Variant alleles in factor V, prothrombin, plasminogen activator inhibitor-1, methylenetetrahydrofolate reductase and risk of thromboembolism in metastatic colorectal cancer patients treated with first-line chemotherapy plus bevacizumab.

Single-nucleotide polymorphisms (SNPs) related to hereditary thrombophilia were investigated as risk factors for thromboembolism in cancer patients. Their effect in metastatic colorectal cancer (mCRC) has never been explored so far. Our aim was to analyse the effect of coagulation factor V (FVL G1691A), prothrombin (PT G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and plasminogen activator inhibitor type 1 (PAI-1 5G/4G) allelic variants in this setting. Fifty-two patients treated with first-line chemotherapy plus bevacizumab who developed a thromboembolic event in their lifetime were initially genotyped. A contemporary cohort of 127 patients who did not experience any thromboembolic event was also analysed. DNA was extracted from peripheral blood and genotypes were determined by real-time PCR, using LightSNiP (TIB MOLBIOL) on LightCcler 480 (Roche). The association between thromboembolism and SNPs was investigated by univariable and multivariable analyses. All SNPs were in Hardy-Weinberg equilibrium (χ2 test P>0.20). FVL G1691A and PT G20210A were present only in heterozygosis in 4 (2.2%) and 7 (3.9%) patients, respectively; MTHFR C677T in homozygosis in 29 (16.2%), MTHFR A1298C in homozygosis in 13 (7.3%); PAI-1 5G/4G in 98 (54.7%) and 4G/4G in 41 (23%) patients. At univariable analysis, treatment duration was significantly associated with thromboembolism (P<0.001), whereas gender, age, obesity, platelets count and chemotherapy backbone were not. Similarly, FVL G1691A and PT G20210A as well as MTHFR C677T and PAI-1 4G allele were significantly associated, whereas MTHFR A1298C was not. At multivariable model including PT G20210A, MTHFR C677T and PAI-1 4G (age, obesity, treatment duration and chemotherapy backbone were included as adjustment factors), the three SNPs were significantlty associated with higher risk of thromboembolism (P=0.025, <0.0001 and P=0.033, respectively). Further validation studies are warranted in order to design a prospective trial of thromboprophylaxis in mCRC patients with high-risk genotypes.

HER2 loss in HER2-positive gastric or gastroesophageal cancer after trastuzumab therapy: Implication for further clinical research.

Mechanisms of acquired resistance to trastuzumab-based treatment in gastric cancer are largely unknown. In this study, we analyzed 22 pairs of tumor samples taken at baseline and post-progression in patients receiving chemotherapy and trastuzumab for advanced HER2-positive [immunohistochemistry (IHC) 3+ or 2+ with in-situ hybridization (ISH) amplification] gastric or gastroesophageal cancers. Strict clinical criteria for defining acquired trastuzumab resistance were adopted. Loss of HER2 positivity and loss of HER2 over-expression were defined as post-trastuzumab IHC score <3+ and absence of ISH amplification, and IHC "downscoring" from 2+/3+ to 0/1+, respectively. HER2 IHC was always performed, while ISH was missing in 3 post-progression samples. Patients with initial HER2 IHC score 3+ and 2+ were 14 (64%) and 8 (36%), respectively. Loss of HER2 positivity and HER2 over-expression was observed in 32 and 32% samples, respectively. The chance of HER2 loss was not associated with any of the baseline clinicopathological variables. The only exception was in patients with initial IHC score 2+ versus 3+, for both endpoints of HER2 positivity (80 vs. 14%; p = 0.008) and HER2 over-expression (63 vs. 14%; p = 0.025). As already shown in breast cancer, loss of HER2 may be observed also in gastric cancers patients treated with trastuzumab-based chemotherapy in the clinical practice. This phenomenon may be one of the biological reasons explaining the failure of anti-HER2 second-line strategies in initially HER2-positive disease.

Toward the molecular dissection of peritoneal pseudomyxoma.

BACKGROUND: Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. PATIENTS AND METHODS: Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). RESULTS: KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not-being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. CONCLUSIONS: Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.

Italian law no. 1/2021 on the subject of vaccination against Covid-19 in people with mental disabilities within the nursing homes.

