MiRNA-21-5p as a biomarker in EBV-associated oropharyngeal cancer.

INTRODUCTION AND OBJECTIVE: Epstein-Barr virus (EBV) is associated with cancers of the head and neck, including oropharyngeal cancer, which is increasing in incidence, and biomarker studies have potential in diagnostics and therapy. One of the most commonly deregulated microRNAs in cancers is miR-21-5p. It has been implicated in neoplastic transformation related to EBV infection in several investigations. The aim of this study was to determine the level of miR-21-5p in the serum of EBV (+) and EBV (-) oropharyngeal cancer patients. MATERIAL AND METHODS: The study was carried out on 78 patients with confirmed OPSCC. Statistical analysis was used to investigate the relationship between clinical and demographic characteristics of patients. Enzyme immunoassays were used to determine the levels of miRNA, TLR9 and MMPs and cytokines. Statistical analysis was used to determine the relationship between miR21-5p and TLR9, MMP3, MMP9 levels, and the cytokines studied. RESULTS: Significantly higher values of all tested parameters for miR-21-5p levels and grading as well as TN stage were found in the EBV (+) group. There was no statistically significant correlation between the miR-21-5p level and the levels of TNFα, VEGF, and TGFß. Positive correlations were shown between miR-21-5p and IL-10, MMP-3 and -9. There was a negative correlation between the level of miR-21-5p and TLR9. CONCLUSIONS: The present study showed that in EBV (+) patients the level of miR-21-5p in the serum was significantly higher than in EBV (-) patients. Our study results could influence future strategies for the diagnosis, prevention and treatment of oropharyngeal cancers.

The Cognitive Aspect of Hope in the Semantic Space of Male Patients Dying of Cancer.

The aim of this study is to characterize the cognitive aspect of the semantic space of hope in patients in the terminal stage of cancer. This was confirmed in the research on hope by C. R. Snyder and B. Schrank. Hope is of great importance in all the great world religions and belief systems, both as regards a personal God or impersonal deities. Hoping is a human capacity with varying affective, cognitive and behavioral dimensions. Psychological, pedagogical (particularly in the framework of special needs pedagogy and thanatological pedagogy) and theological reflection on hope can provide support for dying people. In order to conduct the research, the semantic differential research method was selected. The research technique employed was a therapeutic conversation, and the research tool was the B.L. Block's DSN-3 test. The DSN-3 test allows one to assess hope in the semantic space in three aspects: cognitive, emotional and functional. For the purposes of this study, only the cognitive aspect was taken into account. The study was begun on 1 April 2010 and ended in the last days of December 2020. It included 110 male patients in the terminal stage of cancer. The youngest respondent was 19 years old and the oldest was 94 years old. The surveyed men most often perceived hope in the semantic space in the cognitive aspect as more true, wise, meaningful and real than false, stupid, meaningless and deceptive. Their attitude to hope was, therefore, more affirmative than negative. The research did not reveal the importance of the age of the respondents on the degree of affirmation/negation of hope in the cognitive aspect in the semantic space; however, men in the period of late maturity and professional activity expressed the lowest level of the affirmation of hope. It is worthwhile to conduct further research concerning hope in other aspects (especially emotional and functional) in the semantic space in order to use the obtained results to consider what to take into account when providing patients in the terminal stage of cancer with better personalized holistic care than before.

Oral Contraceptive Use and Breast Cancer Risk Assessment: A Systematic Review and Meta-Analysis of Case-Control Studies, 2009-2020.

