In 2006, the Mauritanian Ministry of Health adopted a new therapeutic strategy based on the systematic use of artemisinin-based combination therapy (ACT), artesunate-amodiaquine and artemether-lumefantrine, for the first- and second-line treatment of uncomplicated malaria, respectively, regardless of Plasmodium spp. In the Saharan zone of the country, recent studies have shown that Plasmodium vivax largely predominates over Plasmodium falciparum. Anti-malarial drug response of P. vivax has not been evaluated in Mauritania. The aim of the present study was to evaluate the clinical efficacy and tolerance of chloroquine to treat P. vivax malaria in Mauritanian patients.
Plasmodium vivax-infected patients aged > 6 months old were enrolled in Nouakchott and Atar in September–October 2013. Chloroquine was administered at the standard dose of 25 mg base/kg body weight over three days. Patients were followed until day 28, according to the standard 2009 World Health Organization protocol.
A total of 128 patients (67 in Nouakchott and 61 in Atar) were enrolled in the study. Seven patients (5.5%) were either excluded or lost to follow-up. Based on the per protocol analysis, chloroquine efficacy (adequate clinical and parasitological response) was 100%. Treatment was well-tolerated. One patient was excluded on day 1 due to urticaria and treated with artesunate-amodiaquine.
Although the current national treatment guideline recommends artesunate-amodiaquine for the first-line treatment of uncomplicated malaria, including P. vivax malaria, chloroquine may still have an important role to play in anti-malarial chemotherapy in Mauritania. Further epidemiological studies are required to map the distribution of P. vivax and P. falciparum in the country.
Drug resistance; Plasmodium vivax; Artemisinin; 4-aminoquinolines; Clinical trial
Genetic improvement for enhanced disease resistance in fish is an increasingly utilized approach to mitigate endemic infectious disease in aquaculture. In domesticated salmonid populations, large phenotypic variation in disease resistance has been identified but the genetic basis for altered responsiveness remains unclear. We previously reported three generations of selection and phenotypic validation of a bacterial cold water disease (BCWD) resistant line of rainbow trout, designated ARS-Fp-R. This line has higher survival after infection by either standardized laboratory challenge or natural challenge as compared to two reference lines, designated ARS-Fp-C (control) and ARS-Fp-S (susceptible). In this study, we utilized 1.1 g fry from the three genetic lines and performed RNA-seq to measure transcript abundance from the whole body of naive and Flavobacterium psychrophilum infected fish at day 1 (early time-point) and at day 5 post-challenge (onset of mortality). Sequences from 24 libraries were mapped onto the rainbow trout genome reference transcriptome of 46,585 predicted protein coding mRNAs that included 2633 putative immune-relevant gene transcripts. A total of 1884 genes (4.0% genome) exhibited differential transcript abundance between infected and mock-challenged fish (FDR < 0.05) that included chemokines, complement components, tnf receptor superfamily members, interleukins, nod-like receptor family members, and genes involved in metabolism and wound healing. The largest number of differentially expressed genes occurred on day 5 post-infection between naive and challenged ARS-Fp-S line fish correlating with high bacterial load. After excluding the effect of infection, we identified 21 differentially expressed genes between the three genetic lines. In summary, these data indicate global transcriptome differences between genetic lines of naive animals as well as differentially regulated transcriptional responses to infection.
Flavobacterium psychrophilum; bacterial cold water disease; selective breeding; disease resistance; aquaculture; immune gene; tnfrsf; rainbow trout genome
A regular evaluation of therapeutic efficacy in sentinel sites and a system of surveillance are required to establish treatment guidelines and adapt national anti-malarial drug policy to the rapidly changing epidemiology of drug-resistant malaria. The current anti-malarial treatment guideline in Mauritania, officially recommended since 2006, is based on artemisinin-based combination therapy. The aim of the present study was to evaluate clinical efficacy and tolerance of artesunate-amodiaquine, the first-line treatment for acute uncomplicated malaria, in Mauritanian paediatric and adult patients to validate its continued use in the country.
Plasmodium falciparum-infected symptomatic patients aged > six months were enrolled in Kobeni and Timbedra in southern Mauritania in September to October 2013. Co-formulated artesunate-amodiaquine was administered at the recommended dose over three days. Patients were followed until day 28. Parasitological and clinical response was classified according to the standard 2009 World Health Organization protocol.
