Autologous stem-cell transplantation in refractory autoimmune diseases after in vivo immunoablation and ex vivo depletion of mononuclear cells

Autoimmune diseases that are resistant to conventional treatment cause severe morbidity and even mortality. In the present study we demonstrate that complete remissions can be achieved in refractory polychondritis and systemic lupus erythematosus (SLE), even at advanced stage, with the use of autologous stem-cell transplantation (SCT). Remissions persisted after reconstitution of the immune system. In the treatment of advanced systemic sclerosis (SSc), stable disease may be achieved with autologous SCT.     

Stem-cell transplantation for autoimmune diseases

The use of intensive immunosuppressive treatment coupled with BM stem-cell transplantation (SCT) to treat human autoimmune diseases (AID) follows anecdotal observations of responses of AID to allogeneic SCT and an extensive background of experience with SCT in animals with AID. In the last decade, numerous clinical trials have been initiated to explore a potential benefit of (mainly autologous) SCT in advanced and debilitating cases of rheumatoid arthritis, scleroderma, systemic lupus erythematosis and multiple sclerosis. In this review the etiology of AID and the experimental basis of SCT is presented, together with recent clinical results of SCT for AID. While much has been learned about the risks and benefits of SCT in AID, the underlying mechanisms regulating remission and relapse of AID after treatment remain largely unknown.     

Primary Treatment Response Rather than Front Line Stem Cell Transplantation Is Crucial for Long Term Outcome of Peripheral T-Cell Lymphomas

Outcome of systemic peripheral T-cell lymphomas (PTCL) is unsatisfactory and no controlled clinical study guides the therapy. Phase II studies suggest to consolidate response achieved after front-line treatment with stem cell transplant (SCT). We retrospectively evaluate the impact of front-line SCT consolidation in a single Center cohort of 209 patients treated during the last two decades. Median age was 49 years (range 15-85) with a prevalence of male sex (61%), advanced stage (68%) while IPI was >2 in 44%. Primary treatment was MACOP-B (39%) CHO(E)P (39%), intensive regimens (18%) or others (4%). Complete response to primary treatment (i.e. before SCT) was 60% (5% partial remission). Forty-four patients further proceeded to SCT while 92 did not receive consolidation. Outcome of primary responders was good, with a 3-year overall survival of 74% (82% in ALCL ALK+ and 69% for the other histologies). By multivariate analysis a better overall survival was significantly associated with IPI<2 (P=0.001), primary response (P=0.000), and ALCL ALK+ (P=0.012). The multivariate analysis performed on responders, showed that only IPI was predictive of a better survival while ALCL ALK+ and undergoing SCT were not. Response to primary treatment rather than post-remission programs is the crucial determinant of PTCL outcome.     

Effects of progestagens and Org OD14 in in vitro and in vivo tumor models

Sex steroids, in particular estradiol (E2) and progesterone (P4), play, together with other hormones and growth factors, a role in the development of normal breast tissue. The effect of four progestagens (norethisterone, 3-ketodesogestrel, gestodene and P4) and Org OD14, a steroid with weak estrogenic, progestagenic and androgenic properties were studied on growth of breast tumor cells in vitro using two subclones of MCF-7 (H and A) and T47D (S and A) cells. In addition, we investigated the effects of 3-ketodesogestrel, gestodene and Org OD14 on the growth of 7,12-dimethyl-benz(a)anthracene(DMBA)-induced mammary tumors in rats. In the in vitro assays with MCF-7 cells norethisterone, 3-ketodesogestrel and gestodene stimulated growth only at high doses (10⁻⁷ M), whereas P4 had no effect. Gestodene was more potent than 3-ketodesogestrel and norethisterone. Org OD14, stimulated cell growth at a dose of 10⁻⁸ M, while E2 is active at 10⁻¹⁰ M. In T47D-A cells similar effects were found, but the subclone S did not respond to the progestagens and Org OD14. The two T47D subclones also reacted differently to progestagens during growth stimulation with E2. In T47D-S the progestagens and Org OD14 inhibited, while in T47D-A these compounds did not modulate the effect of E2. In the DMBA model we found that gestodene and 3-ketodesogestrel were able to inhibit tumor growth to the same extent. Surprisingly, Org OD14 was even more effective in the DMBA model using the therapeutic approach. Using the prophylaxic approach tumor development was delayed and tumor growth was strongly suppressed. The inhibitory effects of Org OD14 on tumor growth in the DMBA model may be attributed to its mixed hormonal profile. From these studies we conclude that different cell lines and even subclones thereof respond quite differently to steroids. Both in vitro and in vivo studies are required to judge whether synthetic steroids might be involved in an increased risk for the development of breast tumors.     

