Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

Sting jets in simulations of a real cyclone by two mesoscale models

The existence of sting jets as a potential source of damaging surface winds during the passage of extratropical cyclones has recently been recognized However, there are still very few published studies on the subject Furthermore, although ills known that other models are capable of reproducing sting jets, in the published literature only one numerical model [the Met Office Unified Model (MetUM)] has been used to numerically analyze these phenomena This article alms to improve our understanding of the processes that contribute to the development of sting jets and show that model differences affect the evolution of modeled sting jets A sting jet event during the passage of a cyclone over the United Kingdom on 26 February 2002 has been simulated using two mesoscale models namely the MetUM and the Consortium for Small Scale Modeling (COSMO) model to compare their performance Given the known critical importance of vertical resolution in the simulation of sting jets the vertical resolution of both models has been enhanced with respect to their operational versions Both simulations have been verified against surface measurements of maximum gusts, satellite imagery and Met Office operational synoptic analyses, as well as operational analyses from the ECMWF It is shown that both models are capable of reproducing sting jets with similar, though not identical. features Through the comparison of the results from these two models, the relevance of physical mechanisms, such as evaporative cooling and the release of conditional symmetric instability, in the generation and evolution of sting jets is also discusse

Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the a-chain of the human interleukin-2 receptor

The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the a-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2Rcc chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy

Providing universal access to antiretroviral therapy in Thyolo, Malawi through task shifting and decentralization of HIV/AIDS care.

Objective  To describe how district-wide access to HIV/AIDS care was achieved and maintained in Thyolo District, Malawi. Method  In mid-2003, the Ministry of Health and Médecins Sans Frontières developed a model of care for Thyolo district (population 587 455) based on decentralization of care to health centres and community sites and task shifting. Results  After delegating HIV testing and counseling to lay counsellors, uptake of testing increased from 1300 tests per month in 2003 to 6500 in 2009. Shifting responsibility for antiretroviral therapy (ART) initiations to non-physician clinicians almost doubled ART enrolment, with a majority of initiations performed in peripheral health centres. By the end 2009, 23 261 people had initiated ART of whom 11 042 received ART care at health-centre level. By the end of 2007, the universal access targets were achieved, with nearly 9000 patients alive and on ART. The average annual cost for achieving these targets was €2.6 per inhabitant/year. Conclusion  The Thyolo programme has demonstrated the feasibility of district-wide access to ART in a setting with limited resources for health. Expansion and decentralization of HIV/AIDS service-capacity to the primary care level, combined with task shifting, resulted in increased access to HIV services with good programme outcomes despite staff shortages

LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation

Carefully orchestrated intercellular communication is an essential prerequisite for the development of multicellular organisms. In recent years, tunneling nanotubes (TNT) have emerged as a novel and widespread mechanism of cell-cell communication. However, the molecular basis of their formation is still poorly understood. In the present study we report that the transmembrane MHC class III protein LST1 induces the formation of functional nanotubes and is required for endogenous nanotube generation. Mechanistically, we found LST1 to induce nanotube formation by recruiting the small GTPase RalA to the plasma membrane and promoting its interaction with the exocyst complex. Furthermore, we determined LST1 to recruit the actin-crosslinking protein filamin to the plasma membrane and to interact with M-Sec, myosin and myoferlin. These results allow us to suggest a molecular model for nanotube generation. In this proposal LST1 functions as a membrane scaffold mediating the assembly of a multimolecular complex, which controls the formation of functional nanotubes

Positive Outcomes of HAART at 24 Months in HIV-Infected Patients in Cambodia.