A total of 130 patients (65 in Kobeni and 65 in Timbedra) were enrolled in the study. Seventeen patients (13.1%) were either excluded (before PCR correction) or lost to follow-up. Based on the per protocol analysis, artesunate-amodiaquine efficacy (i.e., the proportion of adequate clinical and parasitological response) was 96.6% in Kobeni and 98.2% in Timbedra before PCR correction. Late clinical failure was observed in two patients in Kobeni and one patient in Timbedra. After PCR correction, the efficacy rate in the two study sites was 98.2%. On day 3, all patients were afebrile and had negative smears. Treatment was well tolerated.
Artesunate-amodiaquine is well tolerated and highly efficacious for the treatment of uncomplicated P. falciparum malaria. In the majority of patients, fever and parasitaemia were rapidly cleared before day 3. The results support the national anti-malarial drug guideline for a continued use of artesunate-amodiaquine as a first-line drug for uncomplicated malaria in southern Mauritania.
Drug resistance; Plasmodium falciparum; Artemisinin; 4-aminoquinolines; Clinical trial
Acetaminophen (APAP)-induced toxicity is a predominant cause of acute hepatic and renal failure. In both humans and rodents toxicity begins with a reactive metabolite that binds to proteins. This leads to mitochondrial dysfunction and nuclear DNA fragmentation resulting in necrotic cell death. Pleurotus ostreatus (an edible oyster mushroom) is well recognized as a flavourful food, as well as a medicinal supplement. In the present study, we evaluated the role of Pleurotus ostreatus in the protection against APAP-induced hepato-renal toxicity. We also explored the mechanism by which Pleurotus ostreatus exerts its effects.
Ninety adult male Swiss albino mice were divided into three groups (30 mice/group). Mice were offered normal diet (control and APAP groups), or diet supplemented with 10% Pleurotus ostreatus (APAP + Pleurotus ostreatus) for 10 days. Mice were either treated with vehicle (control group, single intra-peritoneal injection.), or APAP (APAP and APAP + Pleurotus ostreatus groups, single intra-peritoneal injection, 500 mg/kg), 24 hours after the last meal.
APAP increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) glutamate dehydrogenase (GDH), creatinine, blood urea nitrogen (BUN), urinary kidney injury molecule-1 (KIM-1), and hepatic and renal malondialdehyde (MDA) content. APAP decreased hepatic and renal glutathione (GSH) content, as well as glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities. Supplementation with Pleurotus ostreatus significantly reduced APAP-induced elevated levels of ALT, AST, GDH, creatinine, BUN, KIM-1and MDA, while GSH level, and GSH-Px and SOD activities were significantly increased. Our findings were further validated by histopathology; treatment with Pleurotus ostreatus significantly decreased APAP-induced cell necrosis in liver and kidney tissues.
We report here that the antioxidant effect of Pleurotus ostreatus opposes mitochondrial dysfunction and oxidative stress accompanying APAP over-dose, with subsequent clinically beneficial effects on liver and kidney tissues.
Pleurotus ostreatus; Oxidative stress; Acute hepato-renal injury; Mitochondrial dysfunction; Acetaminophen; Antioxidant
Resistance against diseases affects profitability of rainbow trout. Limited information is available about functions and mechanisms of teleost immune pathways. Immunogenomics provides powerful tools to determine disease resistance genes/gene pathways and develop genetic markers for genomic selection. RNA-Seq sequencing of the rainbow trout spleen yielded 93,532,200 reads (100 bp). High quality reads were assembled into 43,047 contigs. 26,333 (61.17%) of the contigs had hits to the NR protein database and 7024 (16.32%) had hits to the KEGG database. Gene ontology showed significant percentages of transcripts assigned to binding (51%), signaling (7%), response to stimuli (9%) and receptor activity (4%) suggesting existence of many immune-related genes. KEGG annotation revealed 2825 sequences belonging to “organismal systems” with the highest number of sequences, 842 (29.81%), assigned to immune system. A number of sequences were identified for the first time in rainbow trout belonging to Toll-like receptor signaling (35), B cell receptor signaling pathway (44), T cell receptor signaling pathway (56), chemokine signaling pathway (73), Fc gamma R-mediated phagocytosis (52), leukocyte transendothelial migration (60) and NK cell mediated cytotoxicity (42). In addition, 51 transcripts were identified as spleen-specific genes. The list includes 277 full-length cDNAs. The presence of a large number of immune-related genes and pathways similar to other vertebrates suggests that innate and adaptive immunity in fish are conserved. This study provides deep-sequence data of rainbow trout spleen transcriptome and identifies many new immune-related genes and full-length cDNAs. This data will help identify allelic variations suitable for genomic selection and genetic manipulation in aquaculture.
spleen transcriptome; annotation; KEGG; immune-related genes; spleen-specific genes; full-length cDNA
The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Mauritania. The present study examined and compared the genetic diversity of P. falciparum isolates in Mauritania.
