In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents.

The Effects of Physical and Chemical Modulating Agents on the Induction of Vaccine-Induced Immune Responses

After the development of the viral-based prostate cancer vaccine, Ad5-PSA, much research has been orientated to help enhance the induced immune response by combining the vaccine with physical and chemical modulating agents, more specifically the polymers polyethylenimine (PEI), chitosan, and chitosan coated with CD3 complex antibodies; all previously shown to stimulate an immune response as isolated gene carriers. To compare the vaccine-induced immune responses between the naked vaccine and the polymer-vaccine combinations, a mouse model using the ovalbumin- specific Ad-OVA vaccine was tested using intracellular cytokine staining (ICS), tetramer staining, and cytotoxic T-cell lymphocyte assays to measure the activation of CD8+ T-cells, interferon gamma proteins (INFƒ×), and the induced cytotoxicity to ovalbumin. The Ad-OVA vaccine combined with both chitosan and chitosan with CD3 complex antibodies, both natural polymers, were found to induce similar immune responses to the naked vaccine while the vaccine combined with the synthetic polymer, PEI, diminished the immune response.

Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels.

BACKGROUND The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR). RESULTS Our aim was to analyze and compare angiogenic factor expression levels in both subcutaneous (SC) and omentum (OM) adipose tissues from morbidly obese patients (n = 26) with low (OB/L-IR) (healthy obese) and high (OB/H-IR) degrees of IR, and lean controls (n = 17). Another objective was to examine angiogenic factor correlations with obesity and IR.Here we found that VEGF-A was the isoform with higher expression in both OM and SC adipose tissues, and was up-regulated 3-fold, together with MMP9 in OB/L-IR as compared to leans. This up-regulation decreased by 23% in OB/-H-IR compared to OB/L-IR. On the contrary, VEGF-B, VEGF-C and VEGF-D, together with MMP15 was down-regulated in both OB/H-IR and OB/L-IR compared to lean patients. Moreover, MMP9 correlated positively and VEGF-C, VEGF-D and MMP15 correlated negatively with HOMA-IR, in both SC and OM. CONCLUSION We hereby propose that the alteration in MMP15, VEGF-B, VEGF-C and VEGF-D gene expression may be caused by one of the relevant adipose tissue processes related to the development of IR, and the up-regulation of VEGF-A in adipose tissue could have a relationship with the prevention of this pathology.

Study of the effectiveness of first-line treatment in renal cell carcinoma.

The emergence of novel drugs corresponds with the determination of the effectiveness of the current treatments used in clinical practice. A retrospective observational study was conducted to evaluate the effectiveness of first-line treatments and to test the influence of the prognostic factors established using the Memorial Sloan-Kettering Cancer Center (MSKCC) and the analysis of Mekhail's study for two or more metastatic sites. The primary endpoints were median progression-free survival (mPFS) and median overall survival (mOS) times. A total of 65 patients were enrolled and the mPFS and mOS of the patients treated with sunitinib (n=51) were 9.0 and 20.1 months, respectively, and for the patients treated with temsirolimus (n=14) these were 3.0 and 6.2 months, respectively. In the poor-prognosis (PP) group, a difference of 1.2 months (P=0.049) was found in mPFS depending on the first-line treatment. A difference of 4.1 months (P=0.023) was also found in mPFS when classified by histology (clear verses non-clear cell) in the sunitinib-treatment group. When stratified by the prognostic group, differences of >7 months (P<0.001) were found between the groups. Therefore, it was concluded that the effectiveness of the treatments was reduced compared to previous studies and differences were found in the PP group when classified by first-line drug and histology. Additionally, the influence of prognostic factors on OS and the value of stratifying patients using these factors have been confirmed.

Evaluation of the efficacy and safety of lanreotide in combination with targeted therapies in patients with neuroendocrine tumours in clinical practice: a retrospective cross-sectional analysis.

