Efficacy of different gonadotropin combinations to support ovulation induction in WHO type I anovulation infertility: clinical evidences of human recombinant FSH/human recombinant LH in a 2:1 ratio and highly purified human menopausal gonadotropin stimulation protocols.

BACKGROUND: The World Helath Organization (WHO) Group I anovulation, or hypogonadotropic hypogonadism (HH), is characterized by reduced hypothalamic/pituitary activity which results in abnormally low serum FSH and LH levels and negligible estrogen activity. AIM: To compare the efficacy of human recombinant FSH (r-hFSH) plus human recombinant LH (r-hLH) in a 2:1 ratio with highly purified human menopausal gonadotropin (hMG-HP) urinary extract, containing LH-like activity, in women with HH. SUBJECTS AND METHODS: This two-arm randomized open-label study included 35 HH women (aged 25-36 yr) attending our Center. Eighteen patients received 150 IU hMG-HP (150 IU FSH + 150 IU LH-like activity) and seventeen received 150IU r-hFSH/75IU rhLH daily for a maximum of 16 days. Ovulation was induced by a single administration of hCG on the day after the last hMG-HP or r-hFSH/r-hLH. RESULTS: The primary efficacy endpoint was ovulation induction as measured by follicle ≥17 mm, pre-ovulatory estradiol (E 2 ) ≥400 pmol/l and mid-luteal phase progesterone (P 4 ) ≥25 nmol/l. Secondary efficacy endpoints included E 2 levels/follicle at mid-cycle, number of follicles at mid-cycle and pregnancy rate (PR). Following a total of 70 cycles, 70% of r-hFSH/r-hLH treated patients met the primary endpoint vs 88% in hMG-HP group (p=0.11). However, PR in r-hFSH/r-hLH group was 55.6% compared to 23.3% in hMG-HP group (p=0.01). CONCLUSIONS: The primary endpoint achievement did not correlate with PR. This study has shown the superiority of LH compared to hCG in supporting FSH-induced follicular development in HH women.

Apoptotic effects of a chimeric plant virus carrying a mimotope of the hepatitis C virus hypervariable region 1: role of caspases and endoplasmic reticulum-stress.

The role of apoptosis in the persistence of hepatitis C virus (HCV) infection is controversial. Moreover, conflicting data on the modulation of this process by HCV proteins have been provided. We evaluated the susceptibility of peripheral lymphocytes from patients with chronic hepatitis C to apoptosis both spontaneous and after incubation with a chimeric Cucumber mosaic virus (CMV) carrying 180 copies of the synthetic R9 mimotope obtained from more than 200 hypervariable region-1 sequences of HCV. Resting T lymphocytes were found to be sensitized to apoptosis as a result of chronic HCV infection. The plant virus-derived vector R9-CMV displayed a strong pro-apoptotic effect associated with activation of both caspase-8 and -9, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. A parallel R9-CMV-mediated activation of endoplasmic reticulum-stress was suggested by the significant induction of BiP/GRP78, GADD153 and caspase-12. These data contribute to define the complex HCV/host interaction, and open new prospects for developing a plant-derived antigen-presenting system to strengthen host defences against persistent pathogens.

Cucumber mosaic virus as the expression system for a potential vaccine against Alzheimer's disease.

A primary therapeutic goal in Alzheimer's disease (AD) is to reduce the quantity of amyloid ß protein (Aß) present in the brain. To develop an effective, safe system for vaccination against Alzheimer's disease, the plant virus Cucumber mosaic virus (CMV) was engineered genetically to express Aß-derived fragments that stimulate mainly humoral immune responses. Six chimeric constructs, bearing the Aß1-15 or the Aß4-15 sequence in positions 248, 392 or 529 of the CMV coat protein (CP) gene, were created. Viral products proved to be able to replicate in their natural host. However, only chimeric Aß1-15-CMVs were detected by Aß1-42 antiserum in Western blot analysis. Experimental evidence of Immunoelectron microscopy revealed a complete decoration of Aß1-15-CMV(248) and Aß1-15-CMV(392) following incubation with either anti-Aß1-15 or anti-Aß1-42 polyclonal antibodies. These two chimeric CMVs appear to be endowed with features making them possible candidates for vaccination against Alzheimer's disease.