Plasmodium falciparum isolates blood samples were collected from 113 patients attending health facilities in Nouakchott and Hodh El Gharbi regions. K1, Mad20 and RO33 allelic family of msp-1 gene were determined by nested PCR amplification.
K1 family was the predominant allelic type carried alone or in association with Ro33 and Mad20 types (90%; 102/113). Out of the 113 P. falciparum samples, 93(82.3%) harboured more than one parasite genotype. The overall multiplicity of infection was 3.2 genotypes per infection. There was no significant correlation between multiplicity of infection and age of patients. A significant increase of multiplicity of infection was correlated with parasite densities.
The polymorphism of P. falciparum populations from Mauritania was high. Infection with multiple P. falciparum clones was observed, as well as a high multiplicity of infection reflecting both the high endemicity level and malaria transmission in Mauritania.
Plasmodium falciparum; Malaria; Genetic diversity; Multiplicity of infection; msp-1 gene; Mauritania
Recently, we showed that post cyclophosphamide (CTX) microenvironment benefits the function of transferred T cells. Analysis of the kinetics of cellular recovery after CTX treatment showed that a single 4 mg/mouse CTX treatment decreased the absolute number of leukocytes in the peripheral blood (PBL) at days 3-15, and in the spleen and bone marrow (BM) at days 3-6. The absolute numbers of CD11c+CD11b− and CD11c+CD11b+ dendritic cells (DCs), CD11b+ and Ly6G+ myeloid cells, T and B cells, CD4+CD25+ T regulatory (Treg) cells, and NK1.1+ cells also decreased. The cell numbers returned to control levels during the recovery phase. The absolute numbers of B cells remained low for 3 weeks. The numbers of DCs increased in PBL and spleen at day 9 but returned to control levels at day 15. These data indicate that CTX alters the cellular microenvironment in kinetics that might be precisely targeted to benefit the host.
Cyclophosphamide; Chemotherapy; Dendritic cells; Lymphocytes; Regulatory T cells; Natural Killer cells; Blood; Bone marrow; Spleen
Muscle degradation occurs as a response to various physiological states that are regulated by specific molecular mechanisms. Previously, we characterized the metabolic changes of muscle deterioration of the female rainbow trout at full sexual maturity and spawning (Salem et al., Physiol. Genomics 2006;28:33–45; J. Proteomics 2010;73:778–789). Muscle deterioration in this model represents nutrient mobilization as a response to the energetic overdemands of the egg/ovarian growth phase. Our recent studies showed that most of the changes in muscle growth and quality start 2–3 months before spawning. Gravid fish exhibited reduced intramuscular fat that is lower in saturated and monounsaturated fatty acids and higher in polyunsaturated fatty acids compared to sterile fish. In this study, RNA-Seq was used to explain the mechanisms underlying changes during this phase of sexual maturity. Furthermore, to minimize changes due to nutrient deficits, fish were fed on a high-plane of nutrition. The RNA-Seq technique identified a gene expression signature that is consistent with metabolic changes of gravid fish. Gravid fish exhibited increased abundance of transcripts in metabolic pathways of fatty acid degradation and up-regulated expression of genes involved in biosynthesis of unsaturated fatty acids. In addition, increased expression of genes involved in the citric acid cycle and oxidative phosphorylation was observed for gravid fish. This muscle transcriptomic signature of fish fed on a high nutritional plane is quite distinct from that previously described for fish at terminal stages of maturity and suggest that female rainbow trout approaching spawning, on high nutritional planes, likely mobilize intramuscular fat rather than protein to support gonadal maturation.
muscle; rainbow trout; reproduction; RNA-Seq
Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.
We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.
We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.
Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.