To perform a meta-analysis of case-control studies that addressed the association between oral contraceptive pills (OC) use and breast cancer (BrCa), PubMED (MEDLINE), Embase, and the Cochrane Library were searched to identify case-control studies of OC and BrCa published between 2009 and 2020. We used the DerSimonian-Laird method to compute pooled odds ratios (ORs) and confidence intervals (CIs), and the Mantel-Haenszel test to assess the association between OC use and cancer. Forty-two studies were identified that met the inclusion criteria and we included a total of 110,580 women (30,778 into the BrCa group and 79,802 into the control group, of which 15,722 and 38,334 were using OC, respectively). The conducted meta-analysis showed that the use of OC was associated with a significantly increased risk of BrCa in general, OR = 1.15, 95% CI: 1.01 to 1.31, p = 0.0358. Regarding other risk factors for BrCa, we found that increased risk was associated significantly with early menarche, nulliparous, non-breastfeeding, older age at first parity, postmenopause, obesity, smoking, and family history of BrCa. Despite our conclusion that birth control pills increase the cancer risk being supported by extensive previous studies and meta-analyzes, further confirmation is required.

SARS-CoV-2 Infection Prevalence in the Population of South-Eastern Poland.

COVID-19 outbreak began in Wuhan, China, and has spread to other continents, including Europe, placing pressure on healthcare systems. Poland is one of the European countries with the highest number of SARS-CoV-2 infections and COVID-19-related deaths. The aim of this study was to analyze the presence of SARS-CoV-2 in the population of south-eastern Poland. The correlation between viral infection and demographic data (gender, age, place of residence) and cancer was also investigated. A total of 44,801 samples were tested, of which 4862 cases were diagnosed with SARS-CoV-2 infections. A total of 14,970 samples were tested in cancer patients. The RT-PCR method was used to detect viral nucleic acid. In this study, significantly, the highest rate of virus detection was among people living in Lublin and the lowest among people living in a small town (p < 0.0001). Moreover, there was no significant relationship between sex and the frequency of virus detection. The highest number of SARS-CoV-2 infections was observed in the age groups 10-19, 20-29, 30-39, and 90+ (p = 0.0001). In cancer patients, the percentage of positive cases was significantly lower than in the rest (p = 0.0001).

The newly synthesized thiazole derivatives as potential antifungal compounds against Candida albicans.

Recently, the occurrence of candidiasis has increased dramatically, especially in immunocompromised patients. Additionally, their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. A series of nine newly synthesized thiazole derivatives containing the cyclopropane system, showing promising activity against Candida spp., has been further investigated. We decided to verify their antifungal activity towards clinical Candida albicans isolated from the oral cavity of patients with hematological malignancies and investigate the mode of action on fungal cell, the effect of combination with the selected antimycotics, toxicity to erythrocytes, and lipophilicity. These studies were performed by the broth microdilution method, test with sorbitol and ergosterol, checkerboard technique, erythrocyte lysis assay, and reversed phase thin-layer chromatography, respectively. All derivatives showed very strong activity (similar and even higher than nystatin) against all C. albicans isolates with minimal inhibitory concentration (MIC) = 0.008-7.81 µg/mL Their mechanism of action may be related to action within the fungal cell wall structure and/or within the cell membrane. The interactions between the derivatives and the selected antimycotics (nystatin, chlorhexidine, and thymol) showed additive effect only in the case of combination some of them and thymol. The erythrocyte lysis assay confirmed the low cytotoxicity of these compounds as compared to nystatin. The high lipophilicity of the derivatives was related with their high antifungal activity. The present studies confirm that the studied thiazole derivatives containing the cyclopropane system appear to be a very promising group of compounds in treatment of infections caused by C. albicans. However, this requires further studies in vivo. KEY POINTS: • The newly thiazoles showed high antifungal activity and some of them - additive effect in combination with thymol. • Their mode of action may be related with the influence on the structure of the fungal cell wall and/or the cell membrane. • The low cytotoxicity against erythrocytes and high lipophilicity of these derivatives are their additional good properties.

Serum and Tissue Level of TLR9 in EBV-Associated Oropharyngeal Cancer.

