In vivo Assessment of the Impact of Molecular Weight on Constructs of 68Ga-DOTA-Manocept in a Syngeneic Mouse Tumor Model.

PURPOSE: Manocept™ constructs are mannosylated amine dextrans (MADs) that bind with high affinity to the mannose receptor, CD206. Tumor-associated macrophages (TAMs) are the most numerous immune cells in the tumor microenvironment and a recognized target for tumor imaging and cancer immunotherapies. Most TAMs express CD206, suggesting utility of MADs to deliver imaging moieties or therapeutics to TAMs. The liver Kupffer cells also express CD206, making them an off-target localization site when targeting CD206 on TAMs. We evaluated TAM targeting strategies using two novel MADs differing in molecular weight in a syngeneic mouse tumor model to determine how varying MAD molecular weights would impact tumor localization. Increased mass dose of the non-labeled construct or a higher molecular weight (HMW) construct were also used to block liver localization and enhance tumor to liver ratios. PROCEDURES: Two MADs, 8.7 kDa and 22.6 kDa modified with DOTA chelators, were synthesized and radiolabeled with 68Ga. A HMW MAD (300 kDa) was also synthesized as a competitive blocking agent for Kupffer cell localization. Balb/c mice, with and without CT26 tumors, underwent dynamic PET imaging for 90 min followed by biodistribution analyses in selected tissues. RESULTS: The new constructs were readily synthesized and labeled with 68Ga with ≥ 95% radiochemical purity in 15 min at 65 °C. When injected at doses of 0.57 nmol, the 8.7 kDa MAD provided 7-fold higher 68Ga tumor uptake compared to the 22.6 kDa MAD (2.87 ± 0.73%ID/g vs. 0.41 ± 0.02%ID/g). Studies with increased mass of unlabeled competitors showed reduced liver localization of the [68Ga]MAD-8.7 to varying degrees without significant reductions in tumor localization, resulting in enhanced tumor to liver signal ratios. CONCLUSION: Novel [68Ga]Manocept constructs were synthesized and studied in in vivo applications, showing that the smaller MAD localized to CT26 tumors more effectively than the larger MAD and that the unlabeled HMW construct could selectively block liver binding of [68Ga]MAD-8.7 without diminishing the localization to tumors. Promising results using the [68Ga]MAD-8.7 show a potential path to clinical applications.

Sc-HOPO: A Potential Construct for Use in Radioscandium-Based Radiopharmaceuticals.

Three isotopes of scandium─43Sc, 44Sc, and 47Sc─have attracted increasing attention as potential candidates for use in imaging and therapy, respectively, as well as for possible theranostic use as an elementally matched pair. Here, we present the octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO), an effective chelator for hard cations, as a potential ligand for use in radioscandium constructs with simple radiolabeling under mild conditions. HOPO forms a 1:1 Sc-HOPO complex that was fully characterized, both experimentally and theoretically. [47Sc]Sc-HOPO exhibited good stability in chemical and biological challenges over 7 days. In healthy mice, [43,47Sc]Sc-HOPO cleared the body rapidly with no signs of demetalation. HOPO is a strong candidate for use in radioscandium-based radiopharmaceuticals.

Evaluation of 68Ga-Radiolabeled Peptides for HER2 PET Imaging.

One in eight women will be diagnosed with breast cancer in their lifetime and approximately 25% of those cases will be HER2-positive. Current methods for diagnosing HER2-positive breast cancer involve using IHC and FISH from suspected cancer biopsies to quantify HER2 expression. HER2 PET imaging could potentially increase accuracy and improve the diagnosis of lesions that are not available for biopsies. Using two previously discovered HER2-targeting peptides, we modified each peptide with the chelator DOTA and a PEG2 linker resulting in DOTA-PEG2-GSGKCCYSL (P5) and DOTA-PEG2-DTFPYLGWWNPNEYRY (P6). Each peptide was labeled with 68Ga and was evaluated for HER2 binding using in vitro cell studies and in vivo tumor xenograft models. Both [68Ga]P5 and [68Ga]P6 showed significant binding to HER2-positive BT474 cells versus HER2-negative MDA-MB-231 cells ([68Ga]P5; 0.68 ± 0.20 versus 0.47 ± 0.05 p < 0.05 and [68Ga]P6; 0.55 ± 0.21 versus 0.34 ± 0.12 p < 0.01). [68Ga]P5 showed a higher percent injected dose per gram (%ID/g) binding to HER2-positive tumors two hours post-injection compared to HER2-negative tumors (0.24 ± 0.04 versus 0.12 ± 0.06; p < 0.05), while the [68Ga]P6 peptide showed significant binding (0.98 ± 0.22 versus 0.51 ± 0.08; p < 0.05) one hour post-injection. These results lay the groundwork for the use of peptides to image HER2-positive breast cancer.