G-CSF; Myeloid cells; Antibody/cytokine complexes; Pegylation; Neutrophils; Neupogen; Neulasta; Protein therapeutics
Background. In patients with hepatocellular carcinoma, selection criteria for transarterial hepatic selective internal radiotherapy are imprecise. Additionally, radiographic parameters to predict outcome of transarterial hepatic selective internal radiotherapy have not been fully characterized. Patients and methods. Computed tomography (CT) scans of 23 patients with unresectable primary hepatocellular carcinoma before and after transarterial hepatic selective internal radiotherapy with yttrium-90 microspheres were retrospectively reviewed. Selected radiographic parameters were evaluated and correlated with progression-free survival and overall survival. Response to treatment was assessed with Response RECIST 1.1 and Morphology, Attenuation, Size, and Structure (MASS) criteria. Results. On the post-SIRT CT, 68% of tumors demonstrated decreased size (median decrease of 0.8 cm, P = 0.3); 64% had decreased attenuation (median decrease 5.7 HU, P = 0.06), and 48% demonstrated increased tumor necrosis (P < 0.001). RECIST-defined partial response was seen in 10% patients, stable disease in 80%, and 10% had disease progression. Median progression-free survival was 3.9 months (range, 3.3 to 7.3), and median overall survival was 11.2 months (7.1 to 31.1). Pretreatment lower hepatopulmonary shunt fraction, central hypervascularity, and well-defined tumor margins were associated with improved progression-free survival. Conclusion. In patients with unresectable hepatocellular carcinoma, pretreatment CT parameters may predict favorable response to SIRT and improve patient selection.
Clinical application of adoptive T cell therapy (ACT) has been hindered by an inability to generate adequate numbers of non-tolerized, functionally active tumor-specific T cells which can persist in vivo. In order to address this, we evaluated the impact of IL-12 signaling during tumor-specific CD8+ T cell priming in terms of persistence and anti-tumor efficacy using an established B16 melanoma tumor adoptive therapy model.
B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, i.p.), and one day later, treated by adoptive transfer of tumor-specific pmel-1 CD8+ T cells primed ex vivo 3 days earlier with (i) both IL-12 and antigen (hGP10025–33 peptide) or (ii) antigen only. Tumors were measured biweekly and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function.
Adoptive transfer of tumor-specific CD8+ T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced anti-tumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ).
Our findings demonstrate that ex vivo priming of tumor-specific CD8+ T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability upon transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.
Malignant epithelioid hemangioendothelioma (EH) is a rare tumor of vascular origin. We report a case of a woman who was found to have multiple hepatic masses in the right lobe of the liver on radiologic investigations, initially misdiagnosed as a metastatic carcinoma. The diagnosis of EH was made on histopathological study and confirmed by immunohistochemistry, which showed diffuse response for CD34 marker and no response to tissue CEA, HMB-45 or S-100 protein. Partial hepatectomy was made with good results.
Epithelioid; Liver; Histopathology; Immunohistochemistry
Abnormal accumulation of myeloid-derived suppressor cells (MDSC) is an important mechanism of tumor immune evasion. Cyclophosphamide (CTX) has also been shown in non-tumor bearing animals to cause transient surges in MDSC. Knowledge of MDSC is primarily based on preclinical work, and to date only few published studies have involved cancer patients. The goal of this study was to test the hypothesis that circulating MDSC levels correlate with clinical cancer stage, CTX-based chemotherapy, and metastatic tumor burden. Whole blood was collected from 106 newly diagnosed solid tumor patients (stages I–IV). Percentages of circulating MDSC (Lin−/Lo, HLA DR−, CD33+CD11b+) were determined prior to initiation of systemic therapy. In 17 early stage breast cancer patients receiving doxorubicin–cyclophosphamide chemotherapy every 14 days (ddAC) blood was collected on day 1 of each cycle. Circulating MDSC were significantly increased in cancer patients of all stages relative to healthy volunteers. A significant correlation between circulating MDSC and clinical cancer stage was also observed. Moreover, among stage IV patients, those with extensive metastatic tumor burden had the highest percent and absolute number of MDSC. Significant increases in circulating MDSC were observed with ddAC when compared with pretreatment levels. Circulating MDSC levels correlate with clinical cancer stage, ddAC, and metastatic tumor burden. This information must be incorporated into the design of future trials exploring immune-based therapeutic strategies. Pharmacologic modulation of MDSC should also be tested in future clinical trials.
