Paediatric meningitis in the conjugate vaccine era and a novel clinical decision model to predict bacterial aetiology.

OBJECTIVES: The aims of this study were to assess aetiology and clinical characteristics in childhood meningitis, and develop clinical decision rules to distinguish bacterial meningitis from other similar clinical syndromes. METHODS: Children aged <16 years hospitalised with suspected meningitis/encephalitis were included, and prospectively recruited at 31 UK hospitals. Meningitis was defined as identification of bacteria/viruses from cerebrospinal fluid (CSF) and/or a raised CSF white blood cell count. New clinical decision rules were developed to distinguish bacterial from viral meningitis and those of alternative aetiology. RESULTS: The cohort included 3002 children (median age 2·4 months); 1101/3002 (36·7%) had meningitis, including 180 bacterial, 423 viral and 280 with no pathogen identified. Enterovirus was the most common pathogen in those aged <6 months and 10-16 years, with Neisseria meningitidis and/or Streptococcus pneumoniae commonest at age 6 months to 9 years. The Bacterial Meningitis Score had a negative predictive value of 95·3%. We developed two clinical decision rules, that could be used either before (sensitivity 82%, specificity 71%) or after lumbar puncture (sensitivity 84%, specificity 93%), to determine risk of bacterial meningitis. CONCLUSIONS: Bacterial meningitis comprised 6% of children with suspected meningitis/encephalitis. Our clinical decision rules provide potential novel approaches to assist with identifying children with bacterial meningitis. FUNDING: This study was funded by the Meningitis Research Foundation, Pfizer and the NIHR Programme Grants for Applied Research.

Surgical Techniques for Performing a Through-Knee Amputation: A Systematic Review and Development of an Operative Descriptive System.

BACKGROUND: Through-knee amputation (TKA) carries potential biomechanical advantages over above knee amputation (AKA) in patients unsuitable for a below-knee amputation. However, concerns regarding prosthetic fit, cosmesis and wound healing have tempered enthusiasm for the operation. Furthermore, there are many described surgical techniques for performing a TKA. This frustrates attempts to compare past and future comparative data, limiting the opportunity to identify which procedure is associated with the best patient centered outcomes. The aim of this systematic review is to identify all the recognized operative TKA techniques described in the literature and to develop a clear descriptive system to support future research in this area. METHODS: A systematic review was performed, searching the OVID, PubMed, and Cochrane Library databases, according to Cochrane and PRISMA guidelines. Papers of any design were included if they described an operative technique for a TKA. Key operative descriptions were captured and used to design a classification system for surgical techniques. RESULTS: A total of 906 papers were identified, of which 28 are included. The most important distinctions in operative technique were the level of division of the femur (disarticulation without bone division, transcondylar amputation, with or without shaving of the medial, lateral, and posterior condyles and supracondylar amputation), management of the patella (kept whole, partially preserved, completely removed), use of a muscular gastrocnaemius flap, and skin incisions. A 4-component classification system was developed to be able to describe TKA operative techniques. A suggested shorthand nomenclature uses the first letter of each component (FPMS; Femur, Patella, Muscular flap, Skin incision), followed by a number, to describe the operation. Patient outcomes were poorly reported, and therefore outcomes for different types of TKA are not addressed in this review. CONCLUSIONS: A novel descriptive system for describing different techniques for performing a TKA has been developed. This classification system will help in reporting, comparing, and interpreting past and future studies of patients undergoing TKA.

Junior doctors' wellbeing at peak and post-peak pandemic: a repeated cross-sectional study.

