From Myo-inositol to D-chiro-inositol molecular pathways.

OBJECTIVE: Inositol is a carbocyclic sugar polyalcohol. By epimerization of its hydroxyl groups, nine possible stereoisomers can be generated, two of major physiological and clinical relevance: myo-inositol and D-chiro-inositol. Myo-inositol and D-chiro-inositol are normally stored in kidney, brain and liver and are necessary for functions, such as signal transduction, metabolic flux, insulin signaling, regulation of ion-channel permeability, stress response and embryo development. In this narrative review, we summarize the mechanisms by which myo-inositol and D-chiro-inositol can be synthesized and absorbed and their possible role in the etiopathogenesis of neural tube defects. MATERIALS AND METHODS: We performed an online search in the PubMed database using the following keywords: "inositol", "D-chiro-inositol", "myo-inositol", "neural tube defects and inositol". RESULTS: Inositol requirements are partly met by dietary intake, while the rest is synthesized endogenously. Inositol deficiency may be involved in the pathogenesis of diseases, such as metabolic syndrome, spina bifida (a neural tube defect), polycystic ovary syndrome and diabetes. Supplementation of the two inositol stereoisomers, D-chiro-inositol and myo-inositol is important to prevent these conditions. CONCLUSIONS: Inositol is fundamental for signal transduction in the brain, kidneys, reproductive organs and other tissues in response to neurotransmitters, hormones and growth factors. Various genes are involved in inositol metabolism and associated pathways. Altered inositol concentrations are observed in several diseases. Analysis of the genes involved in inositol metabolism may provide important information for the clinical management of these conditions.

Treatment of the Mayer-Rokitansky-Küster-Hauser syndrome with autologous in vitro cultured vaginal tissue: descriptive study of long-term results and patient outcomes.

OBJECTIVE: Evaluating sexual function and quality of life (QoL) in patients treated with a modified Abbé-McIndoe technique using in vitro cultured autologous vaginal mucosa. DESIGN: Descriptive study. SETTING: Policlinico Umberto I, Sapienza University of Rome. POPULATION: From 2006 to 2016, 39 women affected by Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) underwent vaginoplasty at our centre using a modified Abbé-McIndoe technique with in vitro cultured autologous vaginal tissue. METHODS: For each patient, vaginal tissue was obtained by full-thickness biopsy of the vaginal vestibule. Following enzymatic dissociation, cells were cultured for 2-3 weeks before the transplant. MAIN OUTCOME MEASURES: Each patient completed two validated questionnaires to quantify sexual function and QoL: the Female Sexual Function Index (FSFI), administered at 12, 36, and 60 months, and the Psychological General Well Being Index (PGWBI) administered at 0, 6, and 36 months after surgery. RESULTS: Twelve months after surgery, 29 patients were engaging in regular sexual activity. The FSFI test results showed a satisfactory sexual function compared to the general population, with median values of 25.85 (range 4.6-30.5) at 12 months, 27.2 (range 4.4-33.6) at 36 months, and 29.6 (range 23.9-33.6) at 60 months. The PGWBI questionnaire showed a median score of 420.5 (range 108-540) before surgery, and 459 (range 252-533) at the 60-month follow-up. CONCLUSIONS: Vaginoplasty performed with the use of autologous vaginal tissue, besides ensuring a long-term satisfying sex life, helps in achieving an improvement in QoL that is maintained over time. TWEETABLE ABSTRACT: Vaginoplasty using in vitro vaginal tissue ensures a satisfactory sexual function and improves quality of life.

Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.

