Recurrent CDK1 overexpression in laryngeal squamous cell carcinoma.

In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis.

Asymptotic dynamics of some t-periodic one-dimensional model with application to prostate cancer immunotherapy.

In the case of some specific cancers, immunotherapy is one of the possible treatments that can be considered. Our study is based on a mathematical model of patient-specific immunotherapy proposed in Kronik et al. (PLoS One 5(12):e15,482, 2010). This model was validated for clinical trials presented in Michael et al. (Clin Cancer Res 11(12):4469-4478, 2005). It consists of seven ordinary differential equations and its asymptotic dynamics can be described by some t-periodic one-dimensional dynamical system. In this paper we propose a generalised version of this t-periodic system and study the dynamics of the proposed model. We show that there are three possible types of the model behaviour: the solution either converges to zero, or diverges to infinity, or it is periodic. Moreover, the periodic solution is unique, and it divides the phase space into two sub-regions. The general results are applied to the PC specific case, which allow to derive conditions guaranteeing successful as well as unsuccessful treatment. The results indicate that a single vaccination is not sufficient to cure the cancer.

A mathematical model of low grade gliomas treated with temozolomide and its therapeutical implications.

Low grade gliomas (LGGs) are infiltrative and incurable primary brain tumours with typically slow evolution. These tumours usually occur in young and otherwise healthy patients, bringing controversies in treatment planning since aggressive treatment may lead to undesirable side effects. Thus, for management decisions it would be valuable to obtain early estimates of LGG growth potential. Here we propose a simple mathematical model of LGG growth and its response to chemotherapy which allows the growth of LGGs to be described in real patients. The model predicts, and our clinical data confirms, that the speed of response to chemotherapy is related to tumour aggressiveness. Moreover, we provide a formula for the time to radiological progression, which can be possibly used as a measure of tumour aggressiveness. Finally, we suggest that the response to a few chemotherapy cycles upon diagnosis might be used to predict tumour growth and to guide therapeutical actions on the basis of the findings.

Linking persistent negative symptoms to amygdala-hippocampus structure in first-episode psychosis.

Early persistent negative symptoms (PNS) following a first episode of psychosis (FEP) are linked to poor functional outcome. Reports of reduced amygdalar and hippocampal volumes in early psychosis have not accounted for heterogeneity of symptoms. Age is also seldom considered in this population, a factor that has the potential to uncover symptom-specific maturational biomarkers pertaining to volume and shape changes within the hippocampus and amygdala. T1-weighted volumes were acquired for early (N=21), secondary (N=30), non-(N=44) PNS patients with a FEP, and controls (N=44). Amygdalar-hippocampal volumes and surface area (SA) metrics were extracted with the Multiple Automatically Generated Templates (MAGeT)-Brain algorithm. Linear mixed models were applied to test for a main effect of group and age × group interactions. Early PNS patients had significantly reduced left amygdalar and right hippocampal volumes, as well as similarly lateralized negative age × group interactions compared to secondary PNS patients (P<0.017, corrected). Morphometry revealed decreased SA in early PNS compared with other patient groups in left central amygdala, and in a posterior region when compared with controls. Early and secondary PNS patients had significantly decreased SA as a function of age compared with patients without such symptoms within the right hippocampal tail (P<0.05, corrected). Significant amygdalar-hippocampal changes with age are linked to PNS after a FEP, with converging results from volumetric and morphometric analyses. Differential age trajectories suggest an aberrant maturational process within FEP patients presenting with PNS, which could represent dynamic endophenotypes setting these patients apart from their non-symptomatic peers. Studies are encouraged to parse apart such symptom constructs when examining neuroanatomical changes emerging after a FEP.

New application of carbon nanotubes in haemostatic dressing filled with anticancer substance.