The Epstein-Barr virus (EBV) is associated with the development of various epithelial malignancies including cancer in the head and neck region. Several studies have shown that Toll-like receptors (TLRs) are required for an innate immune response to infection with human DNA viruses, e.g., EBV. During viral infections, TLR response may influence the transformation to malignancy. The aim of the study was to assess TLR9 serum and tissue level in EBV(+) and EBV(-) oropharyngeal cancer patients. The study involved 78 patients: 42 EBV(+) and 36 EBV(-). EBV DNA was detected in fresh frozen tumor tissue. TLR9 level was measured in homogenate of tumor tissue and in serum. Moreover, in serum samples IL-10, VEGF, TGFß, TNFα and antibodies against EBV were detected using ELISA test. TLR9 level was significantly lower in EBV(+) patients, both in tissue and serum, while EBVCA, EBNA and VEGF level was statistically higher in EBV(+) patients. An increase in EBVCA and EBNA antibodies titer was correlated with a TLR9 level decrease. TLR9 level was higher in poorly-differentiated tumors (G3), in tumor of larger dimensions (T3-T4) and with lymph nodes involvement (N3-N4) but without statistical significance. High levels of anti-EA antibodies in the majority of EBV(+) patients may point to the reactivation of EBV infection.

Use of Oral Contraceptives as a Potential Risk Factor for Breast Cancer: A Systematic Review and Meta-Analysis of Case-Control Studies Up to 2010.

Despite numerous studies evaluating the risk of breast cancer among oral contraception users, the effect of oral contraceptive on developing breast cancer remains inconclusive. Therefore, we conducted a systematic review of literature with meta-analysis in order to quantitative estimate this association. The bibliographic database MEDLINE and EMBASE, and reference lists of identified articles were searched, with no language restrictions, from the start of publication to August 2010. We performed a reanalysis and overall estimate of 79 case-control studies conducted between 1960-2010, including a total of 72,030 incidents, histologically confirmed cases of breast cancer and 123,650 population/hospital controls. A decrease was observed in cancer risk in OC users before age 25 years (0.91, 0.83-1.00). However, the use of OCs before the first full-term pregnancy had a significant increased risk of breast cancer (OR, 1.14, 1.01-1.28, p = 0.04), as did OC use longer than 5 years (1.09, 1.01-1.18, p = 0.02). Pooled crude odds ratios of breast cancer in ever-users of oral contraceptives was 1.01 [95% confidence interval (CI), 0.95-1.07], compared with never-users. There was no significant increase in risk among premenopausal women (1.06, 0.92-1.22), postmenopausal women (0.99, 0.89-1.10), or nulliparous women (1.02, 0.82-1.26). Oral contraceptives do not appear to increase the risk of breast cancer among users. However, OC use before a first full-term pregnancy or using them longer than 5 years can modify the development of the breast cancer.

Hope in Advanced Cancer Patients in the Terminal Phase of Neoplastic Disease and Stability of Basic Mood.

The aim of this research is to compare the hope experienced by advanced cancer patients in the terminal phase of neoplastic disease in relation to the stability of their basic mood. The study group consisted of 246 patients, average age 59.5. The youngest respondent was 18 and the oldest was 90. The diagnostic tools used in the work comprised the Personal Card designed by T. Witkowski (PC) and an NCN-36 test (Block's Hope test), designed by B.L. Block to measure the strength of hope in people struggling with serious life-threatening diseases. The test consists of 4 subscales distinguished by factor analysis. Each subscale consists of 8 items. The test allows an evaluation of hope in the following dimensions: situational dimension (health, thelic-temporal dimension), goals to be achieved in the future, spiritual dimension (spirituality), religious beliefs, and emotional-motivational (affective) dimension (motivations). In cheerful patients who are in the terminal phase of cancer, mood stability does not constitute a major differentiating factor for experiencing hope. In sad people, on the other hand, mood stability affects the intensity of hope-those with an unstable mood are more likely to have a stronger emotional-motivational dimension of hope than sad people with a balanced mood.

Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) in Oropharyngeal Cancer Associated with EBV and HPV Coinfection.