INTRODUCTION: The impact of the COVID-19 pandemic on healthcare professionals has been significant. The aim of this study was to explore the mental state and wellbeing of UK junior doctors at different phases of the initial outbreak. METHODS: This is a cross-sectional study of UK-based junior doctors' perceptions of threat and support during and after the first wave of the COVID-19 pandemic. Levels of anxiety, depression, post-traumatic stress disorder symptoms and use of coping mechanisms were explored through a Google questionnaire. RESULTS: 196 participants were included in this study (93 in period A and 103 in period B). Junior doctors reported feeling increased risk (p=0.001) and increased fear of contracting the virus (p<0.001) during period A. Increased levels of severe anxiety (Generalized Anxiety Disorder-7 score >15) along with increased cases level of depression (Patient Health Questionnaire-9 score >10) were reported for both periods. Junior doctors described suffering more frequently with flashbacks (p=0.006) and nightmares (p=0.024) in comparison with senior colleagues during period A. During period A, 21.4% of participants felt isolated at work (p<0.001), whereas 13% reported being easily annoyed on a daily basis, 11.7% reported very low morale (p<0.001) and 66% were not aware of any psychological support being available. The use of exercise, peer support and mindfulness apps increased during period B (p=0.023). CONCLUSIONS: Healthcare systems need to urgently establish robust psychological support mechanisms and infrastructure to protect junior doctors and provide institutional resilience against the adverse consequences of the long physical and mental battle with COVID-19.

Physiological performance and inflammatory markers as indicators of complications after oesophageal cancer surgery.

BACKGROUND: The extent to which physiological factors influence outcome following oesophageal cancer surgery is poorly understood. This study aimed to evaluate the extent to which cardiorespiratory fitness and selected metabolic factors predicted complications after surgery for carcinoma. METHODS: Two hundred and twenty-five consecutive patients underwent preoperative cardiopulmonary exercise testing to determine peak oxygen uptake ( V ˙ o2peak ), anaerobic threshold and the ventilatory equivalent for carbon dioxide ( V ˙ e/ V ˙ co2 ). Cephalic venous blood was assayed for serum C-reactive protein (CRP) and albumin levels, and a full blood count was done. The primary outcome measure was the Morbidity Severity Score (MSS). RESULTS: One hundred and ninety-eight patients had anatomical resection. A high MSS (Clavien-Dindo grade III or above) was found in 48 patients (24·2 per cent) and was related to an increased CRP concentration (area under the receiver operating characteristic (ROC) curve (AUC) 0·62, P = 0·001) and lower V ˙ o2peak (AUC 0·36, P = 0·003). Dichotomization of CRP levels (above 10 mg/l) and V ˙ o2peak (below 18·6 ml per kg per min) yielded adjusted odds ratios (ORs) for a high MSS of 2·86 (P = 0·025) and 2·92 (P = 0·002) respectively. Compared with a cohort with a low Combined Inflammatory and Physiology Score (CIPS), the OR was 1·70 (95 per cent c.i. 0·85 to 3·39) for intermediate and 27·47 (3·12 to 241·69) for high CIPS (P < 0·001). CONCLUSION: CRP and V ˙ o2peak were independently associated with major complications after potentially curative oesophagectomy for cancer. A composite risk score identified a group of patients with a high risk of developing complications.

ANTECEDENTES: El grado en el que los factores fisiológicos influyen en el resultado tras la cirugía del cáncer de esófago no se conoce bien. Este estudio tuvo como objetivo evaluar en qué medida el estado cardiorrespiratorio y los factores metabólicos seleccionados predecían complicaciones después de cirugía por cáncer. MÉTODOS: Pacientes consecutivos fueron sometidos a una prueba de ejercicio cardiopulmonar preoperatoria para determinar el consumo pico de oxígeno (peak oxygen uptake, V ̇ O2Peak ), el umbral anaeróbico (anaerobic threshold, AT) y el equivalente ventilatorio de dióxido de carbono (ventilatory equivalent for carbon dioxide, V ̇ E / V ̇ CO2 ). Se extrajo sangre de la vena cefálica para analizar la proteína C reactiva (C-reactive protein, CRP) sérica, albumina y hemograma completo. La medida de resultado primario fue la puntuación de la gravedad de la morbilidad (Morbidity Severity Score, MSS). RESULTADOS: Se observó MSS (Clavien-Dindo > 2) en 33 (17,7%) pacientes, relacionándose con CRP elevada (AUC 0,69, P = 0,001) y V ̇O2Peak baja (AUC 0,33, P = 0,003). La dicotomización de la CRP (por encima de 10 mg/L) y V ̇O2Peak (por debajo de 18,6 mL/kg/min) se asociada a una razón de oportunidades (odds ratio, OR) de 4,01 (P = 0,002) y 3,74 (P = 0,002) para MSS y CD > 2, respectivamente. En comparación con la cohorte con una puntuación combinada inflamatoria y fisiológica (Combined Inflammatory and Physiology Score, CIPS) baja, el OR fue de 1,70 (i.c. del 95% 0,85-3,39) para una CIPS intermedia y de 27,47 (3,12-241,69, P < 0,001) para CIPS elevada. CONCLUSIÓN: CRP y V ̇O2Peak se asociaron de forma independiente con complicaciones mayores tras esofaguectomía potencialmente curativa por cáncer. Una puntuación combinada de riesgo identificó a un grupo de pacientes con un riesgo elevado de desarrollar complicaciones.