Novel multimodal molecular imaging of Vitamin H (Biotin) transporter activity in the murine placenta.

Vitamin H (biotin) is delivered to the fetus transplacentally by an active biotin-transport mechanism and is critical for fetal development. Our objective was to develop a comprehensive MRI technique for mapping biotin transporter activity in the murine placenta. Visualization of transporter activity can employ MRI's unique T2*-dependent signal 'off-switch', which is triggered by transporter mediated aggregation of biotinylated contrast agent (b-BSA-Gd-DTPA). MRI data were collected from pregnant mice after administration of b-BSA-Gd-DTPA and analyzed using a new sub-voxel biophysical signal model. Validation experiments included competition with native biotin, comparative tests using PET, histology, and ICPMS. MRI signal was governed by binding, aggregation, and clearance of biotin (confirmed by histology). Signal dynamics reflected the placenta's perfusion pattern modulated by biotin transporter activity and trophoblast mediated retention, and were in congruence with a three-compartment sub-voxel model. Pre-saturation of the transporters with free biotin suppressed b-BSA-Gd-DTPA uptake. The results were confirmed by PET, histology and ICPMS. The presented MRI-based platform allows to track activity of essential molecular transporters in the placenta, reflecting a transporter-mediated uptake, followed by retention and aggregation, and recycling associated with the large b-BSA-Gd-DTPA conjugate. The presented DCE-MRI technique can furthermore be used to map and characterize microstructural compartmentation and transporter activity without exposing the fetus to contrast media.

Factors related to the development of CMV-specific CD8+ T cell response in CMV-seropositive solid organ transplant candidates.

This cross-sectional study analyzes factors associated with the development of CMV-specific CD8+ response, measured by IFNg production after cytomegalovirus (CMV) peptide stimulation, in CMV-seropositive solid organ transplantation candidates. A total of 114 candidates were enrolled, of whom 22.8% (26/114) were nonreactive (IFNγ < 0.2 IU/mL). Multivariate logistic regression analysis showed that age, HLA alleles and organ to be transplanted were associated with developing CMV-specific CD8+ immunity (reactive; IFNγ ≥ 0.2 IU/mL). The probability of being reactive was higher in candidates over 50 than in those under 50 (OR 6.33, 95%CI 1.93-20.74). Candidates with HLA-A1 and/or HLA-A2 alleles had a higher probability of being reactive than those with non-HLA-A1/non-HLA-A2 alleles (OR 10.97, 95%CI 3.36-35.83). Renal candidates had a higher probability of being reactive than lung (adjusted OR 8.85, 95%CI 2.24-34.92) and liver candidates (OR 4.87, 95%CI 1.12-21.19). The AUC of this model was 0.84 (p < 0.001). Positive and negative predictive values were 84.8% and 76.9%, respectively. In renal candidates longer dialysis was associated with an increased frequency of reactive individuals (p = 0.040). Therefore, although the assessment of CMV-specific CD8+ response is recommended in all R+ candidates, it is essential in those with a lower probability of being reactive, such as non-renal candidates, candidates under 50 or those with non-HLA-A1/non-HLA-A2 alleles.

Pre-transplant thymic function is associated with the risk of cytomegalovirus disease after solid organ transplantation.

Cytomegalovirus (CMV) disease is an important complication in solid organ transplant recipients. Thymic function in adults is associated with specific T-cell immunity. Pre-transplant thymic function was analysed in 75 solid organ transplant patients by the use of nested PCR. The primary outcome was the incidence of CMV disease 12 months after transplantation. Using multivariable logistic regression, we studied whether pre-transplant thymic function is an independent risk factor for CMV disease after transplantation. Thymic function was related to the risk of CMV disease in CMV-seropositive recipients. In these recipients, pre-transplant thymic function of <9.5 (OR 11.27, 95% CI 1.11-114.43, p 0.040) and the use of thymoglobulin (OR 8.21, 95% CI 1.09-61.84, p 0.041) were independent risk factors for CMV disease at 12 months after transplantation. Patients with pre-transplant thymic function values of <9.5 had a higher subsequent incidence of CMV disease (24%) than patients with values of ≥ 9.5 (3%) (log-rank test: 5.727; p 0.017). The positive and negative predictive values of these pre-transplant thymic function cut-offs were 0.24 (95% CI 0.10-0.45) and 0.97 (95% CI 0.82-1.00), respectively. Pre-transplant thymic function in CMV-seropositive candidates could be useful in determining the risk of post-transplant CMV disease in solid organ transplant patients, selecting a group of low-risk candidates.