Recent reports have pointed to the link between persistent inflammation, oxidative stress, and carcinogenesis; however most of the studies concerning the role of viruses in head and neck cancer (HNC) are focused mainly on one type of virus. Our present study aimed to study the relationship between Epstein-Barr virus/human papilloma virus (EBV/HPV) coinfection and glutathione peroxidase (GPx) and superoxide dismutase (SOD) level in oropharyngeal cancer. Fresh-frozen tumor tissue samples were collected from 128 patients with oropharyngeal cancer infected with EBV or HPV or with EBV/HPV coinfection. After DNA extraction, EBV and HPV DNA was detected using a polymerase chain reaction (PCR) assay. GPx and SOD activity was determined in homogenates of cancer tissue using diagnostic kits produced by Randox Laboratories. Both GPx and SOD activity was statistically lower in patients with EBV/HPV coinfection than in a single EBV or HPV infection. Analysis of GPx and SOD activity in relation to histological grading and tumor, node (TN) classification revealed that in poorly-differentiated tumors, the level of antioxidant enzymes was lower compared with well-differentiated lesions and in cases with greater tumor dimensions and lymph-node involvement, both GPx and SOD activity was decreased. Further studies are necessary to clarify the influence of interplay between EBV, HPV, and oxidative stress on malignant transformation of upper aerodigestive tract epithelial cells.

Hope of Recovery in Patients in the Terminal Phase of Cancer under Palliative and Hospice Care in Poland.

INTRODUCTION: The objective of the presented research is to characterize hope in the situational dimension, i.e., health, in the patients with cancer in the terminal phase of the disease, being treated in hospices and palliative care centers. Hope is very important for all the patients, especially for patients with cancer in various phases of the disease. Giving up on oncologic therapy and causal treatment is often associated with a transition into palliative care. When death and a loss of values become a threat, the individual has got hope to rely on. Material and Methods. The study relies on the Test to Measure Hope in the Health Context (NCN-36) by B.L. Block. 246 patients in the terminal phase of cancer participated in the study. RESULTS: The internal structure of hope of recovery in the patients' group was varied. The patients showed low levels of hope of recovery since they do not believe in the effectiveness of treatment. They were also not convinced of the effectiveness of modifications in dieting, lifestyle, or the use of nonconventional medicine. They trusted the doctor in charge and were moderately satisfied with the therapy in use. The intensity of hope of recovery was on the low level in the patients in the terminal phase of cancer. Age, sex, place of living, and marital status had a significant influence on the level of hope of recovery. Variables such as living on one's own or living with one's family, socioeconomic status, education, or profession did not affect the level of hope of recovery. CONCLUSIONS: The presented results allowed as to conclude that the assessment of hope in terminally ill cancer patients can be considered as one of the important tools enabling the personalization and the improvement of palliative care.

Anthropometric, body composition and behavioural predictors of bioelectrical impedance phase angle in Polish young adults - preliminary results.

INTRODUCTION AND OBJECTIVE: Bioimpedance analysis (BIA) phase angle (PhA) is an index of the integrity of cells and cellular membranes. The aim of the study was identification of behavioural and anthropometric predictors of PhA in a group of young adults. MATERIAL AND METHODS: A cross-sectional observational study of health behaviours, anthropometric indicators and body composition assessed by the BIA method was conducted in a group of Polish young adults (n=92) aged 18 - 24 (mean - 19.33, STD - 0.915). Behavioural variables included: level of physical activity, eating behaviours and nutritional knowledge. Body composition was analysed by means of BIA phase-sensitive 8-electrode medical SECA mBCA 525 device. RESULTS: The mean PhA value in the examined cohort was 6.38±0.75 (males - 7.22±0.72; females - 6.13±0.57). Males also showed higher statistically significant other body composition indices, excluding fat mass. The multiple regression model, including anthropometric variables and gender, which explained the effect of these variables on PhA, occurred to be significant (p<0.0000) and allowed explanation of the 82.49% of PhA variability. PhA was significantly predicted from body mass index (BMI), absolute fat mass, visceral adipose tissue value, skeletal muscle mass value and gender. The regression model, including behavioural predictors and gender, allowed explanation of the lower percentage of PhA variability (42.75%; p<0.0000) and included general intensity of health behaviours, level of nutritional knowledge, and gender. A regression model which would consider simultaneously anthropometric and behavioural variables could not be constructed. CONCLUSIONS: In the examined cohort, anthropometric and body composition variables showed a stronger predictive value with respect to PhA, compared to behavioural variables.