OBJECTIVES: African and Asian cohort studies have demonstrated the feasibility and efficacy of HAART in resource-poor settings. The long-term virological outcome and clinico-immunological criteria of success remain important questions. We report the outcomes at 24 months of antiretroviral therapy (ART) in patients treated in a Médecins Sans Frontières/Ministry of Health programme in Cambodia. METHODS: Adults who started HAART 24 +/- 2 months ago were included. Plasma HIV-RNA levels were assessed by real-time polymerase chain reaction. Factors associated with virological failure were analysed using logistic regression. RESULTS: Of 416 patients, 59.2% were men; the median age was 33.6 years. At baseline, 95.2% were ART naive, 48.9% were at WHO stage IV, and 41.6% had a body mass index less than 18 kg/m. The median CD4 cell count was 11 cells/microl. A stavudine-lamivudine-efavirenz-containing regimen was initiated predominantly (81.0%). At follow-up (median 23.8 months), 350 (84.1%) were still on HAART, 53 (12.7%) had died, six (1.4%) were transferred, and seven (1.7%) were lost to follow-up. Estimates of survival were 85.5% at 24 months. Of 346 tested patients, 259 (74.1%) had CD4 cell counts greater than 200 cells/microl and 306 (88.4%) had viral loads of less than 400 copies/ml. Factors associated with virological failure at 24 months were non-antiretroviral naive, an insufficient CD4 cell gain of less than 350 cells/microl or a low trough plasma ART concentration. In an intention-to-treat analysis, 73.6% of patients were successfully treated. CONCLUSION: Positive results after 2 years of advanced HIV further demonstrate the efficacy of HAART in the medium term in resource-limited settings

Children and Adolescents with Obsessive-Compulsive Disorder: Comorbidity and Quality of Life

The overall aim of the present thesis was to investigate important aspects of childhood obsessive–compulsive disorder (OCD) such as comorbidity and quality of life (QoL). Frequency and continuity of autistic symptoms from preschool age to later childhood in children with OCD were explored as a contribution to the ongoing nosological debate on whether there is an empirical base for summarizing conditions such as OCD, Tourette’s syndrome, eating disorders and autism spectrum disorders as parts of a so-called obsessive– compulsive spectrum. The establishment of proper outcome measures for the evaluation of treatment interventions has been a concern in the field (Pallanti et al., 2002). The measurement of symptom reduction only, without any assessment of subjective well-being, has been criticized (Macy et al., 2013). QoL assessment is a well-established outcome measure in somatic as well as in psychiatric treatment studies. However, in contrast to adult OCD, little is known about QoL in children with OCD and next to nothing is known about the role treatment plays in their QoL. To investigate QoL in children with OCD under different conditions (with and without comorbidity, before and after treatment) is relevant because of the paucity of studies concerning children. In the first study, comorbidity in terms of autistic traits was assessed in children and adolescents seeking treatment in a Swedish OCD Clinic (Gothenburg) and compared with controls from a general population sample. In the second study, QoL was assessed at baseline, and in the third study, both at baseline and after treatment with cognitive behavioural therapy (CBT) in children and adolescents with OCD enrolled in the Nordic Long-term OCD Treatment Study (NordLOTS) and compared with the general population. The NordLOTS is a Nordic multicentre study in which OCD treatment units for children and adolescents collaborated on common procedures for assessment, treatment and outcome evaluation (Thomsen et al., 2013). A manual for CBT with exposure and response prevention was established (Weidle et al., 2014). The QoL study included in this thesis comprised a sample of 135 children and adolescents, 7–17 years of age, with moderate to severe OCD according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (4th edition, text rev.) (DSM-IV-TR) (2000), including nine individuals with Asperger’s syndrome/high-functioning autism. QoL was assessed at baseline and after treatment with 14 sessions of CBT by self-report and caregiver proxy reports on the Questionnaire for Measuring Health-related Quality of Life in Children and Adolescents (KINDL-R) and compared with an age- and gender-matched sample from the general population. Social competence and school functioning were assessed with the Child Behaviour Checklist, comorbidity with the Kiddie Schedule for Affective Disorders and Schizophrenia (Present and Lifetime Version), severity of OCD with the Children’s Yale–Brown Obsessive Compulsive Scale and the families’ involvement with the child’s OCD symptoms with the Family Accommodation Scale. In addition, the significance of potential factors such as onset and duration of symptoms regarding QoL change were explored. Autism spectrum disorder (ASD) symptoms occurred in about one-fifth of children with OCD and were much more common than in the general population. However, the majority of OCD patients did not have these traits, indicating that ASD and OCD co-occur in a subgroup of cases rather than in OCD as a whole. In general, ASD symptoms reported in children with OCD did not show a strong enough relationship between OCD and ASD to support a classification of ASD as a part of an obsessive–compulsive spectrum group. However, a subgroup of paediatric OCD patients had significant subclinical ASD symptom levels. This should be considered when tailoring individual treatment interventions. QoL and social competence were markedly reduced in children with OCD, both in self-reports and in parents’ reports, compared with the general population. Children with higher comorbidity had lower QoL in parents’ proxy reports. After treatment, QoL ratings in treatment responders were in the same range as in the general population, while non-responders rated no change in QoL. Comorbidity, family accommodation and psychosocial functioning were not associated with changes in QoL after treatment. To the best of our knowledge, this is the largest QoL study of paediatric patients with OCD and the first one based on the assessment of OCD and comorbid disorders by standardized semi-structured diagnostic interviews, comparing patients with matched controls from the general population. It is also the first study assessing QoL changes after treatment in paediatric OCD. QoL assessment with the KINDL-R supported outcome measures for symptom reduction used in the study to define treatment outcomes. Based on our findings, we suggest employing QoL assessment in order to have a more comprehensive understanding of childhood OCD