The Ag specificity of the CTL response against CMV is directed almost entirely to a single CMV tegument protein, the phosphoprotein pp65. We report the identification of three peptides derived from the protein pp65 that displayed a high or intermediate binding to HLA-A*0201 molecules, which were also able to induce an in vitro CTL response in peripheral blood lymphocytes from CMV seropositive individuals. The peptide-specific CTLs generated were capable of recognizing the naturally processed pp65 either presented by CMV-infected cells or by cells infected with an adenovirus construct expressing pp65 in an HLA-A*0201-restricted manner. Thus, we were able to demonstrate responses to subdominant CTL epitopes in CMV-pp65 that were not detected in polyclonal cultures obtained by conventional stimulations. We also found that the amino acid sequences of the three peptides identified as HLA-A*0201-restricted CTL epitopes were conserved among different wild-type strains of CMV obtained from renal transplant patients, an AIDS patient, and a congenitally infected infant, as well as three laboratory strains of the virus (AD169, Towne and Davis). These observations suggest that these pp65 CTL peptide epitopes could potentially be used as synthetic peptide vaccines or for other therapeutic strategies aimed at HLA-A*0201-positive individuals, who represent approximately 40% of the European Caucasoid population. However, strain variation must be taken in consideration when the search for CTL epitopes is extended to other HLA class I alleles, because these mutations may span potential CTL epitopes for other HLA molecules, as it is described in this study.

Phosphorylation of Shc proteins in human sperm in response to capacitation and progesterone treatment.

Several authors have demonstrated the involvement of tyrosine kinases during sperm capacitation and acrosome reaction. Shc proteins (p46Shc, p52Shc, and p66Shc) are cytoplasmic substrates of activated tyrosine kinases and are widely expressed in mammalian somatic tissues. Experiments were designed to demonstrate the presence of Shc in spermatozoa and to study its involvement in the signal transduction events leading to acrosome reaction. Anti-Shc antibodies strongly reacted with the acrosomal region of methanol-fixed human sperm. Only one Shc isoform (p52Shc) was detected on Western blot. To study the degree of phosphorylation of Shc during capacitation and acrosome reaction, sperm samples were divided into two groups: noncapacitated and capacitated/progesterone treated. Lysates from both groups were immunoprecipitated with anti-phosphotyrosine antibodies and the precipitated (i.e., phosphorylated) proteins were tested with anti-Shc antibodies. The intensity of p52Shc was clearly increased in capacitated/progesterone-stimulated cells.

Human sperm coating antigen from seminal plasma origin.

Sperm surface glycoproteins may be involved in sperm-zona pellucida recognition. Some of these coating proteins are of seminal plasma origin and their expression may change in the process of capacitation and acrosome reaction. Sperm specific monoclonal antibodies (mAb) define the presence and role of sperm membrane associated proteins. We have isolated a monoclonal antibody (SEM-12) specific for human sperm that shows, by indirect immunofluorescence, a discontinuous distribution of the antigen on the head and tail surfaces of non capacitated sperm. This antigen is also present in human seminal plasma as detected by ELISA. The antigen is detectable in sperm of goat, ram, and mouse. Two proteins in the range of 80-84 kDa have been isolated by affinity chromatography with SEM-12 mAb. The same result is obtained by immunoprecipitation. This antibody inhibits sperm motility and acrosome reaction (spontaneous and A23187 ionophore induced.

Hexokinase present in human sperm is not tyrosine phosphorylated but its antibodies affect fertilizing capacity.

The present study was conducted to investigate the presence of hexokinase in human sperm cell, its subcellular localization, its modulation and role in capacitation/acrosome reaction, and tyrosine kinase activity, if any. These studies were conducted using antibodies (Ab) raised against rat brain hexokinase (type I isozyme) that have significant cross-reaction with various human tissue hexokinases and neutralize the catalytic activity of the enzyme. Hexokinase Ab reacted with acrosomal, mid-piece and tail regions of methanol-fixed (approximately 70-80%) and live (approximately 35-52%) sperm in the indirect immunofluorescence technique (IFT) and the immunobead binding technique (IBT), respectively. Hexokinase Ab specifically recognized a band of 116 kDa on the Western blot of detergent-solubilized human sperm preparation that was different from the 95-kDa phosphotyrosine protein. Hexokinase Ab caused a significant (P < 0.01 to < 0.001) and concentration-dependent inhibition of human sperm penetration of zona-free hamster oocytes in the sperm penetration assay (SPA). These data indicate that the hexokinase of 116-kDa molecular weight is present in acrosomal, mid-piece, and tail regions of human sperm. The sperm hexokinase is a glycoprotein that is different from the 95-kDa phosphotyrosine protein and is not phosphorylated at tyrosine residues; however, its antibodies cause agglutination and a concentration-dependent inhibition of fertilizing capacity of human sperm.