Immature myeloid cells; Myeloid-derived suppressor cells; Breast cancer; Cancer; Cyclophosphamide; Tumor burden; Cytokines; PBMC; T cells
Intramuscular administration of plasmid DNA vaccines is one of the main delivery approaches that can generate antigen specific T cell responses. However, major limitations of the intramuscular delivery strategy are the low level of myocyte transfection, resulting in a minimal level of protein expression; the inability to directly target antigen presenting cells, in particular dendritic cells, which are critical for establishment of efficacious antigen-specific immune responses. Although several viral vectors have been designed to improve plasmid DNA delivery, they have limitations, including the generation of neutralizing antibodies in addition to lacking the simplicity and versatility required for universal clinical application. We have developed an inexpensive non-viral delivery vector based on the polysaccharide polymer poly-N-acetyl glucosamine with the capability to target dendritic cells. This vector is fully biocompatible, biodegradable, and nontoxic. The advantage of the application of this delivery system relative to other approaches is discussed.
Cytokines; IL-12; paracrine treatment; Schistosoma mansoni; SWAP; systemic treatment; vaccination
Fast growth is an important and highly desired trait, which affects the profitability of food animal production, with feed costs accounting for the largest proportion of production costs. Traditional phenotype-based selection is typically used to select for growth traits; however, genetic improvement is slow over generations. Single nucleotide polymorphisms (SNPs) explain 90% of the genetic differences between individuals; therefore, they are most suitable for genetic evaluation and strategies that employ molecular genetics for selective breeding. SNPs found within or near a coding sequence are of particular interest because they are more likely to alter the biological function of a protein. We aimed to use SNPs to identify markers and genes associated with genetic variation in growth. RNA-Seq whole-transcriptome analysis of pooled cDNA samples from a population of rainbow trout selected for improved growth versus unselected genetic cohorts (10 fish from 1 full-sib family each) identified SNP markers associated with growth-rate. The allelic imbalances (the ratio between the allele frequencies of the fast growing sample and that of the slow growing sample) were considered at scores >5.0 as an amplification and <0.2 as loss of heterozygosity. A subset of SNPs (n = 54) were validated and evaluated for association with growth traits in 778 individuals of a three-generation parent/offspring panel representing 40 families. Twenty-two SNP markers and one mitochondrial haplotype were significantly associated with growth traits. Polymorphism of 48 of the markers was confirmed in other commercially important aquaculture stocks. Many markers were clustered into genes of metabolic energy production pathways and are suitable candidates for genetic selection. The study demonstrates that RNA-Seq at low sequence coverage of divergent populations is a fast and effective means of identifying SNPs, with allelic imbalances between phenotypes. This technique is suitable for marker development in non-model species lacking complete and well-annotated genome reference sequences.
Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.
Duffy blood group polymorphisms are important in areas where Plasmodium vivax is present because this surface antigen is thought to act as a key receptor for this parasite. In the present study, Duffy blood group genotyping was performed in febrile uninfected and P. vivax-infected patients living in the city of Nouakchott, Mauritania.
Plasmodium vivax was identified by real-time PCR. The Duffy blood group genotypes were determined by standard PCR followed by sequencing of the promoter region and exon 2 of the Duffy gene in 277 febrile individuals. Fisher's exact test was performed in order to assess the significance of variables.
In the Moorish population, a high frequency of the FYBES/FYBES genotype was observed in uninfected individuals (27.8%), whereas no P. vivax-infected patient had this genotype. This was followed by a high level of FYA/FYB, FYB/FYB, FYB/FYBES and FYA/FYBES genotype frequencies, both in the P. vivax-infected and uninfected patients. In other ethnic groups (Poular, Soninke, Wolof), only the FYBES/FYBES genotype was found in uninfected patients, whereas the FYA/FYBES genotype was observed in two P. vivax-infected patients. In addition, one patient belonging to the Wolof ethnic group presented the FYBES/FYBES genotype and was infected by P. vivax.
This study presents the Duffy blood group polymorphisms in Nouakchott City and demonstrates that in Mauritania, P. vivax is able to infect Duffy-negative patients. Further studies are necessary to identify the process that enables this Duffy-independent P. vivax invasion of human red blood cells.
Plasmodium vivax; Duffy blood group; Mauritania; polymorphism; malaria
The optimal time sequences for chemotherapy and radiation therapy after breast surgery for patients with breast cancer remains unknown. Most of published studies were done for early breast cancer patients. However, in Egypt advanced stages were the common presentation. This retrospective analysis aimed to assess the optimum sequence for our population.
