Mother and daughter with Kenny-Caffey syndrome: the adult phenotype.

Kenny-Caffey Syndrome (KCS) is a genetic syndrome characterized by growth retardation with short stature, cortical thickening and medullary stenosis of long bones, and hypoparathyroidism with hypocalcemia. KCS and the related but more severe condition osteocraniostenosis are determined by monoallelic variants in the FAM111A gene. Here we describe the KCS phenotype resulting from the monoallelic FAM111A variant p.Y511H in a 31-year-old woman and in her 56-year-old mother, who is one of the oldest affected individuals known so far. To our knowledge, it is also one of the few molecularly confirmed cases of a mother-to-child transmission of KCS.

A patient with limb girdle muscular dystrophy carries a TRIM32 deletion, detected by a novel CGH array, in compound heterozygosis with a nonsense mutation.

Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically highly variable, caused by mutations in the TRIM32 gene. Here we describe a 35-years-old who experienced progressive muscle weakness. The muscle biopsy revealed an unspecific pattern of atrophic and hypertrophic fibers; the immunohistochemistry for several proteins was normal. Comparative genomic hybridization (CGH) analysis showed a heterozygous deletion of the entire TRIM32 gene. On the other allele we identified the R316X nonsense mutation. The genetic diagnosis of LGMD2H in this case was reached by using a novel high throughput diagnostic tool.

Methylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples of infertile couples correlates with recurrent spontaneous abortion.

STUDY QUESTION: Is the methylation status of the methylenetetrahydrofolate reductase (MTHFR) promoter region in semen samples associated with 'recurrent spontaneous abortion' (RSA)? SUMMARY ANSWER: MTHFR promoter hypermethylation is more frequent in semen samples from RSA couples than in semen samples from infertile couples with no history of RSA (NRSA) and affects the whole sperm population significantly more often. WHAT IS KNOWN ALREADY: Modifications to the MTHFR gene such as polymorphisms and promoter methylations are associated with male infertility. STUDY DESIGN, SIZE AND DURATION: Retrospective cohort study of semen samples from 20 RSA couples, 147 NRSA couples and 20 fertile men between 2011 and 2012. MATERIALS, SETTING AND METHODS: DNA from the semen samples of RSA, NRSA and fertile men were analyzed by methylation-specific PCR amplification using primers which anneal to the methylated or unmethylated cytosine-phosphodiester bond guanine (CpG) islands within the promoter region of MTHFR. The specificity of the PCR products was assessed by DNA sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: The methylated MTHFR epigenotype (including samples where it co-existed with unmethylated MTHFR epigenotypes) was detected in 75% of RSA men, 54% of NRSA men and 15% of fertile men. MTHFR methylation was observed in the whole sperm population in semen samples from 55% of RSA men compared with 8% in NRSA men (P < 0.05) and 0% in fertile men (P < 0.05). DNA sequencing analysis was fully concordant with the PCR results and revealed that when MTHFR methylation occurred, CpG islands within the promoter region were 100% methylated (hypermethylation of MTHFR promoter). LIMITATIONS, REASONS FOR CAUTION: The relatively small sample size of RSA infertile couples. WIDER IMPLICATIONS OF THE FINDINGS: The hypermethylation of the MTHFR gene promoter should be taken into consideration as a novel putative risk factor in RSA etiology. STUDY FUNDING/COMPETING INTEREST(S): Our institution has received an FAR research grant from the University of Ferrara, Ferrara, Italy. No competing interests declared.

Effects of chemical ischemia in cerebral cortex slices. Focus on nitric oxide.

Superfused rat cerebral cortex slices were submitted to a continuous electrical (5 Hz) stimulation and treated with sodium azide (1-10 mM) in the presence of 2 mM 2-deoxyglucose ("chemical ischemia"). Presynaptic cholinergic activity, evaluated as acetylcholine release, was inhibited depending on the sodium azide concentrations and on the length of application (5-30 min). Following a 5-min treatment with 10 mM sodium azide, acetylcholine release was reduced to 45+/-2.3%; simultaneously, there was a 15- and 10-fold increase in glutamate and nitric oxide effluxes, respectively. After restoring normal superfusion conditions, acetylcholine release recovered to 70+/-3.1% of the controls; the N-methyl-D-aspartate receptor antagonist MK-801 (10 microM) as well as the nitric oxide scavengers, haemoglobin (20 microM) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (150 microM), improved the recovery in presynaptic activity, showing that both glutamate and nitric oxide play detrimental roles in chemical ischemia. On the other hand, the post-ischemic recovery was worsened by the guanylylcyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (10 microM), suggesting that the activation of such a pathway plays a neuroprotective role and that the nitric oxide-induced harmful effects depend on different mechanisms. Chemical ischemia-evoked nitric oxide efflux partly derived from its calcium-dependent endogenous synthesis, since both the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (1 mM), and the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 microM), substantially prevented sodium azide effects. Nitric oxide efflux was only weakly reduced by MK-801 and was not modified by either the L-type calcium channel blocker, nifedipine (10 microM) or the N-type calcium channel blocker omega-conotoxin (0.5 microM), thus suggesting a prevailing intracellular calcium-dependence of nitric oxide production, although a partial extracellular calcium source cannot be ruled out. These findings show that sodium azide plus 2-deoxyglucose treatment is a useful protocol to induce brain ischemia in vitro and underline the involvement of nitric oxide in the complex events following the ischemic insult.