In vitro stability of Cucumber mosaic virus nanoparticles carrying a Hepatitis C virus-derived epitope under simulated gastrointestinal conditions and in vivo efficacy of an edible vaccine.

The Cucumber mosaic virus (CMV) is an isodiametric plant virus with an extremely wide host range, present worldwide. CMV chimeric particles (R9-CMV), engineered to express a 27-aa synthetic peptide derived from Hepatitis C virus (HCV), were demonstrated to be stable under simulated gastric and intestinal conditions. Then the possibility of inducing a humoral immune response in rabbits fed with R9-CMV infected lettuce plants was demonstrated, suggesting that this system could function as a confirming tool of a bioreactor for the production of a stable edible vaccine against HCV.

Structural and biological properties of Cucumber mosaic virus particles carrying hepatitis C virus-derived epitopes.

The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. The CP gene was then engineered to express one or two copies of a synthetic peptide derived from many hypervariable region 1 (HVR1) sequences of the Hepatitis C virus (HCV) envelope protein E2 (the so-called R9 mimotope). Study of the symptoms pattern displayed in tobacco by these chimeric CMV particles, together with determination of their structural characteristics, assessed by circular dichroism (CD) spectroscopy and electron microscopy, revealed a possible relationship between the biological behavior and the structural properties of virus components.

Cucumber mosaic virus as a presentation system for a double hepatitis C virus-derived epitope.

Chimeric plant viruses are emerging as promising vectors for use in innovative vaccination strategies. In this context, cucumber mosaic virus (CMV) has proven to be a suitable carrier of the hepatitis C virus (HCV)-derived R9 mimotope. In the present work, a new chimeric CMV, expressing on its surface the HCV-derived R10 mimotope, was produced but lost the insert after the first passage on tobacco. A comparative analysis between R10- and R9-CMV properties indicated that R9-CMV stability was related to structural features typical of the foreign insert. Thus, in order to combine high virus viability with strong immuno-stimulating activity, we doubled R9 copies on each of the 180 coat protein (CP) subunits of CMV. One of the chimeras produced by this approach (2R9-CMV) was shown to systemically infect the host, stably maintaining both inserts. Notably, it was strongly recognized by sera of HCV-infected patients and, as compared with R9-CMV, displayed an enhanced ability to stimulate lymphocyte IFN-gamma production. The high immunogen levels achievable in plants or fruits infected with 2R9-CMV suggest that this chimeric form of CMV may be useful in the development of oral vaccines against HCV.

[Laser assisted intracytoplasmic sperm injection: a more effective and faster technique of immobilization of spermatozoa than traditional one]. / Impiego del laser per l'immobilizzazione degli spermatozoi: un'alternativa efficace e più rapida alla tecnica tradizionale.

AIM: Intracytoplasmic sperm injection (ICSI) is employed routinely in the assisted reproductive technique, in particular in case of male factor infertility. Recently, some options have been introduced in the micromanipulation system employed in ICSI, improving the performance of this technique. One of these is a no-contact laser, mostly used for the assisted hatching and the blastomere biopsy in preimplantation genetic diagnosis. One of the most important step of the ICSI technique is the sperm immobilization. Normally, this is performed by the operator and needs some time. METHODS: A one-year perspective-comparative study has been carried out and the results obtained with the traditional technique of immobilization of spermatozoa have been compared with those obtained with the laser technique (spermatozoa previously immobilized by no-contact laser). We have evaluated 3 parameters: 1) fertilization rate; 2) cleavage rate and 3) time needed to immobilize sperm. RESULTS: Some interesting findings have been observed: the embryo quality, fertilization rate and cleavage rate seem the same in both groups. The most interesting finding is the drastic reduction of the time needed for microinjection when the laser technique is used. CONCLUSIONS: This technique will reduce the total time needed to perform the ICSI, decreasing the time of exposure of the gametes outside the incubator and reducing the possibility of irreversible damage.