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 andCD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.     

Efficacy and safety of autologous stem cell transplantation for decompensated liver cirrhosis: A retrospective cohort study

AIM To evaluate the long-term efficacy and safety of autologous stem cell transplantation(SCT) for decompensated liver cirrhosis.METHODS Consecutive patients with decompensated liver cirrhosis were included and assigned into the SCT group and non-transplantation(non-SCT) group according to whether they received SCT treatment. Patients werefollowed up for ten years. The long-term survival rate and incidence of hepatocellular carcinoma(HCC) were compared between groups.     

In vitro and in vivo interactions between nuclear receptors at estrogen response elements

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.     

Comparison of the effect of stem cell therapy and percutaneous transluminal angioplasty on diabetic foot disease in patients with critical limb ischemia

Background aimsThe aim of our study was to compare the effect of autologous stem cell therapy (SCT) and percutaneous transluminal angioplasty (PTA) on diabetic foot disease (DFD) in patients with critical limb ischemia (CLI).MethodsThirty-one patients with DFD and CLI treated by autologous stem cells and 30 patients treated by PTA were included in the study; 23 patients with the same inclusion criteria who could not undergo PTA or SCT formed the control group. Amputation-free survival, transcutaneous oxygen pressure (TcPO₂) and wound healing were assessed over 12 months.ResultsAmputation-free survival after 6 and 12 months was significantly greater in the SCT and PTA groups compared with controls (P = 0.001 and P = 0.0029, respectively) without significant differences between the active treatment groups. Increase in TcPO₂ did not differ between SCT and PTA groups until 12 months (both Ps < 0.05 compared with baseline), whereas TcPO₂ in the control group did not change over the follow-up period. More healed ulcers were observed up to 12 months in the SCT group compared with the PTA and control groups (84 versus 57.7 versus 44.4 %; P = 0.042).ConclusionsOur study showed comparable effects of SCT and PTA on CLI, a major amputation rate that was superior to conservative therapy in patients with diabetic foot and an observable effect of SCT on wound healing. Our results support SCT as a potential promising treatment in patients with CLI and diabetic foot.     

Experimental cryosurgery investigations in vivo

Cryosurgery is the use of freezing temperatures to elicit an ablative response in a targeted tissue. This review provides a global overview of experimentation in vivo which has been the basis of advancement of this widely applied therapeutic option. The cellular and tissue-related events that underlie the mechanisms of destruction, including direct cell injury (cryolysis), vascular stasis, apoptosis and necrosis, are described and are related to the optimal methods of technique of freezing to achieve efficacious therapy. In vivo experiments with major organs, including wound healing, the putative immunological response following thawing, and the use of cryoadjunctive strategies to enhance cancer cell sensitivity to freezing, are described.     

Effect of physostigmine on relative acetylcholine output induced by systemic treatment with scopolamine in in vivo microdialysis of rat frontal cortex

By means of an in vivo brain microdialysis, the effect of different concentrations of physostigmine on the acetylcholine level in the dialysate of rat frontal cortex was studied. Perfusion of the various degrees of physostigmine (eserine) concentration (10 nM−10 μM) into the cortex through the dialysis membrane increased the basal acetylcholine level in a dose-dependent manner. In the presence of 10 nM, 0.1 μM and 10 μM physostigmine in the perfusate, systemic treatment with scopolamine (0.5 mg/kg, i.p.) increased 200, 270 and 510%, respectively, the relative acetylcholine level in the dialysates in comparison with the corresponding basal levels, while in the absence of physostigmine the treatment increased it only 40%. From these results, it appears that perfusion of physostigmine at a variety of concentrations, changes not only the basal level of acetylcholine induced by the inhibition of acetylcholinesterase but also the relative acetylcholine output induced by systemic treatment with scopolamine.     