Mice genetic immunization with plasmid DNA encoding a secreted form of HSV-1 gB induces a protective immune response against herpes simplex virus type 1 infection.

Intramuscularly (i.m.) delivered plasmid DNA encoding a secreted form of glycoprotein B of herpes simplex virus type 1 (HSV-1 gB1s) was evaluated for the ability to elicit a protective immune response in Balb/c mice. Animals received three i.m. injections of a gB1s expression plasmid (pRP-RSV-gB1s) or of a wild-type transmembrane gB1 coding plasmid (pRP-RSV-gB1), while control mice were injected with the vector alone (pRP-RSV). A specific antibody response was observed in almost all immunized animals, and in most cases antibodies were also detected after 1 month in the absence of further vaccine boosts. Serum antibodies mostly displayed neutralizing activity against HSV-1. Glycoprotein B1s DNA immunization was also effective in protecting animals against the primary infection induced by a subsequent HSV-1 challenge and limited HSV-1 infection of sensitive ganglia.

Nuclear accumulation of c-myc mRNA in phytohaemagglutinin-activated T lymphocytes treated with anti-HLA class I monoclonal antibody.

Anti-HLA class I monoclonal antibody 01.65 inhibits the proliferative response of PHA-activated human T lymphocytes from peripheral blood mononuclear cells. The recruitment rate in the cell cycle is slack and the G1 and S phases are prolonged. Among the early events after PHA activation, only the calcium-dependent PKC activity appears to be modified: particulate PKC is completely depleted while cytosolic residual PKC is reduced by 80% after MAb 01.65 treatment. We have carried out in greater detail the study of c-myc gene regulation by MAb 01.65 and the results are as follows: (i) c-myc RNA transcription is normally initiated and finished, suggesting a post-transcriptional regulation of c-myc gene expression; (ii) no alteration in c-myc mRNA stability has been documented; (iii) steady-state levels of c-myc mRNA expression by Northern blot analysis and PCR amplification are decreased in the cytoplasmic compartment, while in the nuclear compartment they appear to be increased. Nuclear accumulation of mature mRNA after MAb 01.65 and PKC inhibitor (H7 and StSp) treatment appears to be the most probable mechanism involved. The possible implications of this are discussed.

Identification of a novel protein kinase C inhibitor in microsomes from phytohaemagglutinin activated human peripheral blood mononuclear cells.

A peptide inhibiting either corpuscolate or purified PKC has been identified from microsomes of PHA-activated human PBMC but it is not detectable in microsomes of resting PBMC. The peptide was obtained from a microsomal preparation in an oligomeric form that could be transformed into a monomeric form by beta-MSH. The active peptide (IN) was retained on a PC-11 chromatographic column and could be eluted with NaCl. IN is ineffective on PKC-dependent protamine phosphorylation of protamine and on Ca2+ and phospholipid-independent activity generated by mild hydrolysis with trypsin of PKC. Ca2+ binding is permissive for IN activity. IN inhibits particulate PKC in PHA-activated PBMC, but is ineffective after TPA activation. All these data indicate that IN acts at the regulatory domain of PKC.

Anchored anti-HLA class I monoclonal antibody fails to induce inhibition of PHA-activated lymphocytes proliferation.

It is known that anti-HLA Class I antibodies inhibit the proliferative response of PHA-activated T-lymphocytes. We found that plastic- or sepharose-linked anti-HLA Class I monoclonal antibody 01.65 does not inhibit either [3H]Thymidine incorporation or recruitment in the cell cycle, nor does it reduce the expression of c-myc mRNA and the membrane expression of Interleukin-2 Receptor and Transferrin Receptor. Furthermore, particulate Protein Kinase C is not affected by anchored anti-HLA Class I monoclonal antibody 01.65. We suggest that anti-HLA Class I monoclonal antibody may act through crosslinking or internalization of HLA Class I antigens.

Altered proliferative kinetics in PHA-activated human T-lymphocytes treated with the anti-HLA class I monoclonal antibody 01.65.

Anti-HLA class I monoclonal antibody (mAb) 01.65 inhibited phytohaemagglutinin (PHA)-induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G1-phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA-induced proliferation of human T-lymphocytes.

Molecular analysis of cell cycle-related gene expression in anti-HLA class I monoclonal antibody (01.65) treated PHA-activated human T-lymphocytes.

HLA class I antigens seem to be involved in the proliferative response of PHA-activated human T-lymphocytes. We have previously reported that the treatment of PHA-activated peripheral blood mononuclear cells (PBMC) with an anti-HLA class I monoclonal antibody, 01.65, (i) inhibits the tritiated thymidine incorporation, (ii) inactivates cytosolic protein kinase C (PKC) and (iii) causes an increase in the duration of the cell cycle. Northern Blot kinetic analysis of c-fos, c-myc, cdc2, IL-2R, c-myb, ODC, TK and H3, from 10 minutes to 120 hours, was performed in MAb 01.65 treated cultures. We found that the expression of four genes (c-myc, IL-2R, cdc2 and TK) was depressed 24 hours after PHA stimulation.