Neutrophil to lymphocyte ratio as a predictor of response to neoadjuvant chemotherapy and survival in oesophageal adenocarcinoma.

BACKGROUND: Inflammation has an important role in cancer survival, yet whether serum markers of inflammation predict response to potentially curative neoadjuvant chemotherapy (NAC) in oesophageal adenocarcinoma (OAC) is controversial. This study aimed to determine whether the systemic inflammatory response (SIR) is associated with response to NAC and survival. METHODS: Consecutive patients with OAC planned for surgery with curative intent received blood neutrophil and lymphocyte measurements at diagnosis to calculate the neutrophil to lymphocyte ratio (NLR). Pathological variables including pTNM stage, differentiation, vascular invasion and Mandard tumour regression grade (TRG) were recorded. TRGs 1 and 2 were taken to represent a good response, and the primary outcome was overall survival. RESULTS: During follow-up of 136 patients, 36 patients (26·5 per cent) had recurrence and 69 (50·7 per cent) died. Receiver operating characteristic (ROC) curve analysis of NLR before NAC predicted poor TRG (area under the ROC curve 0·71, 95 per cent c.i. 0·58 to 0·83; P = 0·002). In univariable analysis, pT category (P < 0·001), pN category (P < 0·001), poor differentiation (P = 0·006), margin positivity (P = 0·001), poor TRG (P = 0·014) and NLR (dichotomized at 2·25; P = 0·017) were associated with poor overall survival, and NLR retained independent significance in multivariable analysis (hazard ratio 2·26, 95 per cent c.i. 1·03 to 4·93; P = 0·042). CONCLUSION: The pretreatment NLR was associated with a pathological response to NAC and overall survival in patients with OAC. ANTECEDENTES: La inflamación juega un importante papel en la supervivencia por cáncer, aunque aún no se sabe si los marcadores séricos de inflamación predicen la respuesta a la quimioterapia neoadyuvante (neoadjuvant chemotherapy, NAC) potencialmente curativa en el adenocarcinoma de esófago (oesophageal adenocarcinoma, OAC). Este estudio se propuso determinar si la respuesta inflamatoria sistémica (systemic inflammatory response, SIR) estaba asociada con la respuesta a la NAC y a la supervivencia. MÉTODOS: A pacientes consecutivos con OAC en los que se planificó cirugía con intención curativa se les determinó neutrófilos y linfocitos en sangre en el momento del diagnóstico para calcular la tasa neutrófilo-linfocito (neutrophil-lymphocyte ratio, NLR). Se registraron variables patológicas que incluían el estadio pTNM, diferenciación tumoral, invasión vascular y grado de regresión tumoral (tumour regression grade, TRG) de Mandard. Los grados TRG 1 y 2 fueron considerados como una buena respuesta y el resultado primario fue la supervivencia global (overall survival, OS). RESULTADOS: Durante el seguimiento de 136 pacientes, 36 pacientes (26,5%) presentaron recidiva y 69 pacientes (50,7%) fallecieron. El análisis de las características operativas del receptor (receiver-operator-characteristic, ROC) de NLR antes de la NAC predijo una pobre TRG (área bajo la curva ROC, AUC 0,71, i.c. del 95% 0,58-0,83, P = 0,002). En el análisis univariable, el estadio pT (P < 0,001), el estadio pN (P < 0,001), una pobre diferenciación tumoral (P = 0,006), un margen positivo (P = 0,001), una pobre TRG (P = 0,014) y la NLR (dicotomizada a 2,25, P = 0,017) se asociaron con una pobre OS, pero solamente la NLR (cociente de riesgos instantáneos, hazard ratio, HR 2,28, i.c. del 95% 1,03-4,93, P = 0,042) conservó la significación estadística como variable independiente en el análisis multivariable. CONCLUSIÓN: La NLR antes del tratamiento se asoció con respuesta patológica del OAC a la NAC y OS.