The drug-carrier system used as innovative haemostatic dressing with oncostatic action is studied. It is obtained from CDDP (cisplatin) doped SWCNT (single walled carbon nanotubes), modified and purified by H2O2 in hydrothermal treatment process. In the in vivo nephron sparing surgery (NSS) study we used 35 BALB/c nude mice with induced renal cancer using adenocarcinoma 786-o cells. Animals were divided into four groups: CDDP(M-), CDDP(M+), CONTROL(M-) and CONTROL(M+). In CDDP(M-) and CDDP(M+) groups we used, intraoperatively, carbon nanotubes filled with cisplatin (CDDP). In CONTROL(M-) and CONTROL(M+) groups carbon nanotubes were used alone. During NSS free margin (M-) or positive margin (M+) was performed. In the CDDP(M-) group, we do not observe local tumor recurrences. In Group CDDP(M+) only one animal was diagnosed with tumor recurrence. In control groups the recurrent tumor formation was observed. In our study, it is shown that CDDP filled SWCNT inhibit cancer recurrence in animal model NSS study, and can be successfully applied as haemostatic dressings for local chemoprevention.

Heck reactions of steroidal alkenyl iodides.

Heck reactions of some steroid derivatives possessing iodo-alkenyl moiety (17-iodo-androst-16-ene, 1, 17-iodo-4-aza-4-methyl-androst-16-en-3-one, 2, 17-iodo-4-aza-androst-16-en-3-one, 3) were carried out in the presence of palladium catalysts using various olefins (methyl acrylate, ethyl methacrylate, allyl alcohol and allyl acetate) as coupling partners. With methyl acrylate, a side reaction was observed: the coupling product underwent a Diels-Alder reaction with the excess of methyl acrylate resulting in a six-membered carbocyclic E-ring. Reaction conditions of the synthesis of the Heck-product were optimized. Although the coupling with allyl alcohol led to the formation of 21-formyl-16-pregnene derivatives, the synthesis of the corresponding steroidal unsaturated alcohol could be achieved only via hydrolysis of the coupling product of the alkenyl iodide with allyl acetate.

[Continuous administration of vancomycin in patients with severe burns]. / Vancomycine en administration continue chez le grand brûlé.

OBJECTIVES: In the severely burned patient, a marked, rapid fall in serum concentrations is often observed after intermittent infusion of vancomycin at the usual dose of 30 mg/kg. This specific "jagged" pharmokinetic course with inadequate residual concentrations raises the problem of the efficacy of this time-dependent antibiotic. Studies in patients in general resuscitation units have shown the interest of vancomycin administration in continuous infusion. METHODS: We analyzed variations in serum concentrations of vancomycin during continuous infusion in 18 patients with burns involving a mean of 40% total body surface and reported the doses necessary to maintain serum vancomycin at therapeutic levels; the possible correlations between serum vancomycin concentrations, burn parameters, age and renal function; and clinical and biological tolerance. RESULTS: Higher initial doses were required in burn patients (40 mg/kg in patients aged under 60) than in other patients. Impairment of renal function is a contra-indication of continuous infusion. CONCLUSION: This mode of administration has the advantage of ensuring greater efficacy by preventing fluctuations in serum concentrations.

[Experience with Cefobid in severe infections complicating immunodeficiency diseases]. / Cefobiddal szerzett tapasztalatok immundeficiens haematologiai betegek súlyos fertözéseiben.

As a 3rd generation cephalosporin Cefobid monotherapy was applied during 1985-1986 with 16 hematological patients in immunodeficient, immunosuppressive states where the available aimed and combined antibiotic therapy failed to be effective for the treatment of bacterial infections of grave course and septic character. 4 g/day was the average I.V. dose of Cefobid, higher doses were applied only in especially grave septic states. The hematological patients tolerated well the Cefobid in monotherapy. Recovery form the septic state and excellent clinical effect was found with 9 patients, good effect with 4 and satisfactory effect with 1 patient. In 1 case the therapy had to be stopped owing to drug hypersensitivity. Cefobid is regarded as an antibiotic drug that is effective if used in monotherapy for treating grave, septic infections of hematological patients in immunodeficient--immunosuppressive--myelodepressive states having received earlier antineoplasmic polychemotherapy.

[Onset of Philadelphia chromosome negative chronic myeloid leukemia with symptoms of intrahepatic cholestasis]. / Intrahepatikus cholostasis tüneteivel kezdödö Philadelphia chromosoma negatív chronikus myeloid leukaemia.