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

斑马鱼胸苷酸合成酶与自身mRNA的相互作用

斑马鱼(zebrafish,Danio rerio)是生物学领域中公认的研究脊椎类生物的模式生物.胸苷酸合成酶(thymidylate synthase,TS)是DNA从头合成的限速酶,多年来一直作为肿瘤化疗的重要靶酶.前期的研究表明,人和大肠杆菌中TS能与自身的mRNA结合,在翻译水平上具有反馈抑制自调控现象.斑马鱼作为药物模型的研究已成为热点研究领域,为了探讨斑马鱼的胸苷酸合成酶的调控规律,以及与人TS的相关性,利用原核表达,纯化获得高均一性斑马鱼TS蛋白,采用凝胶迁移研究了TS和其mRNA的体外结合,采用免疫共沉淀:RT-PCR技术研究了它们在体内的相互作用,实验结果表明,斑马鱼的TS在体内外均与自身的mRNA存在特异性的相互作用.研究说明,斑马鱼和人的TS具有高度生物学过程相关性,为构建斑马鱼抗肿瘤药理模型提供了理论基础.     

In vivo antitumor efficacy of interleukin-21 in combination with chemotherapeutics

Interleukin-21 (IL-21) is a class I cytokine with antitumor properties due to enhanced proliferation and effector function of CD8⁺ T cells and natural killer (NK) cells. Here we have explored the magnitude and time-course of cytostatics-induced lymphopenia in mice and investigated whether treatment with cytostatics influences the antitumor effect of IL-21 in mouse tumor models. We show that pegylated liposomal doxorubicin (PLD), irinotecan and oxaliplatin induced transient lymphopenia, whereas 5-fluorouracil (5-FU) transiently increased lymphocyte counts. B cells were more sensitive than T cells towards irinotecan and oxaliplatin. Additive antitumor effects were observed after combining IL-21 with PLD, oxaliplatin and to less extent 5-FU but not irinotecan, and larger effect was observed when IL-21 administration was postponed relative to chemotherapy, suggesting that these agents may transiently impair immune function. However, the chemotherapies did not significantly alter the levels of circulating regulatory T cells and only marginally affected the ability of CD8⁺ T cells to respond to IL-21 measured as increased granzyme B mRNA. Our results show that IL-21 therapy can be successfully combined with agents from different chemotherapeutic drug classes, i.e. topoisomerase II inhibitors (PLD), anti-metabolites (5-FU) and platinum analogs (oxaliplatin) provided that IL-21 therapy is delayed relative to chemotherapy.     

Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia: Studies in vivo and in vitro

We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.     

Transplantation of hematopoietic stem cells from the peripheral blood

Hematopoietic stem cells can be collected from the peripheral blood. These hematopoietic stem cells (HSC), or better progenitor cells, are mostly expressed as the percentage of cells than react with CD34 antibodies or that form colonies in semi-solid medium (CFU-GM). Under steady-state conditions the number of HSC is much lower in peripheral blood than in bone marrow. Mobilization with chemotherapy and/or growth factors may lead to a concentration of HSC in the peripheral blood that equals or exceeds the concentration in bone marrow. Transplantation of HSC from the peripheral blood results in faster hematologic recovery than HSC from bone marrow. This decreases the risk of infection and the need for blood-product support. For autologous stem-cell transplantation (SCT), the use of peripheral blood cells has completely replaced the use of bone marrow. For allogeneic SCT, on the other hand, the situation is more complex. Since peripheral blood contains more T-lymphocytes than bone marow, the use of HSC from the peripheral blood increases the risk of graft-versus-host disease after allogeneic SCT. For patients with goodrisk leukemia, bone marrow is still preferred, but for patients with high-risk disease, peripheral blood SCT has become the therapy of choice.     

Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial

In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95 x 10(6) DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4 x 10(6) DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.     

Recent approaches in acute lymphoblastic leukemia in adults

In the last decades outcome of adult acute lymphoblastic leukemia (ALL) has improved considerably. In large multicenter studies remission rates range from 75% to 89%, and long-term leukemia-free survival (LFS) from 28% to 39%. Major progress has also been made regarding better characterization of subtypes of ALL. Complete diagnostic procedures are essential to identify these subtypes which have significant differences in clinical and laboratory features and prognosis. LFS of > 50% can be expected in favorable subtypes such as T-ALL or mature B-ALL, while LFS of < 20% is expected in Ph/BCR-ABL positive ALL. Prognostic factors can be used for risk stratification and selection of treatment strategies can be adapted to the subtype and relapse risk. This includes measurement of minimal residual disease (MRD) to evaluate individualized treatment strategies adapted to the molecular response. Several new approaches for improvement in chemotherapy and stem cell transplantation (SCT) are under investigation. They include the use of intensified anthracyclines, asparaginase, cyclophosphamide or high-dose cytarabine during induction and intensive rotational chemotherapy during consolidation. Also SCT - mainly from sibling donors - is now part of standard treatment of de novo ALL, although it remains open whether indications should be based on prognostic factors or whether SCT should be offered to all patients with sibling donor. However, substantial progress can only be achieved by new, experimental strategies. These include new approaches for SCT, such as nonmyeloablative SCT, measurement of MRD, causal treatment with molecular targeting, e.g. with kinase inhibitors, and antibody therapy.     