Mechanisms of hypertension during and after orthotopic liver transplantation in children

The aim of this study was to assess the hormonal alterations that may mediate the systemic hypertension that develops in patients during the perioperative period of orthotopic liver transplantation. We studied nine pediatric patients without previous hypertension or renal disease during six time points, starting before transplantation and ending at 48 hours after surgery. Hypertension developed in all patients in association with central venous pressures <10 mm Hg. Free water clearance was negative in all nine patients. Vasopressin levels increased intraoperatively but fell as hypertension developed. Atrial natriuretic factor levels increased as systemic blood pressure rose. A high level of plasma renin activity was observed in four patients with renal insufficiency. In six patients, postoperative 24-hour urinary norepinephrine excretion was within the normal age-adjusted range. These findings suggest that the combination of cyclosporine, corticosteroids, and, in some patients, an elevated plasma renin activity prevents the kidney from responding to the acute volume and salt overload with an appropriate diuresis and natriuresis, thus leading to systemic hypertension. The treatment of hypertension after liver transplantation may include salt restriction, diuretics, and, in those patients with a low creatinine excretion index, anglotensin coverting enzyme inhibitors. © 1989 The C. V. Mosby Company

CD147 and MCT1 - potential partners in bladder cancer aggressiveness and cisplatin resistance

"Article first published online: 27 SEP 2014"The relapsing and progressive nature of bladder tumors, and the heterogeneity in the response to cisplatin-containing regimens, are the major concerns in the care of urothelial bladder carcinoma (UBC) patients. The metabolic adaptations that alter the tumor microenvironment and thus contribute to chemoresistance have been poorly explored in UBC setting. We found significant associations between the immunoexpressions of the microenvironment-related molecules CD147, monocarboxylate transporters (MCTs) 1 and 4, CD44 and CAIX in tumor tissue sections from 114 UBC patients. The presence of MCT1 and/or MCT4 expressions was significantly associated with unfavorable clinicopathological parameters. The incidence of CD147 positive staining significantly increased with advancing stage, grade and type of lesion, and occurrence of lymphovascular invasion. Similar associations were observed when considering the concurrent expression of CD147 and MCT1. This expression profile lowered significantly the 5-year disease-free and overall survival rates. Moreover, when selecting patients who received platinum-based chemotherapy, the prognosis was significantly worse for those with MCT1 and CD147 positive tumors. CD147 specific silencing by small interfering RNAs (siRNAs) in UBC cells was accompanied by a decrease in MCT1 and MCT4 expressions and, importantly, an increase in chemosensitivity to cisplatin. Our results provide novel insights for the involvement of CD147 and MCTs in bladder cancer progression and resistance to cisplatin-based chemotherapy. We consider that the possible cooperative role of CD147 and MCT1 in determining cisplatin resistance should be further explored as a potential theranostics biomarker.Grant sponsor: Life and Health Sciences Research Institute (ICVS); Grant sponsor: Portuguese Science and Technology Foundation (FCT); Grant number: SFRH/BD/51997/201