ABSTRACT: The Coronavirus Disease-19 (Covid-19) pandemic, in the last year, has resulted in a significant number of infections and deaths among nursing homes' residents. This phenomenon has set up the necessity to subject these patients, often suffering from mental disabilities to a vaccination against Covid-19. However, vaccination has long been the subject of public atten-tion, being regulated differently in many European countries. In Italy, the Ministry of Health has given priority, vaccination-wise, to health facilities' patients. The government has regulated through-law no. 1 of January 5, 2021, art. 5, the manifestation of consent to be Covid-19 vac-cinated in incapacitated subjects admitted to assisted health facilities. This rule arose from the need to protect fragile individuals as well as providing real dispositions for the involved health professionals. Nursing homes' elderly guests could be divided into four catego-ries: a) subjects capable to express their will (affected by physical problems); b) subjects who, due to varying degrees of incapacitation, have their own legal guardian, curator or support administrator, ap-pointed in accordance with the law; c) incapacitated subjects without legal representatives d) subjects who, pursuant to law no. 219/2017, have appointed their own trustee. This paper provides for a clear exemplification of all the possible scenarios identified by the Italian law no.1/2021.

Recent Judgement of the Italian Judiciary about medical assisted procreation (MAP): is informed consent valid after parents separation?

ABSTRACT: Law No 40/2004 regulates in Italy the matter of medically assisted procreation (MAP). Recently, the Tribunal of Capua Vetere expressed its position on the subject of informed consent in a case of MAP. In the specific case, a couple entered the preliminary stages of the PMA procedures, carrying out the fertilization of the ovum and the embryo production. Afterwards, the couple separated and the man denied consent to the continuation of the MAP. The woman, willing to proceed with the implantation, the woman made an urgent judicial appeal, obtaining the judge's permission to transfer the embryo to the uterus. This paper analyses the different bioethical positions on MAP's informed consent. In fact, on the one hand, the paper highlight what is set out in Law 219/2017 which provides for the possibility of the patient to revoke at any time the consent to the treatment given. On the other hand, it should be noted that Law 40/2004, willing to protect the embryo, establishes the irrevocability of the position of parental consent after fertilization. The judgment in question seems to favour this latter position, placing itself in the protection of the cryopreserved embryo and recall-ing the principle of entrustment following the fertilization of the egg. Nevertheless, the matter is controversial a consistent amount of legal developments are expected to arise in the next future.

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA).

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

The effects of osmotic shock on the organ cultured mammalian ocular lens.

Freshly isolated rabbit lenses were incubated in anosmolar culture media to study the effects of osmotic shock on lens transparency and protein synthesis. The rate of protein synthesis was measured using three markers, namely alpha-crystallin, a water soluble protein, vimentin, a cytoskeletal protein and MP-26, an intrinsic membrane protein. It was found that only when lenses were cultured in a hyperosmolar medium of 450 mOsM, was there a significant decrease in the rate of lens protein synthesis. The hyperosmolar medium inhibited the synthesis of all three marker proteins. Lens clarity also depended on the solute used to prepare the hyperosmolar medium. Our experimental data showed that there is no relation between decreased protein synthesis and the appearance of lens opacity in the organ cultured rabbit lens.

Iris-ciliary complex inhibits protein synthesis by organ cultured mammalian ocular lens cells.

Freshly isolated rabbit ocular lenses with or without adhering iris-ciliary complex were organ cultured. It was found that lenses with iris-ciliary complex showed decreased protein synthetic ability. Vimentin, a cytoskeletal protein and MP-26, a membrane associated protein, were used as markers for protein synthetic activity. Removal of the iris-ciliary complex at least partially restores the protein synthetic capability of the lens. The ocular lenses cultured with iris-ciliary complex for 48 hr showed marked equatorial opacities.

Analysis of a failed alumina THR ball head.