Expression of bovine follicle-stimulating hormone subunits in a Hansenula polymorpha expression system increases the secretion and bioactivity in vivo

Bovine follicle-stimulating hormone (bFSH), a pituitary gonadotropin, is a heterodimer hormone that consists of a common α-subunit non-covalently associated with the hormone-specific β-subunit. Unfortunately, expression levels of recombinant bFSH or its subunits are invariably low. We report here the secretory expression of biologically active bFSHα and bFSHβ subunit in the methylotrophic yeast Hansenula polymorpha. A slightly higher level of expression of recombinant bFSH subunits was achieved by using the Saccharomyces cerevisiae-derived calnexin (ScCne1) as a chaperone in engineered H. polymorpha strains. The preliminary data also suggested that bFSH subunits expressed in H. polymorpha appeared to be less-glycosylated. This isoform had been shown to be 80% increase in in vivo bioactivity compared with the hyperglycosylated Pichia pastoris-derived recombinant bFSHα/β. More sophisticated applications of bFSH would profit from the assembled less-glycosylated heterodimer.     

Antifungal activity of tea tree oil from Melaleuca alternifolia against Trichophyton equinum: An in vivo assay

Dermatophytes are a group of keratinophilic and keratinolytic molds, some of which are responsible for ringworm. Among them Trichophyton equinum, which mostly infects equids, can cause extensive outbreaks in stud farms. The conventional treatment of equine trichophytosis is topic, based upon medicated shampoos to reduce the spread of infection among the animals. Nevertheless the popularity of phytotherapy is at an all-time peak, and the interest for natural alternatives or complements to conventional drug therapy is challenging both in human and veterinary field. Among herbal remedia Tea Tree Oil (TTO) shows a wide range of antimicrobial activities. A randomized open clinical trial was carried out on 60 thoroughbred breeding horses affected by equine ringworm. The animals were randomly divided into 2 groups of 30 subjects. Diagnostic criteria were the presence of clinical signs and positive T. equinum culture. Specificity control using TTO mixture in 5 not dermatophyte affected animals was achieved also. The antimycotic activity against T. equinum of a mixture containing 25% TTO in sweet almond oil, was evaluated in vivo treating 30 subjects, the others were administered enilconazole 2% solution. The animals of both groups were topically treated twice a day for 15 days with a 25% mixture of TTO diluted in sweet almond oil and every 3 days, four times with enilconazole rinses, respectively. The clinical and mycological outcome were evaluated at day 30 from the start of the treatments. Data analysis was performed by chi square test. All the treated animals showed complete clinical and aetiological healing. Part of control subjects also, showed an improvement and none of them exacerbate the lesions. This therapeutic protocol appears to be effective and versatile, being applicable immediately after physical examination, prior to have the laboratory response. It could be an alternative for practitioners interested in herbal medicines, contributing to fulfill the gap existing between in vitro and clinical studies.     

A study of chlorophyll b in vivo

1. The absorption spectrum of chlorophyll b in vivo at 77°K is presented as the difference spectrum between preparations of spinach and chlorophyll b-free Vischeria stellata chloroplasts.

2. A shoulder on this spectrum around 662 nm is due to a component different from chlorophyll b. This component may well be identical with the chlorophyll a form, chlorophyll a (665).

3. The 77°K chlorophyll b absorption spectra in the nonfractionated photosyn-thetic pigment apparatus and in fractions mainly representing Photosystems 1 or 2 are not significantly different.

4. The aerobic irreversible photobleaching of chlorophyll b was studied in the intact pigment complex as well as in fractions mainly consisting of Photosystem 1 or 2. A two-step photobleaching was observed in all cases. The time-course of this bleaching was not significantly different for chlorophyll b in both fractions.

5. These results do not indicate that more than a single chlorophyll b complex occurs in vivo.