Hope in Hospitalized Patients with Terminal Cancer.

: Hope is of great importance for patients diagnosed with cancer, especially those that are terminally ill. The diagnosis often puts an end to the realization of personal, social, and professional goals. The aim of this study was to characterize the hope of hospitalized patients diagnosed with cancer in the terminal phase of the disease. The research tool used in the study was Block's hope test (NCN-36; NCN- Nadzieja Chorych Nowotworowych - Hope of Cancer Patients), designed for patients with life-threatening diseases. The results showed that the patients were characterized by a moderate level of global hope. The highest levels of hope were noted in the spiritual-religious area and the lowest levels of hope concerned curing the disease. Patients exhibited varied levels of hope and varied internal structures of hope. They presented four different types of hope: optimistic, moderate, religious, and weak. Optimistic hope was found most frequently in patients diagnosed with a terminal phase of cancer, while weak hope was represented by the smallest group of these patients.

Rotavirus VP6 as an Adjuvant for Bivalent Norovirus Vaccine Produced in Nicotiana benthamiana.

Rotaviruses (RVs) and noroviruses (NoVs) are major causes of childhood acute gastroenteritis. During development of a combination vaccine based on NoV virus-like particles (VLP) and RV VP6 produced in baculovirus expression system in insect cells, a dual role of VP6 as a vaccine antigen and an adjuvant for NoV-specific immune responses was discovered. Here the VP6 adjuvant effect on bivalent GI.4 and GII.4-2006a NoV VLPs produced in Nicotiana benthamiana was investigated. BALB/c mice were immunized intradermally with suboptimal (0.3 µg) dose of each NoV VLP alone or combined with 10 µg of VP6, or equal doses of NoV VLPs and VP6 (1 µg/antigen). NoV-specific serum IgG antibodies and their blocking activity were analyzed using vaccine-homologous and heterologous NoV VLPs. Immunization with 0.3 µg NoV VLPs alone was insufficient to induce NoV-specific immune responses, but with co-administration of 10 µg of VP6, antibodies against vaccine-derived and heterologous NoV genotypes were generated. Furthermore, corresponding adjuvant effect of VP6 was observed with 1 µg dose. Efficient uptake and presentation of VP6 by dendritic cells was demonstrated in vitro. These results show that adjuvant effect of VP6 on bivalent NoV VLP vaccine is independent of the cell source used for vaccine production.

Identification of a First Human Norovirus CD8+ T Cell Epitope Restricted to HLA-A*0201 Allele.

