimensional crystal structures demonstrate allelic differences in their interaction with beta2m. A comprehensive analysis of the existing murine MHC structures indicates a conformational flexibility on the part of murine beta2m that is not present in beta2m of the human structures. This flexibility is in a region directly interacting with the heavy chain and may relate to its lower affinity for murine heavy chains relative to human beta2m. This defined panel of beta2m mutants of differing affinity may also be useful for subsequent studies of thymic selection, T-cell recognition, and more refined algorithms for protein structure prediction.     

A randomized trial of 2 different 4-drug antiretroviral regimens versus a 3-drug regimen,in advanced human immunodeficiency virus disease

To compare long-term virologic benefits of antiretroviral regimens in persons with advanced human immunodeficiency virus (HIV) disease, a randomized, open-label study was conducted of 517 subjects with no or limited previous experience with antiretroviral therapy. Subjects received lamivudine plus zidovudine and indinavir (indinavir group), efavirenz plus indinavir (efavirenz + indinavir group), or nelfinavir plus indinavir (nelfinavir + indinavir group) and were monitored for 2.1 years. Virologic failure was lower in the efavirenz + indinavir group (P=.04) and higher in the nelfinavir + indinavir group (P=.006), compared with that in the indinavir group. No difference in grade 3 or 4 adverse event rates in the efavirenz + indinavir group (P=.97) and a trend toward an increased rate in the nelfinavir + indinavir group (P=.07), compared with the indinavir group, were noted. A 4-drug regimen containing efavirenz plus indinavir resulted in a superior virologic response, whereas one containing nelfinavir plus indinavir resulted in an inferior response and a greater likelihood of toxicity.     

Gynecologic aspects of Crohn's disease.

Because Crohn's disease has a tendency to be transmural and to form fistulas, it may involve contiguous organs. Gynecologic involvement is frequent, diverse and often difficult to diagnose. Inflammation involving adjacent structures has been reported in as many as one third of patients. Enteric fistulas to the vagina, uterus, ovaries, perineum and vulva also have been reported. Diagnosis may not be obvious if pelvic involvement precedes active bowel disease, or if drainage is clear or mucoid. Abscesses, draining sinuses, edema and ulceration of the perineum or vulva are common, and are caused by direct extension from the involved bowel or by granulomas separated from the bowel by normal tissue. Lesions presenting with vulvar hypertrophy, a fluctuant mass or ulceration are easily misdiagnosed. Menstrual abnormalities are reported in more than one half of patients. Pelvic manifestations of Crohn's disease can be psychologically crippling. Patients or physicians may be hesitant to address serious psychosocial morbidity. To optimize management, physicians must be aware of the diverse manifestations, confusing presentations and psychologic morbidity of Crohn's disease.     

Achieving house staff competence in emergency airway management: results of a teaching program using a computerized patient simulator

OBJECTIVES: Patient simulation is emerging as a training technique in the field of medicine. It has particular application in training responses to high-risk, low-frequency clinical events, of which a typical example is in-hospital cardiac arrest. A critical element of response by the cardiac arrest team is initial airway management. In teaching hospitals, medical interns are first responders to in-hospital cardiac arrests. Our objective was to design and test a program using a computer-controlled patient simulator to train medical interns and demonstrate their competence in initial airway management. DESIGN: Prospective, randomized, controlled, unblinded trial. SETTING: Internal medicine residency training program in an urban teaching hospital. PARTICIPANTS: All 50 starting internal medicine interns in July 2002, all Advanced Cardiac Life Support certified in June 2002. INTERVENTIONS: All interns were tested in initial airway management skills and then were randomly assigned to receive either immediate or delayed individualized training using a computer-controlled patient simulator. The computer-simulated training process consisted of a scenario of respiratory arrest. The interns were challenged with the scenario twice following testing. The interns were debriefed extensively and given hands-on training by the attending using the simulator until they achieved perfect performance. MEASUREMENTS AND MAIN RESULTS: Initial airway management was divided into specific scorable steps. Individual step scores and total scores were recorded for each intern on initial and repeat testing. For 10 months following simulator training, intern airway management skills were scored in actual patient airway events. Despite recent Advanced Cardiac Life Support training and certification, all starting medical interns demonstrated poor airway management skills. The immediate training group showed significant improvement in initial airway management when tested before and 4 wks after training. In contrast, the delayed training group showed no significant improvement. Direct observation of interns in actual initial airway events revealed excellent clinical performance. CONCLUSIONS: Individualized training of medical interns using a computer-controlled patient simulator is an effective means of achieving and measuring competence in initial airway management skills. The improvement appears to be transferable to the bedside of real patients.