The favourable properties, on which is based the successful history of the use of alumina in orthopaedic surgery, are due to the tough control made by manufacturers on the whole process from raw materials purity to the quality controls. The minimum requirements of a medical grade alumina are stated from 1981 in ISO 6474 standard. Nevertheless, due to the voluntary character of standards, and lack of national and international laws, it was possible in the past to introduce into the market materials with characteristics not in compliance with the standard ISO 6474. A THR implanted on a fifty years old patient was revised three years after implantation, due to the failure of the alumina ball head. To understand the causes of such an early failure, the fragments of the retrieved ball head were submitted to a series of non destructive and destructive controls. The results show that the poor quality of the design and manufacture of the implant was the origin of the failure.

The role of specific macronutrient availability in the effect of food restriction on length of lactational diestrus in rats.

In lactating rats, food restriction for the first two weeks postpartum extends the period of lactational diestrus by about 1 week. In these studies we investigated whether this effect results from caloric restriction or the reduced availability of a specific macronutrient. In Experiment 1 lactating rats nursing litters of eight pups were assigned to one of four conditions: 1) ad lib. fed; 2) protein-restricted; 3) carbohydrate-restricted; and 4) fat-restricted. Animals in all the restricted conditions were given access to 50% of ad lib. intake of the appropriate nutrient for Days 1-14 postpartum and ad lib. access to the other two macronutrients. In Experiment 2, ad lib. supplementation from one macronutrient source was provided to lactating rats given restricted access to a composite diet. No differential effect of specific macronutrient deprivation or supplementation on length of lactational diestrus was observed in these studies. Thus, the results of both studies are consistent with the hypothesis that caloric restriction plays a primary role in inducing the prolongation of lactational diestrus in food-restricted rats.

Cocaine use and the mid-latency auditory evoked responses.

To examine the effects of chronic cocaine use on the mid-latency auditory evoked responses (MLAERs), we recorded the evoked responses of 15 cocaine-dependent subjects and 13 age-matched healthy control subjects. Two evoked response paradigms were used: a trains paradigm with four different inter-stimulus intervals (ISIs) and a paired-click paradigm. Our data suggest that cocaine-dependent subjects generate smaller P50 components when long ISIs are used with multiple repetitions (in the trains paradigm). In a single repetition paradigm (paired clicks), a significant decrease in the ability to attenuate the N100 component was seen in the cocaine-dependent subjects.

An improved methodology for the synthesis of enantiomerically pure (S)-2,3-O-cyclohexylideneglyceraldehyde.

An improved methodology to prepare (S)2,3-O-cyclohexylideneglyceraldehyde is described. Starting from the commercially available (L)-cyclohexylidene protected ascorbic acid the enantiomerically pure aldehyde was synthesized in only two steps in 41% overall yield.

[Non-cemented reimplantation of failed hip prosthesis]. / Reimpianti non cementati nei fallimenti delle protesi d'anca.

After reviewing their series of prosthetic revisions and comparing it with other reports in literature, the authors reassess, where possible, classic conservative intervention as well as the indications for cemented prostheses given the poor long-term reliability of the bone-cement interface. The authors emphasize the always more frequent incidence of prosthetic loosening and the resulting need for surgical revision, especially in patients who were under 65 years of age at the time of primary prosthetic replacement. Finally, the authors present their clinical and surgical experience in revision using cementless implants, which they prefer in this type of operation.

[The initial findings in 50 cases of the cementless hip prosthesis]. / Primi rilievi su 50 casi di protesi d'anca non cementate.

The authors, after putting forth several biomechanical premises, discuss their experience in total hip arthroplasty (THA) using a PCA-type press fit cementless prosthesis with partial Madreporic covering. Several cases which demonstrate the versatility of this type of replacement are discussed in depth. Even though the short follow-up precludes definitive conclusions, use of cementless total hip replacements must be considered a valid option in the future, especially in cases of early onset of hip pathology.

Effect of hyperosmolar medium on the protein synthesis and morphology of epithelial cells of the cultured rabbit lens.

The effects of hyperosmolar medium on the lens epithelial cell protein synthesis and morphology were studied. It was found that the hyperosmolar medium inhibits protein synthesis, and the high molecular weight proteins were more susceptible to the osmolar shock than were the low molecular weight proteins. Fine structure analysis of the epithelial cells of the lenses incubated in hyperosmolar medium showed that the osmolar shock caused a reduction in the amount of rough endoplasmic reticulum and polysomes; some chromosome clumping was also evident. Whether the morphological changes observed were directly related to the decrease in the rate of protein synthesis is not clear.