Norovirus (NoV) causes a substantial global burden of acute gastroenteritis in all age groups and the development of NoV vaccine is a high priority. There are still gaps in understanding of protective NoV-specific immunity. Antibody mediated immune responses have been widely studied, but in contrast, the research on NoV-specific human T cell-mediated immunity is very limited. We have recently reported NoV capsid VP1-specific 18-mer peptide (134SPSQVTMFPHIIVDVRQL151) to induce strong CD8+ T cell immune responses in healthy adult donors. This work extends to identify the precise NoV T cell epitope and the restricting human leucocyte antigen (HLA). Pentamer technology was used to detect HLA-A*0201-restricted T cell-mediated responses to 10-mer peptide 139TMFPHIIVDV148 of four healthy adult blood donors. Immunogenicity of the 10-mer epitope was confirmed by ELISPOT IFN-γ and intracellular cytokine staining (ICS) on flow cytometry. A population of CD3+CD8+ T lymphocytes binding to HLA-A*0201/TMFPHIIVDV pentamers was identified in two HLA-A*0201-positive donors. Recognition of the 10-mer epitope by T cells resulted in a strong IFN-γ secretion as shown by ELISPOT assay. In addition, ICS confirmed that high proportion (31 and 59%) of the TMFPHIIVDV epitope-responsive CD3+CD8+ T cells in the two donors had multifunctional phenotype, simultaneously producing IFN-γ, IL-2 and TNF-α cytokines. In the present study novel human NoV HLA-A*0201-restricted minimal 10-mer epitope 139TMFPHIIVDV148 in the capsid VP1 was identified. The HLA-peptide pentamer staining of T cells from healthy donor PBMCs and cytokine responses in ex-vivo ELISPOT and ICS assays suggest that this epitope is recognized during NoV infection and activates memory phenotype of the epitope-specific multifunctional CD8+ T cells. The importance of this epitope in protection from NoV infection remains to be determined.

Parenterally Administered Norovirus GII.4 Virus-Like Particle Vaccine Formulated with Aluminum Hydroxide or Monophosphoryl Lipid A Adjuvants Induces Systemic but Not Mucosal Immune Responses in Mice.

Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH)3) or monophosphoryl lipid A (MPLA) would induce mucosal antibodies in mice. Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)3 and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses.

Functionality and avidity of norovirus-specific antibodies and T cells induced by GII.4 virus-like particles alone or co-administered with different genotypes.

Norovirus (NoV) is the main cause of acute gastroenteritis worldwide across all age groups. Current NoV vaccine candidates are based on non-infectious highly immunogenic virus-like particles (VLPs) produced in cell cultures in vitro. As NoVs infecting human population are highly divergent, it is proposed that the vaccine should contain at least two different NoV genotypes, potentially affecting the immunogenicity of each other. We investigated the immunogenicity of NoV GII.4 VLPs administered by intramuscular (IM) or intradermal (ID) injections to BALB/c mice either alone or co-delivered with genogroup I (GI) and other genogroup GII VLPs. Serum NoV-specific IgG binding antibody titers and antibody functionality in terms of avidity and blocking potential were assessed. Furthermore, the specificity and functional avidity of CD4+ and CD8+ T cell responses were analyzed using synthetic peptides previously identified to contain NoV VP1 P2 domain-specific H-2d epitopes. The results showed that IM and ID immunization induced comparable GII.4-specific antibodies and T cell responses. Similar magnitude and functionality of antibodies and interferon-gamma producing T cells were developed using monovalent GII.4 VLPs or different genotype combinations. For the first time, degranulation assay using multicolor flow cytometry showed that NoV GII.4-specific CD8+ T cells had cytotoxic T lymphocyte phenotype. To conclude, our results demonstrate that there is no immunological interference even if up to five different NoV VLP genotypes were co-administered at the same time. Furthermore, no inhibition of NoV-specific antibody functionality or the magnitude, specificity and affinity of T cell responses was observed in any of the immunized animals, observations relevant for the development of a multivalent NoV VLP vaccine.

Rotavirus Recombinant VP6 Nanotubes Act as an Immunomodulator and Delivery Vehicle for Norovirus Virus-Like Particles.

We have recently shown that tubular form of rotavirus (RV) recombinant VP6 protein has an in vivo adjuvant effect on the immunogenicity of norovirus (NoV) virus-like particle (VLP) vaccine candidate. In here, we investigated in vitro effect of VP6 on antigen presenting cell (APC) activation and maturation and whether VP6 facilitates NoV VLP uptake by these APCs. Mouse macrophage cell line RAW 264.7 and dendritic cell line JAWSII were used as model APCs. Internalization of VP6, cell surface expression of CD40, CD80, CD86, and major histocompatibility class II molecules, and cytokine and chemokine production were analyzed. VP6 nanotubes were efficiently internalized by APCs. VP6 upregulated the expression of cell surface activation and maturation molecules and induced secretion of several proinflammatory cytokines and chemokines. The mechanism of VP6 action was shown to be partially dependent on lipid raft-mediated endocytic pathway as shown by methyl-ß-cyclodextrin inhibition on tumor necrosis factor α secretion. These findings add to the understanding of mechanism by which VP6 exerts its immunostimulatory and immunomodulatory actions and further support its use as a part of nonlive RV-NoV combination vaccine.

Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture.

Hematopoietic stem cell transplantation (HSCT) is followed by a period of immune deficiency due to a paucity in T-cell reconstitution. Underlying causes are a severely dysfunctional thymus and an impaired production of thymus-seeding progenitors in the host. Here, we addressed whether in vitro-derived human progenitor T (proT)-cells could not only represent a source of thymus-seeding progenitors, but also able to influence the recovery of the thymic microenvironment. We examined whether co-transplantation of in vitro-derived human proT-cells with hematopoietic stem cells (HSCs) was able to facilitate HSC-derived T-lymphopoiesis posttransplant. A competitive transfer approach was used to define the optimal proT subset capable of reconstituting immunodeficient mice. Although the 2 subsets tested (proT1, CD34(+)CD7(+)CD5(-); proT2, CD34(+)CD7(+)CD5(+)) showed thymus engrafting function, proT2-cells exhibited superior engrafting capacity. Based on this, when proT2-cells were coinjected with HSCs, a significantly improved and accelerated HSC-derived T-lymphopoiesis was observed. Furthermore, we uncovered a potential mechanism by which receptor activator of nuclear factor κb (RANK) ligand-expressing proT2-cells induce changes in both the function and architecture of the thymus microenvironment, which favors the recruitment of bone marrow-derived lymphoid progenitors. Our findings provide further support for the use of Notch-expanded progenitors in cell-based therapies to aid in the recovery of T-cells in patients undergoing HSCT.

Combining DNA technologies and different modes of immunization for induction of humoral and cellular anti-HIV-1 immune responses.

We show here that it is possible to combine two different genetic immunogens, one designed to induce HIV-1 specific humoral immune responses (pKCMVgp160B) and one designed to induce cellular anti-HIV-1 immune responses (Auxo-GTU-MultiHIV), and still retain the major properties of both vaccine constructs. The two different constructs were delivered using two different methods; the gene-gun and the Biojector, which both are needle-free devices. In BALB/c mice we were able to induce high levels of HIV-1-specific T cell responses as well as high levels of anti-gp160 antibodies by co-administrating the vaccine constructs. The cellular immune responses, but not antibody responses, were moderately compromised from the combination. This study shows that it is a feasible strategy to combine different vaccines and modes of delivery, but that interference as to magnitude may occur to certain gene products.

GTU-MultiHIV DNA vaccine results in protection in a novel P815 tumor challenge model.

A novel animal model for testing the immunogenicity and protective immune response induced by HIV-1 DNA vaccines was developed. DBA/2 mice were immunized with GTU-MultiHIV DNA encoding multigene for Rev, Nef, Tat, optp17/24 and a stretch of Pol/Env epitopes. A single GTU-MultiHIV B-clade specific plasmid or Auxo-GTU-MultiHIV(mix) (mixture of four plasmids with A, B, C and FGH clade specific MultiHIV antigens) were administered via gene gun and cell-mediated and humoral immune responses were analysed. The protective efficacy of the immune response was evaluated by challenging the mice with syngeneic tumor cells (P815) stably transfected with the MultiHIV fusion gene. Our results show that the strong MultiHIV-specific immune response generated by the GTU-MultiHIV vaccines in DBA/2 mice was able to delay the tumor growth substantially, indicating that the CTL response detected in vitro confers protection in vivo. The model described here is a safe and feasible in vivo assay for assessment of the vaccine potency to induce protective cell-mediated immune responses.