BACKGROUND Based on the mechanism of action, combining somatostatin analogues (SSAs) with mTOR inhibitors or antiangiogenic agents may provide synergistic effects for the treatment of patients with neuroendocrine tumours (NETs). Herein, we investigate the use of these treatment combinations in clinical practice. METHODS This retrospective cross-sectional analysis of patients with NETs treated with the SSA lanreotide and targeted therapies at 35 Spanish hospitals evaluated the efficacy and safety of lanreotide treatment combinations in clinical practice. The data of 159 treatment combinations with lanreotide in 133 patients was retrospectively collected. RESULTS Of the 133 patients, with a median age of 59.4 (16-83) years, 70 (52.6 %) patients were male, 64 (48.1 %) had pancreatic NET, 23 (17.3 %) had ECOG PS ≥2, 41 (30.8 %) had functioning tumours, 63 (47.7 %) underwent surgery of the primary tumour, 45 (33.8 %) had received prior chemotherapy, and 115 (86.5 %) had received prior SSAs. 115 patients received 1 lanreotide treatment combination and 18 patients received between 2 and 5 combinations. Lanreotide was mainly administered in combination with everolimus (73 combinations) or sunitinib (61 combinations). The probability of being progression-free was 78.5 % (6 months), 68.6 % (12 months) and 57.0 % (18 months) for patients who only received everolimus plus lanreotide (n = 57) and 89.3 % (6 months), 73.0 % (12 months), and 67.4 % (18 months) for patients who only received sunitinib and lanreotide (n = 50). In patients who only received everolimus plus lanreotide the median time-to-progression from the initiation of lanreotide combination treatment was 25.8 months (95 % CI, 11.3, 40.3) and it had not yet been reached among the subgroup of patients only receiving sunitinib plus lanreotide. The safety profile of the combination treatment was comparable to that of the targeted agent alone. CONCLUSIONS The combination of lanreotide and targeted therapies, mainly everolimus and sunitinib, is widely used in clinical practice without unexpected toxicities and suggests efficacy that should be explored in randomized prospective clinical trials.

Potencial angiogênico de células pulpares humanas em hipóxia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação na linhagem endotelial tEnd dos efeitos diretos da transferência gênica de IFNbeta e p19arf e efeitos parácrinos mediados pela linhagem B16 transduzida pelos mesmos vetores adenovirais

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliais

Inhibition of tumor angiogenesis by non-steroidal anti-inflammatory drugs: emerging mechanisms and therapeutic perspectives.

Chronic intake of non steroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of developing gastrointestinal tumors, in particular colon cancer. Increasing evidence indicates that NSAID exert tumor-suppressive activity on pre-malignant lesions (polyps) in humans and on established experimental tumors in mice. Some of the tumor-suppressive effects of NSAIDs depend on the inhibition of cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxane, which is highly expressed in inflammation and cancer. Recent findings indicate that NSAIDs exert their anti-tumor effects by suppressing tumor angiogenesis. The availability of COX-2-specific NSAIDs opens the possibility of using this drug class as anti-angiogenic agents in combination with chemotheapy or radiotherapy for the treatment of human cancer. Here we will briefly review recent advances in the understanding of the mechanism by which NSAIDs suppress tumor angiogenesis and discuss their potential clinical application as anti-cancer agents.

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.

Clinical presentation and time-course of postoperative venous thromboembolism: Results from the RIETE Registry