Attitudes and barriers to providing dietary advice: perceptions of dental hygienists and oral health therapists.

OBJECTIVES: Effective dietary counselling in a dental setting can significantly reduce the risk of oral disease. However, studies suggest that dental professionals are not instigating dietary advice on a regular basis, and there is a lack of current information of the barriers experienced that limit the delivery of dietary advice. The aim of this study was to investigate the current attitudes and practice behaviours of dental hygienists and oral health therapists in NSW, Australia, regarding dietary advice, and identify the barriers that limit its delivery. METHODS: A convenience sample of dental hygienists and oral health therapists were surveyed using a mail-out questionnaire. The questionnaire investigated the demographic data of participants, the attitudes and practice behaviours of participants, the perceived barriers and current dietary resources accessed by participants. RESULTS: Of 987 dental hygienists and oral health therapists, 426 participants responded. The study results suggest that many dental hygienists and oral health therapists have positive beliefs regarding the importance of dietary counselling. However, there are a multitude of barriers preventing the delivery of dietary advice; these include time, patient compliance, patient knowledge of nutrition topics, personal counselling skills and practitioners' knowledge of nutrition. CONCLUSION: Whilst dental hygienists and oral health therapists recognize the importance of diet and have positive attitudes towards providing dietary advice to patients, this study identified many barriers preventing implementation in practice. This information may be used to develop targeted strategies aimed at overcoming these barriers and improving behaviours.

Anti-tumor activity of motesanib in a medullary thyroid cancer model.

BACKGROUND: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. AIM: The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. METHODS/RESULTS: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. CONCLUSIONS: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer.

Activation of Crtc1 (also known as Mect1/Torc1) by a t(11;19) chromosomal rearrangement underlies the etiology of malignant salivary gland tumors. As LKB1 is a target for mutational inactivation in lung cancer and was recently shown to regulate hepatic Crtc2/CREB transcriptional activity in mice, we now present evidence suggesting disruption of an LKB1/Crtc pathway in cancer. Although Crtc1 is preferentially expressed in adult brain tissues, we observed elevated levels of steady-state Crtc1 in thoracic tumors. In addition, we show that somatic loss of LKB1 is associated with underphosphorylation of endogenous Crtc1, enhanced Crtc1 nuclear localization and enhanced expression of the Crtc prototypic target gene, NR4A2/Nurr1. Inhibition of NR4A2 was associated with growth suppression of LKB1 null tumors, but showed little effect on LKB1-wildtype cells. These data strengthen the role of dysregulated Crtc as a bona fide cancer gene, present a new element to the complex LKB1 tumorigenic axis, and suggest that Crtc genes may be aberrantly activated in a wider range of common adult malignancies.

Sustained expression of Mect1-Maml2 is essential for tumor cell growth in salivary gland cancers carrying the t(11;19) translocation.

Mucoepidermoid (MEC) salivary gland tumors arise from a t(11;19) rearrangement which generates a fusion oncogene, Mect1-Maml2, that functions to activate CREB-responsive target genes. To determine if sustained expression of Mect1-Maml2 is required for tumor cell growth, we first showed that ectopic expression of Mect1-Maml2 in rat epithelial RK3E cells is tumorigenic in vivo in nude mice and that excised xenografts continue to express the fusion oncogene. We then generated a hairpin RNAi vector that selectively suppressed the fusion peptide and showed that ectopic expression in either parotid or pulmonary MEC tumor cell lines containing the t(11;19) rearrangement resulted in at least 90% colony growth inhibition. In contrast, single nucleotide changes within this RNAi sequence abolished the ability to suppress Mect1-Maml2 protein and abolished all growth inhibition of these MEC tumor lines. In addition, the RNAi-specific vector had no effect on colony growth of non-MEC tumors including a lung tumor or two other salivary gland cell lines that do not express Mect1-Maml2. We also generated a mutant Mect1-Maml2 expression plasmid that carried silent nucleotide changes within the RNAi target sequence and observed that co-transfection of this mutant, but not wild-type Mect1-Maml2, could partially rescue RNAi growth inhibition in the MEC tumor line. The recent detection of acquired fusion oncogenes in epithelial solid tumors has suggested new possibilities for the diagnosis and therapy of these cancers. Our data show that the 'gain-of-function' activity from aberrant Mect1-Maml2 expression is a candidate therapeutic target for this group of malignant salivary gland tumors.