Polymorphisms of HLA-A, -B, -Cw and DRB1 antigens in Moroccan patients with ankylosing spondylitis and a comparison of clinical features with frequencies of HLA-B*27.

Ankylosing spondylitis (AS) is very often associated with human leukocyte antigen (HLA), particularly HLA-B*27. However, the strength of this association and clinical features may vary in different ethnic groups. Our study aims to assess the distribution of HLA-A, -B, -Cw and DRB1 alleles in Moroccan patients with AS and to compare the clinical features of AS and the frequencies of HLA-B27 in patients from Morocco with other series. Seventy-five patients diagnosed with AS and assessed for clinical manifestations were selected and compared to 100 healthy controls. HLA class I and II antigens were typed by polymerase chain reaction sequence-specific oligonucleotide. HLA-B27 subtypes were studied by polymerase chain reaction amplification with sequence-specific primers. HLA-B27 was found in 64% of patients. It was positively associated with younger age at disease onset, family history, and uveitis while it had a negative association with late onset. Six B*27 subtypes were identified in the AS group. HLA-B*2705 and B*2702 were the most common observed subtypes. Among other HLA genes, a significant increase in the prevalence of HLA-Cw*02 and HLA-DRB*15 was found in AS patients. HLA-B27 is involved in the predisposition of AS in the Moroccan population. HLA-B*2705 and B*2702 were the predominant subtypes supporting previous reports in Caucasian spondyloarthropathies. Other HLA genes, HLA-Cw*02 and HLA-DRB1*15, seem to confer predisposing effect to the disease. However, the lower frequency of HLA-B27 compared to the literature in our study suggests the existence of different genetic and/or environmental factors in Morocco.

Unknown loads affect force production capacity in early phases of bench press throws.

AIM: Explosive strength training aims to improve force generation in early phases of movement due to its importance in sport performance. The present study examined the influence of lack of knowledge about the load lifted in explosive parameters during bench press throws. METHODS: Thirteen healthy young men (22.8±2.0 years) participated in the study. Participants performed bench press throws with three different loads (30, 50 and 70% of 1 repetition maximum) in two different conditions (known and unknown loads). In unknown condition, loads were changed within sets in each repetition and participants did not know the load, whereas in known condition the load did not change within sets and participants had knowledge about the load lifted. RESULTS: Results of repeated-measures ANOVA revealed that unknown conditions involves higher power in the first 30, 50, 100 and 150 ms with the three loads, higher values of ratio of force development in those first instants, and differences in time to reach maximal rate of force development with 50 and 70% of 1 repetition maximum. CONCLUSION: This study showed that unknown conditions elicit higher values of explosive parameters in early phases of bench press throws, thereby this kind of methodology could be considered in explosive strength training.

Pretransplant interferon-γ secretion by CMV-specific CD8+ T cells informs the risk of CMV replication after transplantation.

In this prospective study we analyzed pretransplant interferon-γ secretion by cytomegalovirus (CMV)-specific CD8+ T cells to assess its possible utility in determining the risk of CMV replication after solid organ transplantation. A total of 113 lung and kidney transplant patients were enrolled in the study but only 55 were evaluable. All CMV-seronegative recipients were pretransplant "nonreactive" (IFNγ <0.2 IU/mL) (11/11), whereas 30/44 (68.2%) CMV-seropositive (R+) recipients were "reactive" (IFNγ ≥0.2 IU/mL) and 14/44 (31.8%) were "nonreactive". In the R(+) "nonreactive" group, 7/14 (50%) developed posttransplant CMV replication, whereas the virus replicated only in 4/30 (13.3%) of the R(+) "reactive" patients (p = 0.021). According to the best multivariate model, pretransplant "nonreactive" recipients receiving an organ from a CMV-seropositive donor had a 10-fold increased risk of CMV replication compared to pretransplant "reactive" recipients (adjusted OR 10.49, 95% CI 1.88-58.46). This model displayed good discrimination ability (AUC 0.80) and calibration (Hosmer-Lemeshow test, p = 0.92). Negative and positive predictive values were 83.7% and 75%, respectively. The accuracy of the model was 82%. Therefore, assessment of interferon-γ secretion by cytomegalovirus (CMV)-specific CD8+ T cells prior to transplantation is useful in informing the risk of posttransplant CMV replication in solid organ transplant patients.

Current concepts on the effect of environmental factors on cleft lip and palate.