There is little literature about the clinical presentation and time-course of postoperative venous thromboembolism (VTE) in different surgical procedures. RIETE is an ongoing, prospective registry of consecutive patients with objectively confirmed, symptomatic acute VTE. In this analysis, we analysed the baseline characteristics, thromboprophylaxis and therapeutic patterns, time-course, and three-month outcome of all patients with postoperative VTE. As of January 2006, there were 1,602 patients with postoperative VTE in RIETE: 393 (25%) after major orthopaedic surgery (145 elective hip arthroplasty, 126 knee arthroplasty, 122 hip fracture); 207 (13%) after cancer surgery; 1,002 (63%) after other procedures. The percentage of patients presenting with clinically overt pulmonary embolism (PE) (48%, 48%, and 50% respectively), the average time elapsed from surgery to VTE (22 +/- 16, 24 +/- 16, and 21 +/- 15 days, respectively), and the three-month incidence of fatal PE (1.3%, 1.4%, and 0.8%, respectively), fatal bleeding (0.8%, 1.0%, and 0.2%, respectively), or major bleeding (2.3%, 2.9%, and 2.8%, respectively) were similar in the three groups. However, the percentage of patients who had received thromboprophylaxis (96%, 76% and 52%, respectively), the duration of prophylaxis (17 +/- 9.6, 13 +/- 8.9, and 12 +/- 11 days, respectively) and the mean daily doses of low-molecular-weight heparin (4,252 +/- 1,016, 3,260 +/- 1,141, and 3,769 +/- 1,650 IU, respectively), were significantly lower in those undergoing cancer surgery or other procedures. In conclusion, the clinical presentation, time-course, and three-month outcome of VTE was similar among the different subgroups of patients, but the use of prophylaxis in patients undergoing cancer surgery or other procedures was suboptimal.

Intrachannel versus interchannel application of angiogenic factors in transmyocardial laser revascularization.

BACKGROUND AND OBJECTIVE: Theoretically myocardial angiogenesis of laser injury can be further enhanced by the addition of angiogenic growth factors. The influence of the way of administration of these factors on vascular growth around the channels is still unclear. MATERIALS AND METHODS: 18 pigs (mean weight 72 +/- 5.2 kg) were randomized to either triads of transmyocardial laser revascularization (TMLR) channels (group 1, n = 6) or isolated channels (group 2, n = 6), or a control group (n = 6). The animals had injections of bovine bone derived growth factor mixture either in the center of the triads in group 1 or within the channels themselves in group 2. Animals were sacrificed one month later for histological analysis. RESULTS: The vascular densities of myocardial areas within the triads of group 1 and around the channels in group 2 were significantly larger than in the control group: 15.2 +/- 3.7/mm2 and 14.2 +/- 3.5/mm2 respectively vs 5.3 +/- 1.6/mm2 (p < 0.001 for both differences). Differences of densities between group 1 and 2 were not statistically significant (p = 0.6). CONCLUSIONS: In this porcine model, the addition of a bovine bone derived growth factor mixture to TMLR significantly stimulates angiogenesis in the areas adjacent to the channels. The place of injection does not influence the angiogenesis intensity.

Modulation of potassium channels inhibits bunyavirus infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Etablierung eines In-vivo-Modells zur Untersuchung der Rolle epidermaler Langerhanszellen bei der Entstehung der Transplantat-gegen-Wirt-Erkrankung nach allogener Blutstammzelltransplantation