Multidimensional scaling of pediatric sleep breathing problems and bio-behavioral correlates.

STUDY OBJECTIVES: Complex relationships exist between pediatric sleep disorders and daytime behavior. Using a multidimensional scaling model, we investigated these relationships in 126 children with sleep breathing disorders (SBD). METHOD: Validated questionnaires on nighttime behavior, daytime behavior, and respiratory health were administered to a large number of school children in Belgium. Children who met the criterion of having at least one sleep-related breathing problem (three or more times per week during the past six months) were selected for further analyses. A total of 26 indicators were defined and modeled, including sleep problems, sleep efficiency, sleep environment, sleep enuresis, internalised and externalised behavioral problems, respiratory health of the child and relatives, smoking exposure, and caffeine consumption. RESULTS: From 3,045 questionnaire responses 4.1% of the children were reported to have a SBD symptom. SBD children differed on sleep and health domains from non-SBD children. Furthermore, through scaling of the (dis)similarities among the 26 indicators the SBD child was able to be modeled. By way of an internal analysis of the data-matrix the following indicators were eliminated: sleep correlates, health of the family, and behavior rated by teachers, followed by caffeine intake, drugs, and behavior rated by the parents. This revealed a two-dimensional model, consisting of primary SBD and secondary SBD. CONCLUSION: Children with SBD differ on many domains from children without such disorders and an underlying two-fold SBD concept was found. Firstly, the SBD-indicator positioned in between investigated correlates with disorders of initiating and maintaining sleep and sleep hyperhydrosis on one hand and with respiratory-related illnesses on the other; this was labeled primary SBD. Secondly, the SDB-indicator not closely associated with any of the investigated correlates can be interpreted as secondary SBD.

How many samples from a drug seizure need to be analyzed?

Recently Aitken introduced an outstanding advance in the approach to decision making regarding drugs sampling. Unfortunately this approach has not, as yet, been widely implemented despite being based on a solid mathematical foundation. In this paper we advocate a Bayesian approach along the lines of that outlined by Aitken but designed to be both easily understood with less mathematical sophistication and implementable using standard EXCEL software. The emphasis is placed on encouraging the application of this methodology to routine case work by explaining the statistics involved. Minor differences exist between this approach and that of Aitken in both the modeling of the prior probability and in dealing with the discrete nature of the samples. These differences in no way detract from the sound mathematical foundation of the approach.

Fc gamma RIII mediates neutrophil recruitment to immune complexes. a mechanism for neutrophil accumulation in immune-mediated inflammation.

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Protein expression of the RB-related gene family and SV40 large T antigen in mesothelioma and lung cancer.

Mutational inactivation of the RB-related gene RBL2/p130 has been reported as a common and important prognostic factor in human lung cancer. To examine the role of the RB-related gene family in lung cancer we analysed the protein expression of the RB gene in cell lines obtained from 83 patients with small cell lung cancer (SCLC) and 114 patients with non-SCLC that included 21 novel lung tumor samples. While we detected five new SCLC with mutant RB expression (RB inactivation in 75/83; 90.4%), we did not detect any RB mutations in the new non-SCLC cell lines (RB inactivation in 13/114 non-SCLC and mesothelioma; 11.4%). In addition, we detected expression of a full-length RBL1/p107 and RBL2/p130 species in every sample tested (RBL1 or RBL2 inactivation in 0/69) and confirmed that both RB-related gene products retain functional binding activity to the E1A viral oncoprotein. Since expression of SV40 Large T antigen (Tag) has been reported in a subset of human lung tumors where it may inactivate RBL1 and RBL2, we also examined mesothelioma and non-mesothelioma lung tumors for Tag expression. Although we detected a faint 85 kDa protein species using specific anti-Tag antibodies, this signal migrated slightly faster than Tag extracted from Cos7 cells and did not exhibit binding activity to the RB or RBL1 proteins. Finally, we subjected 11 lung cancer cell lines to nucleotide sequencing and did not detect mutations within the C-terminal RBL2 exons 19-22 as recently reported. While the RB/p16 tumor suppressor pathway is targeted for mutations in 100% of lung cancers, mutational inactivation of the related RBL1 and RBL2 genes is a rare event.