267 eligible patients planned to receive adjuvant chemotherapy [FAC] and radiotherapy. Majority of patients (87.6%) underwent modified radical mastectomy while, 12.4% had conservative surgery.
We divided the patients into 3 groups according to the sequence of chemotherapy and radiotherapy. Sixty-seven patients (25.1%) received postoperative radiotherapy before chemotherapy [group A]. One hundred and fifty patients (56.2%) were treated in a sandwich scheme (group B), which means that 3 chemotherapy cycles were given prior to radiotherapy followed by 3 further chemotherapy cycles. A group of 50 patients (18.7%) was treated sequentially (group C), which means that radiotherapy was supplied after finishing the last chemotherapy cycle. Patients' characteristics are balanced between different groups.
Disease free survival was estimated at 2.5 years, and it was 83.5%, 82.3% and 80% for patient receiving radiation before chemotherapy [group A], sandwich [group B] and after finishing chemotherapy [group C] respectively (p > 0.5). Grade 2 pneumonitis, which necessitates treatment with steroid, was detected in 3.4% of our patients, while grade 2 radiation dermatitis was 17.6%. There are no clinical significant differences between different groups regarded pulmonary or skin toxicities.
Regarding disease free survival and treatment toxicities, in our study, we did not find any significant difference between the different radiotherapy and chemotherapy sequences.
breast cancer; chemotherapy; radiotherapy; sequence
Adoptive T-cell therapy holds great promise for the treatment of metastatic melanoma. However, prohibitive costs associated with current technology required for culture and expansion of tumor-reactive T-cells, the need for intense preconditioning regimens to induce lymphopenia, and the unpredictable anti-tumor effect of adoptively transferred T-cells remain significant impediments for its clinical implementation. Here we report a simplified combinatorial approach that involves short activation of CD8+ T cells in the presence of IL-12 followed by adoptive transfer into tumor bearing animals after a single injection of cyclophosphamide. This approach resulted in complete eradication of B16 melanoma, and the establishment of long term immunological memory capable of fully protecting mice after a second B16 melanoma challenge. The activated donor cells were unique because they simultaneously exhibited traits for cytotoxic effector function, central memory-like, homing, and senescence. After tumor eradication and within three months after transfer, CD8+ cells exhibited a conventional memory CTL phenotype. Moreover, these memory CTLs acquired functional attributes characteristic of memory stem cells, including the ability to resist chemotherapy-induced toxicity. Our results suggest that short-term T-cell receptor signaling in the presence of IL-12 promotes promiscuous qualities in naïve CTL which – upon transfer into lymphopenic hosts– are sufficient to eradicate tumors and generate life-long tumor-specific memory.
Pmel; melanoma; IL-12; ACT; T cell therapy; memory; CD8
Adoptive T-cell therapy holds great promise for the treatment of metastatic melanoma. However, prohibitive costs associated with current technology required for culture and expansion of tumor-reactive T-cells, the need for intense preconditioning regimens to induce lymphopenia, and the unpredictable anti-tumor effect of adoptively transferred T-cells remain significant impediments for its clinical implementation. Here we report a simplified combinatorial approach that involves short activation of CD8+ T cells in the presence of IL-12 followed by adoptive transfer into tumor bearing animals after a single injection of cyclophosphamide. This approach resulted in complete eradication of B16 melanoma, and the establishment of long term immunological memory capable of fully protecting mice after a second B16 melanoma challenge. The activated donor cells were unique because they simultaneously exhibited traits for cytotoxic effector function, central memory-like, homing, and senescence. After tumor eradication and within three months after transfer, CD8+ cells exhibited a conventional memory CTL phenotype. Moreover, these memory CTLs acquired functional attributes characteristic of memory stem cells, including the ability to resist chemotherapy-induced toxicity. Our results suggest that short-term T-cell receptor signaling in the presence of IL-12 promotes promiscuous qualities in naïve CTL which - upon transfer into lymphopenic hosts- are sufficient to eradicate tumors and generate life-long tumor-specific memory.