Dendritische Zellen der Haut, wie z.B. die Langerhanszellen (LC) der Epidermis, sind potente antigenpräsentierende Zellen (APC). Nach allogener Blutstammzelltransplantation (engl.: hematopoietic stemm cell transplantation, HSCT) persistieren Empfänger-APC und können Spender-T-Zellen aktivieren. Somit spielen dendritische Zellen eine kritische Rolle bei der Initiierung von akuter Transplantat-Gegen-Wirt-Reaktion (engl.: graft-versus-host-disease, GvHD).rnIn der vorliegenden Arbeit wurde ein Modellsystem entwickelt, welches humane Haut in einem Xenotransplantationsmodell nutzt, um die Wechselwirkung dieser gewebsständigen APC mit alloreaktiven T-Zellen zu untersuchen. Dafür wurden humane Resthautpräparate von subkutanem Gewebe befreit und intraskaptulär auf immunsupprimierte NOD/LtSz-scid IL2R#-null Mäuse (NSG) transplantiert. Diesen Tieren fehlen funktionale T-, B- und NK-Zellen, und sie tolerieren somit ein xenogenes Transplantat. Im Vergleich zu anderen immundefizienten Stämmen, haben sie eine erhöhte Lebenserwartung und es ist zudem möglich humane Hämatopoese durch Stammzellgabe zu etablieren.rnPublizierte Methoden der Hauttransplantation wurden für diese Arbeit optimiert und weiterentwickelt. So konnte die Erfolgsrate von 44% auf bis zu 95% gesteigert werden. Erste Untersuchungen fokussierten den Einfluss der Wundheilung auf die Verteilung dermaler Zellpopulationen, wie z.B. CD11c positive APC, und die Population der LC in der Epidermis. Während der ersten Wochen der Wundheilung war ein vorübergehendes Verschwinden der LC aus der Epidermis zu beobachten. Im Gegensatz dazu waren CD11c positive dermale Zellen permanent detektierbar. Die zu späteren Zeitpunkten festgestellte Repopulation der Epidermis mit LC unterstützt die Hypothese einer lokalen Vorläuferzelle. Die vorgelegten Daten und die lokale proliferative Aktivität dieser Zellen unterstreichen ihre Unabhängigkeit vom peripheren Blut. Versuche, eine Depletion der LC mittels UVC-Bestrahlung zu erreichen, gelangen nicht. Auch dies spricht für das Vorhandensein eines lokalen Vorläufers.rnZur Induktion von GvHD in der transplantierten Haut wurden in vitro DC des Hautspenders generiert und damit HLA-disparate T-Zellen stimuliert. Auf diese Weise sollte eine maximale Alloreaktivität gegen das Hauttransplantat generiert werden. In allen vorgestellten Systemen ließ sich nach Infusion der T-Lymphozyten in transplantierte Tiere, eine T-Zellinduzierte inflammatorische Reaktion auslösen. Optisch war eine deutliche Rötung des Transplantats feststellbar. Diese war jedoch nur in den Proben besonders deutlich, welche T-Zellen mit vorheriger in vitro Stimulation durch DC des Hautspenders erhalten hatten. Histologisch konnten Anzeichen einer Entzündung nachgewiesen werden. Neben Akanthose und Hyperparakeratose, waren deutliche T-Zellinfiltrate detektierbar. Auch Spaltbildung und Ablösung der Epidermis, sowie vereinzelte Apoptosen der epidermalen Zellen wiesen auf eine GvHD artige Entzündung hin.rnEine weitere Beobachtung nach T-Zellgabe, war die Depletion der LC aus der Epidermis. Auch konnte durch spätere T-Zellgaben keine weitere Hautrötung ausgelöst werden. Dies belegt die Funktion der LC als primäre Zielzelle der alloreaktiven T-Zellen. Unterstrichen wird dies durch Verwendung einer LC defizienten Haut, welche keine Hautrötung oder Anzeichen einer Entzündung entwickelte.rnZusammenfassend wurde für diese Arbeit ein Modellsystem entwickelt, welches es erlaubt Untersuchungen entzündlicher Hautkrankheiten unter Berücksichtigung hautständiger APC durchzuführen. Dabei kann dieses Modell in Zukunft für die Untersuchung von APC modulierenden Agenzien genutzt werden, da präklinische Modelle für spezies-spezifische Therapien bislang fehlten. Das Entstehen einer Entzündung könnte so verhindert oder eine Behandlung ermöglicht werden.

Radiotherapy and Immunotherapy Combinations in Non-small Cell Lung Cancer: A Promising Future?

The goal of re-programming the host immune system to target malignancy with durable anti-tumour clinical responses has been speculated for decades. In the last decade such speculation has been transformed into reality with unprecedented and durable responses to immune checkpoint inhibitors seen in solid tumours. This mini-review considers the mechanism of action of immune modulating agents and the potential for combination with radiotherapy in the treatment of non-small cell lung cancer.

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.