Cytokine-activated endothelial cells delay neutrophil apoptosis in vitro and in vivo. A role for granulocyte/macrophage colony-stimulating factor.

The activation of endothelium is important in recruiting neutrophils to sites of inflammation and in modulating their function. We demonstrate that conditioned medium from cultured, activated endothelial cells acts to significantly delay the constitutive apoptosis of neutrophils, resulting in their enhanced survival and increased phagocytic function. The antiapoptotic activity is, in part, attributable to granulocyte/macrophage colony-stimulating factor (GM-CSF) secreted by activated endothelial cells. The in vivo relevance of these findings was investigated in a cytokine-induced model of acute meningitis in mice. Peripheral blood neutrophils (PBNs) from mice with meningitis exhibited a delay in apoptosis compared with untreated mice. Furthermore, neutrophils recovered from the inflamed cerebrospinal fluid (CSF) exhibited enhanced survival compared with neutrophils isolated from the peripheral blood of the same animals. In unchallenged GM-CSF-deficient mice, the apoptosis of circulating PBNs was similar to wild-type animals; however, after cytokine-induced meningitis, the delay in neutrophil apoptosis typically observed in wild-type mice was attenuated. In contrast, the apoptosis of neutrophils recovered from the CSF of mice of both genotypes was comparable. Taken together, these studies suggest that neutrophil apoptosis is regulated during an inflammatory response, in both intravascular and extravascular compartments. GM-CSF released by activated endothelium can act to increase neutrophil survival and function in the peripheral blood, whereas other factor(s) appear to perform this function in the extravascular space.

Temperature-sensitive RB mutations linked to incomplete penetrance of familial retinoblastoma in 12 families.

The tumor-suppressor activity of the retinoblastoma protein (RB) is encoded within a protein-binding ("pocket") domain that is targeted for mutations in all cases of familial retinoblastoma and in many common adult cancers. Although familial retinoblastoma is a paradigm for a highly penetrant, recessive model of tumorigenesis, the molecular basis for the phenotype of incomplete penetrance of familial retinoblastoma is undefined. We studied the RB pocket-binding properties of three independent, mutant RB alleles that are present in the germline of 12 kindreds with the phenotype of incomplete penetrance of familial retinoblastoma. Each arises from alterations of single codons within the RB pocket domain (designated "delta 480," "661W," or "712R"). Under the same conditions, we studied the properties of wild-type (WT) RB, an RB point mutant isolated from a lung carcinoma sample (706F) and an adjacent, in vitro-generated point mutant (707W). The delta 480, 661W, and 712R mutants lack pocket protein-binding activity in vitro but retain the WT ability to undergo cyclin-mediated phosphorylation in vivo. Each of the low-penetrant RB mutants exhibits marked enhancement of pocket protein binding when the cells are grown at reduced temperature. In contrast, in this temperature range, no change in binding activity is seen with WT RB, the 706F mutant, or the 707W mutant. We have demonstrated that many families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles with temperature-sensitive pocket protein-binding activity. The variable frequency for tumor development in these families may result from reversible fluctuations in a threshold level of RB pocket-binding activity.

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.

Parallel accounts? Discrepancies between self-report (diary) and recall (questionnaire) measures of the same sexual behaviour.