Autoantigenicity of human sperm: molecular identities of sperm antigens recognized by sera of immunoinfertile men in the immunoprecipitation procedure.

Autoantigenicity of human sperm as related to immunoinfertility was investigated using the immunoprecipitation procedure. Sera from immunoinfertile men specifically immunoprecipitated six specific bands of 135, 95, and 65 kD and double bands of 47, 41, and 23 kD that were not recognized by the sera from fertile men. Of these six specific bands, three bands of 65-, 47-, and 23-kD molecular identities were recognized by > 60% of immunoinfertile sera, and 47 and 23 kD may correspond to the dimeric and monomeric forms of FA-1 antigen, respectively. Seminal plasma from immunoinfertile men specifically recognized three specific bands of 95, 37, and 16 kD; only the 95-kD band was also recognized with the immunoinfertile sera. These specific antigens may find applications in immunocontraception and immunoinfertility in humans.

Characterization and regional binding of human sperm monoclonal antibodies.

Five monoclonal antibodies (MAb) with specific binding for tail, midpiece, equatorial region, and postacrosome of human sperm were selected. The reactivity of the MAbs was tested by three techniques: an ELISA with human sperm, an indirect immunostaining analyzed by a cell sorter, and immunofluorescence on the cell where the antigens were localized. The localization or exposure of the antigens changes throughout the in vitro capacitation. These antigens are distributed among other mammalian species: mouse, ram, and goat. Their presence was also analyzed in some human tissues: peripheral blood mononuclear cells, spleen, pancreas, thyroid, heart, skin, intestine, and lung. No cross-reactivity was detected. Some of the MAbs showing tail staining severely inhibited sperm motility after 5 hr of incubation with sperm, although no agglutination was present. This immobilizing activity of MAbs would prevent spermatozoa from reaching the oocyte in vivo.

Characterization of a sperm-specific monoclonal antibody and isolation of 95-kilodalton fertilization antigen-2 from human sperm.

Monoclonal antibodies (mAbs) were raised in mice against human sperm. Of the eight hybridomas secreting mAbs that react with human sperm, one, the Vic-1 antibody, was selected for detailed analysis because of its high degree of tissue specificity. The Vic-1 antibody was of the IgG1 subclass and demonstrated binding predominantly with the acrosomal regions of viable but not methanol-fixed noncapacitated and capacitated human sperm cells. It also reacted with the acrosomal and mid-piece regions of viable capacitated as well as noncapacitated murine sperm, but not with methanol-fixed murine sperm. The Vic-1 antibody was germ-cell specific as it did not react with any human somatic cell, tissue, or secretion examined including seminal plasma. The Vic-1 antibody significantly (p = 0.0006) inhibited human sperm penetration of zona-free hamster oocytes in a concentration-dependent manner; at 15 g% concentration it almost completely blocked sperm penetration. The antibody significantly reduced the acrosome reaction and the release of acrosin activity in human sperm cells. There was no effect of the Vic-1 antibody on percentage of motile sperm, although it significantly affected motility characteristics such as linearity, amplitude of lateral head displacement, and beat frequency; motility parameters involved in the hyperactivation phenomenon related to capacitation and the acrosome reaction. The Vic-1 antibody recognized a predominant antigen of 95 kDa, designated fertilization antigen-2 (FA-2), in Western blot and immunoprecipitation procedures using human sperm preparations. The FA-2 antigen was isolated from human sperm preparations by using an immunoaffinity column containing the Vic-1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)