The aim of this study was to investigate the effect of environmental factors, such as tobacco, alcohol and folic acid intake, obesity, stressful events, low blood levels of zinc and fever during pregnancy, on the incidence of cleft lip and/or palate (CL±P). An electronic search was performed in the Cochrane Reviews, the ISI Web of Knowledge, PubMed and Scopus, along with a manual search to identify other relevant case-control and cohort studies. Quality assessments and an evaluation of publication bias were undertaken. Statistical heterogeneity was examined, and odds ratios (ORs) and 95% confidence intervals (CI) estimated using the random effects model. Of 372 articles initially retrieved, 28 studies were selected as eligible for meta-analysis. No evidence of publication bias was found using funnel plot analysis and the Egger linear regression method. Many studies were classified as low quality due to inadequate case-control data. On the basis of this research, maternal factors most associated with CL±P were: tobacco (OR 1.48), alcohol (OR 1.28), folic acid intake (OR 0.77), obesity (OR 1.26), stressful events (OR 1.41), low blood zinc levels (OR 1.82), and fever during pregnancy (OR 1.30). Folic acid intake by the mother reduced the risk of CL±P in offspring (OR 0.77).

Role of renal microcirculation in experimental renovascular disease.

BACKGROUND: Renal artery stenosis (RAS) causes renal injury partly via microvascular (MV) endothelial dysfunction and damage. Vascular endothelial growth factor (VEGF) is crucial for preservation of microvasculature and promotes vascular proliferation and endothelial repair. We have previously shown that MV rarefaction is associated with decreased VEGF in the kidney exposed to chronic RAS, accompanied by deteriorated renal function and fibrosis. We hypothesized that preserving the renal microcirculation in the stenotic kidney will halt the progression of renal damage. METHODS: Unilateral RAS was induced in 16 pigs. In eight, VEGF (0.05 micrograms/kg) was infused intra-renally at the onset of RAS. After 6 weeks, single-kidney haemodynamics and function were assessed using in vivo multi-detector computed tomography (CT). Renal microvessels, angiogenic pathways and morphology were investigated ex vivo using micro-CT, real-time PCR and histology. RESULTS: Blood pressure and degree of RAS was similar in RAS and RAS + VEGF pigs. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in RAS compared to Normal (221.1 +/- 46.5 and 29.9 +/- 3.8 vs. 522.5 +/- 60.9 and 49.3 +/- 3.4 mL/min, respectively, P < 0.05), accompanied by decreased cortical MV density and increased renal fibrosis. Pre-emptive administration of VEGF preserved MV architecture, attenuated fibrosis and normalized RBF and GFR (510.8 +/- 50.9 and 39.9.1 +/- 4.1 mL/min, P = not significant vs. Normal). CONCLUSIONS: This study underscores the importance of the renal microcirculation in renovascular disease. Intra-renal administration of VEGF preserved renal MV architecture and function of the stenotic kidney, which in turn preserved renal haemodynamics and function and decreased renal fibrosis. These observations suggest that preventing renal MV loss may be a potential target for therapeutic approaches for patients with chronic renovascular disease.

Impaired response of HIV type 1-specific CD8(+) cells from antiretroviral-treated patients.

Impairment of the response of HIV-specific CD8(+) T cells, in spite of the high frequency of occurrence of these cells even in the advanced phase of HIV-1 infection, has been demonstrated. It is also known that new antiretroviral treatments are able to reduce the viral load and partially repair the immunological damage caused by HIV-1, but it is not clear whether the extent of these changes affects the functional profile of HIV-specific CD8(+) T cells. We evaluated, in HIV-1(+) patients undergoing antiretroviral therapy, the HIV-specific CD8(+) subset distribution and their functional capacity as intracellular expression of IFN-gamma, TNF-alpha, and perforin after PMA stimulation. Our results indicate that HIV-1(+)-treated individuals show distributions of HIV-specific CD8 subsets similar to nontreated patients, while the frequency of HIV-specific CD8 cells expressing IFN-gamma and perforin after stimulation is lower in HAART-treated patients. This indicates that HAART, which controls viral replication, may impair the HIV-specific CD8(+) response.