Questionnaires and diaries have complementary biases and advantages for obtaining information on sexual behaviour but self-completed sexual diaries have the advantage of reducing retrospective bias. In a validation study of homosexual behaviour, sexual diary counts and subsequent questionnaire estimates (together with ratings of the certainty of the estimates) referring to the same month are compared and the discrepancies analyzed. Main findings include: questionnaire data yield consistently higher average estimates than diary counts, but have the same ordinal profile; individual difference (diary-questionnaire) scores show that 55% of questionnaire estimates of acts are higher than diary counts, 20% are identical and 25% are under-estimates; discrepancies are differentially located in different sexual acts. Masturbation and fellatio are systematically over-estimated in questionnaires and anal intercourse without a condom is the major source of inaccuracies, but in different directions: active partners under-estimate and passive partners over-estimate the amount of highest-risk sex. A strategy of joint use is discussed.

Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury.

BACKGROUND AND PURPOSE: Macrophage-1 antigen (Mac-1) (CD11b/CD18), a leukocyte beta2 integrin, facilitates neutrophil adhesion, transendothelial migration, phagocytosis, and respiratory burst, all of which may mediate reperfusion-induced injury to ischemic brain tissue in conditions such as stroke. To determine the role of Mac-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in mice genetically engineered with a specific deficiency in Mac-1. METHODS: Transient focal ischemia/reperfusion was induced by occluding the left middle cerebral artery for 3 hours followed by a 21-hour reperfusion period in Mac-1-deficient (n=12) and wild-type (n=11) mice. Regional cerebral blood flow was determined with a laser-Doppler flowmeter. Brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride to determine the infarct volume. Neutrophil accumulation was determined by staining the brain sections with dichloroacetate esterase to identify neutrophils. RESULTS: Compared with the wild-type cohort, Mac-1-deficient mice had a 26% reduction in infarction volume (P<0.05). This was associated with a 50%, but statistically insignificant, reduction in the number of extravasated neutrophils in the infarcted areas of the brains in the mutant mice. There were no differences in regional cerebral blood flow between the 2 groups. CONCLUSIONS: Mac-1 deficiency reduces neutrophil infiltration and cerebral cell death after transient focal cerebral ischemia. This finding may be related to a reduction in neutrophil extravasation in Mac-1-deficient mice.

Impaired mast cell development and innate immunity in Mac-1 (CD11b/CD18, CR3)-deficient mice.

Mac-1 (CD11b/CD18, CR3), a beta2 integrin expressed on leukocytes, is important in leukocyte migration. We demonstrate that Mac-1 is also expressed on peritoneal mast cells and LPS stimulated bone marrow-derived cultured mast cells, and that Mac-1-deficient mice, which lack this receptor, have significant reductions in the numbers of mast cells resident in the peritoneal cavity, peritoneal wall, and dorsal skin. The reduced numbers of mast cells in Mac-1-deficient mice may have important functional consequences, in that Mac-1-deficient mice exhibit significantly increased mortality after cecal ligation and puncture, a model of acute septic peritonitis in which host resistance has been shown to be dependent on both mast cells and complement. These findings demonstrate that Mac-1 is required for the expression of normal levels of mast cells in the peritoneal cavity, peritoneal wall, and certain areas of the skin, as well as for maintaining adequate mast cell-dependent host defense against bacterial infection.

RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer.

Mice bred to carry germline Rb and p53 null alleles are associated with a tumor spectrum that overlaps with the inherited multiple endocrine neoplasia-1 (MEN1) and MEN2 syndromes in humans, including medullary thyroid cancer (MTC). To study the genetic basis for these tumors, we microdissected MTC specimens or obtained fresh MTC tissue from nine independent Rb(+/-) p53(+/-) mice, amplified the region of the Ret gene known to be mutated in human MTC, and detected acquired missense Ret mutations in four different mice. These mutations were localized to a group of tandem cysteines which are analogous to activating germline mutations observed in human MEN2A and familial MTC (FMTC). To determine whether the remaining wild type Rb allele was inactivated in these murine MTC samples, we subjected tumor tissue to immunohistochemical staining with an Rb antibody, and demonstrated the absence of RB staining in murine MTC, while normal tissue retained RB nuclear staining. These findings demonstrate the ability of the gene knockout model to recapitulate somatic multi-step tumorigenesis and suggest that the development of a murine neuroendocrine tumor requires mutational dysregulation within both receptor tyrosine kinase and nuclear tumor suppressor gene pathways.