Pmel; melanoma; IL-12; ACT; T cell therapy; memory; CD8
We have recently reported that the toll-like receptor 3 (TLR3) agonist poly(I:C) induces adjuvant effects to post vaccination CD8+ T cells responses through rapid induction of innate mediators, including NK cells, macrophages, dendritic cells (DCs), and inflammatory cytokines. However, whether this TLR3 agonist directly targets CD8+ T cells needs to be carefully investigated. In this study, we found that optimal post vaccination CD8+ T cell responses to ex vivo DC-based vaccination requires triggering of TLR3 signaling pathway in DCs in vitro as well as in the recipient host, indicating a role for other cell types. Real-time PCR analysis revealed that TLRs (TLR1–TLR13) are expressed in purified (>99% pure) CD4+ and CD8+ T cells from C57BL/6 and BALB/c mice, where the magnitude of the expression was strain and cell type dependent. In vitro, treatment of these purified T cells with poly(I:C) modulated the expression of TLRs including TLR3. Furthermore, non-specific and antigen-specific stimulation of CD8+ T cells by phorbol myristate acetate and MHC class I peptide-pulsed splenocytes, respectively, modulated TLR expression in purified CD4+ and CD8+ T cells. Importantly, brief conditioning of purified naïve TCR transgenic OT-1 (CD8+) T cells in vitro with poly(I:C) induced activation of these cells in absence of antigen stimulation. Interestingly, when these in vitro poly(I:C)-conditioned OT-1 cells were adoptively transferred into naïve recipient followed by peptide vaccination, they showed superior expansion and activation to their naïve counterparts. These results suggest that CD8+ T cells can be activated by triggering their TLR3. Furthermore, the data support the notion of direct involvement of TLRs in adaptive immune responses.
Adoptive transfer; BALB/c; CD4; CD8; TLR; C57B6/L; OT-1; OVA peptide; Poly(I:C); TLR3 agonist; TLR3 ligand; T cells; Vaccination
Adoptive transfer of autologous tumor-reactive T cells holds promise as a cancer immunotherapy. In this approach, T cells are harvested from a tumor-bearing host, expanded in vitro and infused back to the same host. Conditioning of the recipient host with a lymphodepletion regimen of chemotherapy or radiotherapy before adoptive T cell transfer has been shown to substantially improve survival and anti-tumor responses of the transferred cells. These effects are further enhanced when the adoptive T cell transfer is followed by vaccination with tumor antigens in combination with a potent immune adjuvant. Although significant progress has been made toward an understanding of the reasons underlying the beneficial effects of lymphodepletion to T cell adoptive therapy, the precise mechanisms remain poorly understood. Recent studies, including ours, would indicate a more central role for antigen presenting cells, in particular dendritic cells. Unraveling the exact role of these important cells in mediation of the beneficial effects of lymphodepletion could provide novel pathways toward the rational design of more effective anti-cancer immunotherapy. This article focuses on how the frequency, phenotype, and functions of dendritic cells are altered during the lymphopenic and recovery phases post-induction of lymphodepletion, and how they affect the anti-tumor responses of adoptively transferred T cells.
Adoptive T cell transfer; Cancer; Chemotherapy; Dendritic cells; Lymphodepletion; Tumor; Vaccination
Preconditioning a recipient host with lymphodepletion can markedly augment adoptive T cell therapy. However, the precise mechanisms involved are poorly understood. In a recent study, we observed a significant increase in the circulating levels of dendritic cells (DCs; CD11c+CD11b+) during the recovery from cyclophosphamide (CTX)-induced lymphodepletion. Herein, we demonstrate that the CTX-induced DC expansion was not altered by adjuvant chemotherapy or tumor burden but was augmented by coadministration of granulocyte-colony stimulating factor. Although the increase in the number of DCs was preceded by a systemic expansion of a population expressing the phenotype of myeloid-derived suppressor cells (Gr-1+CD11b+), depletion of these Gr-1+ cells had no effect on the noted expansion. Moreover, when Gr-1highCD11bhigh cells were sorted from CTX-treated mice and adoptively transferred into control or CTX-treated recipients, they did not differentiate into DCs. Post-CTX expansion of DCs was associated with proliferation of DCs in bone marrow (BM) during the lymphopenic phase and in the blood and spleen during the recovery phase. Furthermore, adoptive transfer of BM cells from CTX-treated mice produced equal numbers of DCs in the blood of either CTX-treated or untreated recipients. CTX induced a dynamic surge in the expression of growth factors and chemokines in BM, where CCR2 and Flt3 signaling pathways were critical for DC expansion. In sum, our data suggest that CTX induces proliferation of DCs in BM prior to their expansion in the periphery. Targeting DCs at these phases would significantly improve their contribution to the clinical application of lymphodepletion to adoptive immunotherapy