Competitive effect of anti-HLA class I monoclonal antibody (01.65) and N-N-staurosporine on prolipherative response of PHA activated T-lymphocytes.

N-N-Staurosporine (STAR) inhibits in a dose dependent manner Tritiated-Thymidine (3H-TdR) incorporation in phytohemagglutinin (PHA) activated Peripheral Blood Mononuclear Cells (PBMC) treated with anti-HLA class I monoclonal antibody (MAb) 01.65 and its effect results competitive with MAb 01.65. Cytosolic and particulate Protein Kinase C (PKC) have been studied. Only when PKC particulate activity is no more detectable, the effect of STAR on 3H-TdR incorporation is evident.

C-fos, c-myc and IL-2R mRNA expression in PHA activated T lymphocytes treated with a monoclonal anti-HLA class I antibody (MAb 01.65).

Monoclonal anti HLA class I antibodies inhibit the proliferative response of PHA-stimulated T lymphocytes. We studied the effects of MAb 01.65 anti-HLA class I on c-fos, c-myc and IL-2R mRNA expression. We found that MAb treatment does not modify either c-fos mRNA levels observed after 10 minutes to 3 hrs or the early c-myc mRNA expression revealed after 1 to 6 hrs, but decreases the intensity of autoradiographic signals of late c-myc and IL-2R mRNA expression. Since we had previously ascertained that MAb 01.65 treatment induces a decrease in PKC enzymatic activity after few minutes, the correlation of that result with the data presented in this paper will be discussed.

PKC cellular distribution in TPA activated human peripheral blood mononuclear cells, treated with an anti-HLA class I monoclonal antibody.

Cytosolic and Particulate Protein Kinase C has been studied in Peripheral Blood Mononuclear Cells activated with 12-O-Tetradecanoyl phorbol 13-acetate and treated with the anti-HLA Class I Monoclonal Antibody 01.65. No effects on the cellular distribution of PKC activity nor to the proliferative response has been found. In phytohemagglutinin stimulated PBMC cultures treated with MoAb 01.65 total PKC activity depletion and 3H-Thymidine incorporation inhibition has been found. In PBMC cultures activated with both PHA and TPA, the proliferative response was similar to cultures activated with PHA alone, while the PKC cellular distribution was similar to the one detected in TPA stimulated cultures. Addition of the MoAb 01.65 was ineffective on both PKC activity and 3H-Thymidine incorporation. These data indicate that anti-HLA Class I MoAb induced 3H-Thymidine incorporation inhibition may be related to low levels of PKC activity.

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes.

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.

Anti HLA class I monoclonal antibody effect on PKC kinetics in PHA activated human peripheral blood mononuclear and E+ cells.

Cytoplasmic protein kinase C (PKC) has been studied in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) and macrophage depleted E+ cell culture. Within 10' after contemporanous addition of PHA and anti HLA class I monoclonal antibody 01.65 (MoAb) PKC is depleted in both cell types. Enzyme activity recovers in the following hours however at 72 hours is at control values in E+ cultures while in PBMC cultures it is still depleted at 68% of the control. Anti HLA class I MoAb induced tritiated lymidine (3H-TdR) incorporation inhibition appears to be related to low levels of PKC activity.

Co-operation in cell transformation between BK virus and the human c-Harvey-ras oncogene.

Early-passage hamster embryo cells were transformed by recombinant DNA molecules containing BK virus (BKV) early-region gene and either the activated c-Ha-ras oncogene (pBK/c-rasA) or the normal c-Ha-ras proto-oncogene (pBK/c-rasN). The recombinant DNAs had a greater transforming ability and converted hamster cells to a more malignant phenotype than the single genes transfected separately. pBK/c-rasA was significantly more powerful than pBK/c-rasN in conferring to cells all the characteristics of transformation. Transfected DNA sequences were integrated mostly as single insertions into cellular DNA. Specific c-Ha-ras and BKV transcripts as well as c-Ha-ras p21 and BKV T antigen were detected in transformed cells. Although stimulation of c-Ha-ras expression by BKV enhancers cannot be excluded in recombinants, super-transfection and co-transfection experiments in hamster embryo cells and pre-neoplastic cell lines showed that BKV early-region and c-Ha-ras co-operate in transformation by contributing separate and independent functions.

Depletion of protein kinase C induced by an anti HLA class I monoclonal antibody in phytohemagglutinin activated human T cells.

Anti HLA Class I Monoclonal Antibody depletes Protein Kinase C (PKC) to 20% of control value in PHA activated human T cells. The effect is reversible: in 24 hours the enzymatic activity returns to 58% of control value. Removal of antibody from the culture medium increases the rate of recovery. Implications of this finding for the modulation by HLA Class I antigens of the proliferative response of T cells to lectins are discussed.