Inmunobiología e inmunopatología de las células NKT humanas / Human NKT cells in health and disease

Las células T "Natural killer" (NKT) constituyen un nuevo linaje de linfocitos fenotípicamente y funcionalmente diferente de los linfocitos T alfa /beta convencionales. Las células NKT se caracterizan por una expresión restringida de la cadena alfa del TCR codificada por los segmentos génicos V alfa24/J alfaQ en seres humanos y V alfa14/J alfa281 en ratones, así como por la expresión de los receptores asociados a células NK, CD161 en humanos y NK1.1 en ratones. Tanto las células NKT humanas como las murinas interaccionan específicamente con glicolípidos presentados en el contexto de la molécula CD1d y juegan un papel inmunorregulador importante mediante la producción de Interferon-alfa e Interleuquina-4. Recientemente, el desarrollo de ratones manipulados genéticamente, que presentan pérdida selectiva de células NKT V alfa14, ha permitido elucidar la función de las células NKT en la respuesta frente a diferentes infecciones y tumores en diversos modelos experimentales. Se ha demostrado la existencia de cambios en el número y función de las células NKT en diferentes enfermedades que apoyan la participación de estas células en la regulación de la respuesta inmune particularmente en la regulación de la homeostasis de las respuestas autoinmunes y en la inmunidad tumoral (AU)

El amor y la consulta. Relaciones amorosas y problemas médicos / The love and the consult. Loving relations and medical problems

El artículo trata de la articulación entre la psicología y los problemas de las relaciones amorosas y la sintomatología física. La evolución social está cambiando las relaciones familiares y las relaciones amorosas. Al tiempo los problemas, conflictos o trastornos de personalidad están aumentando. Ambas cosas alteran la capacidad de establecer relaciones amorosas sólidas y estables. Mucho del sufrimiento físico que llega a la consulta del médico tiene que ver con problemas de amor y desamor. El médico debe de conocer estos problemas para ayudar a sus pacientes a interpretar su patología y buscar soluciones (AU)

Role of the carboxy-termini of tubulin on its chaperone-like activity.

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of alpha-crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone-like activity as demonstrated by prevention of DTT-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, betagamma-crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subunits has been investigated. We observe that the digestion of C-terminus of beta-subunit with subtilisin causes loss of chaperone-like activity of tubulin. The contribution of C-terminus of alpha-subunit is difficult to establish directly as subtilisin cleaves C-terminus of beta-subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C-terminus of alpha-subunit. We have shown that the binding of P2 peptide to alphabeta-tubulin causes complete loss of its chaperone-like activity. NMR and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of alpha-subunit compared to that of beta-subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Implications for the chaperone functions in vivo have been discussed.

Células NK en la infección VIH-1 / NK cells in HIV-1 infection

La inmunodeficiencia producida por el virus de la inmunodeficiencia humana (VIH-1), puede ser una consecuencia de la estrategia que el virus lleva a cabo para sobrevivir en el huésped. La infección por VIH-1 no sólo afecta a las células CD4+ sino también a otros componentes de la respuesta adaptativa e innata. Cada día mayor número de trabajos apoyan la idea de que la función de las células NK, que son un componente esencial del sistema inmunitario innato y también del adaptativo, están dañadas durante la infección por VIH-1. Sin embargo, no se conoce ni la magnitud del deterioro de estas células ni su contribución al progreso de la enfermedad. En nuestro laboratorio, estamos analizado las bases celulares y moleculares del defecto funcional de las células NK de individuos infectados por VIH-1. Consideramos que el conocimiento del papel de las células NK en la infección VIH-1 contribuirá a un mejor entendimiento de la patogénesis del síndrome de inmunodeficiencia adquirida (SIDA). (AU)

HLA-B2702 (77-83/83-77) peptide binds to beta-tubulin on human NK cells and blocks their cytotoxic capacity.

It has been described that peptides derived from a highly conserved region of the alpha1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity blocking both T and NK cell cytotoxicity. In vivo treatment with these peptides prolongs survival of MHC-mismatched allografts. However, the molecular bases of these effects are still unclear. In this study, we further analyze the mechanisms by which the dimeric peptide HLA-B2702 (77-83/83-77) induces suppression of NK cell cytotoxicity. This peptide inhibits natural and redirected lysis mediated by NK cells without significantly affecting effector-target cell binding. We have also isolated and sequenced a protein that binds this inhibitory peptide, which structurally corresponds to beta-tubulin. Tubulin is the major protein of microtubules and is involved in target cell killing. Furthermore, B2702 peptide promotes GTP-independent tubulin assembly, producing aggregates that cannot be depolymerized by cold. Treatment of NK cells with Taxol or demecolcine, which interfere with microtubule organization, also prevents NK cell cytotoxicity. Taken together, these results support the hypothesis that the peptide B2702 (77-83/83-77) exerts its inhibitory effect on NK cell cytotoxicity by inducing polymerization of microtubules and interfering with their normal assembly/disassembly dynamics.