The role of the iris-ciliary complex in the control of protein synthesis by the organ cultured rabbit ocular lens.

When freshly isolated rabbit lenses were co-cultured with iris-ciliary body (IC), the protein synthetic activity of these lenses decreased significantly. The inhibitory effect is temporal as longer incubation showed greater inhibition. it was also found that the presence of IC in the culture medium increases the sodium ion levels in the lenses and causes some decrease in the potassium ion levels. Both the protein synthetic activity and cation levels of the lenses cocultured with IC are at least partially reversible. It seems that the IC-derived factor(s) has a molecular weight of more than 1 kDa. It is not clear from these experiments whether the altered cation balance is responsible for the inhibition of the protein synthesis.

[The P loop in atrioventricular septal defect. Embryologico-vectorcardiographic correlation]. / El asa P en el defecto del septum atrio-ventricular. Correlación embriológico-vectocardiográfia.

Atrial vectorcardiograms (vcg) were analysed in 23 patients with partial (n = 15) or complete (n = 8) forms of atrioventricular septal defect (AVSD). The rotation and the projection of the maximum left atrial vector (MLAV) on the frontal plane (FP) and on the horizontal plane (HP) were noted. To assess the possible influence of the degree of left-to-right shunting and the right atrial and right ventricular pressures on the rotation and MLAV projection, these vcg data were correlated with hemodynamic values obtained at the time of preoperative cardiac catheterization. Vcg characteristics of the patients were also compared with those of 25 healthy individuals. No significant differences were noted between the vcg of patients with the partial form and those with the complete form of AVSD. Also, there was no apparent influence of the hemodynamic values on the loop rotation or the MLAV projection in either plane among the patients. However, the MLAV showed a more posteriorly and superior location in the FP and HP than in normal subjects (p = 0.0001). Moreover, 67% of the cases showed a clockwise rotation of the loop in the HP and 33% in the FP; in contrast, normal subjects always showed a counterclockwise rotation in both planes. A more posterior and superior MLAV is consistent with direct observations in human embryos with AVSD which have shown that deficiencies of the posterior portion of the interventricular septum are the basic feature in all cases. The adaptation of the conductive tissue to such deficiency occurs in early phases of cardiac development.

Study of experimental cataract produced by sugar alcohols in the organ-cultured mammalian ocular lens.

Freshly isolated rabbit lenses were cultured in isosmolar TC-199 medium or hyperosmolar medium containing 180 mM sorbitol or mannitol. These experiments were performed to investigate the probable effects of hyperosmolar media on lens clarity and the ability of lens fiber cells to synthesize membrane intrinsic protein, MP-26. The data from these experiments show that incubation in hyperosmolar medium causes depressed MP-26 synthesis, whereas the presence of sugar alcohols in the culture medium induced anterior and posterior subcapsular opacities. The cation levels of lenses incubated in iso- and hyperosmolar medium were also measured. Data from these experiments revealed that although the experimental lenses display prominent opacities, their cation levels are generally similar to those of control lenses. It is proposed that the observed lens opacities are due to the presence of sugar alcohols in the culture medium and not to hyperosmolar shock.

[Long-term scintigraphic follow-up study of the hip prosthesis]. / L'indagine scintigrafica nel controllo a distanza delle protesi d'anca.

The authors emphasize the importance of the bone scan in long-term follow-ups of total hip arthroplasty and describe some technical details of the examination. The changes revealed in the bone scan are confirmed by the operative report. Even though the number of patients in the study was relatively small, the fact that the bone scan taken of patients with cementless total hip replacements revealed no pathological changes is considered significant.

Vimentin synthesis by ocular lens cells.

A study of the synthesis of the intermediate filament protein, vimentin, is reported here. The following systems were examined: the epithelial cells of the organ-cultured rabbit lens, the epithelial and cortical fiber cells of the organ-cultured adult chicken lens and the epithelial cells of the rabbit lens grown in tissue culture. Vimentin is actively synthesized by all of the above mentioned cells.