The case of a chronic myelogenous leukemia (CML) starting in an unusual form in a young woman is reported. Rapidly progressing icterus was the first and leading symptom of the disease. Simultaneously with the exclusion of the possibility of hepatitis and extrahepatic obstruction of the bile duct the qualitative blood picture roused the suspicion of a myeloproliferative disease. Detailed hematological examinations confirmed Philadelphia chromosome (Ph1) negative CML. Besides the histologically diffuse leukemic infiltration intrahepatic cholostasis could be demonstrated in the background of the icterus. In the chronic and accelerated phase clinical symptoms developing as a consequence of hepatic organic manifestation were dominating. In the authors's case the moderate leukocytosis, initial thrombocytopenia, absence of splenomegaly, early blast-phase and short survival were atypical, characteristic of Ph1 negative CML. The diagnosis and the absence of other associated hepatopathies was supported also by the post-mortem examination. CML beginning with icteric symptoms due to intrahepatic cholostasis is considered as rarity in the literature.

How to isolate urothelial cells? Comparison of four different methods and literature review.

The aim of this study is to present the comparison of four different methods for urothelial cell isolation and culture and compare them to methods cited in the literature. Four different techniques were examined for urothelium isolation from rat bladders. Isolation effectiveness was calculated using trypan blue assay. Confirmation of isolated cell phenotype and comparison with native bladder tissue was confirmed using immunohistochemical (IHC), immunocytochemical (ICC) and immunofluorescence (IF) analysis. The method with bladder inversion and collagenase P digestion resulted in the highest number of isolated cells. These cells showed positive expression of cytokeratin 7, 8, 18, α6-integrin and p63. Our results and the literature review showed that the best method for urothelium bladder isolation is dissection of the epithelium layer from other bladder parts and digestion of mechanically prepared tissue in a collagenase solution.

Facial orf.

Orf is an occupational dermatosis caused by a poxvirus that infects sheep or goats. Human transmission typically occurs in people in contact with the infected animals or by handling contaminated animal products such as wool or meat. The infection in humans is classically characterized by a solitary papule on the fingers or hands. Involvement of the face or head has rarely been reported. We report orf in a young woman with multiple nodules on the face.

Paget's disease of the male breast associated with intraductal carcinoma.

Paget's disease of the breast is a rare condition with an incidence of 3% to 5% of all mammary malignancies. Of all malignant breast cancer, 1% occurs in male patients, and thus, Paget's disease of the male breast is extremely rare. We present a case of intraductal carcinoma of the male breast presenting as Paget's disease.

Optociliary shunts and sickle retinopathy in a woman with sickle cell trait.

We report a case of unilateral optociliary shunt vessels and sickle cell retinopathy in a patient with sickle cell trait. Sickle cell retinopathy has rarely been reported in patients with sickle cell trait hemoglobinopathy. To our knowledge, this is the first report of the association of sickle cell trait, unilateral sickle cell retinopathy, and ipsilateral optociliary shunt vessels.

DNA binding activity is required for EBNA 1-dependent transcriptional activation and DNA replication.

Epstein-Barr virus nuclear antigen 1 (EBNA 1) has been shown to be a sequence-specific DNA binding protein that is required for the replication of episomal elements carrying the viral origin of DNA replication, oriP, as well as for the activation of a specific transcriptional enhancer. We have constructed and analyzed a series of deletion and nonsense mutants in a cloned copy of the EBNA 1 gene and have tested mutant peptides for the ability (a) to bind to a synthetic oligonucleotide containing a consensus EBNA 1 binding site, (b) to activate the EBNA 1-specific enhancer, and (c) to drive replication of an oriP-bearing plasmid in a transient replication assay. The presence of a DNA binding domain in the carboxy-terminal third of the protein was confirmed. Interestingly, neither the acidic tail nor the Gly-Ala copolymer of EBNA 1 contributes significantly to binding. In addition to sequences in the carboxy-terminal portion of the protein, our data indicate that sequences in the amino-terminal portion of the polypeptide affect the binding of EBNA 1 to its target sequence. Further, we show that EBNA 1 binds to its recognition sequence as a dimer. Results of transient expression assays indicate that the ability of EBNA 1 species to activate the transcriptional enhancer and to drive the replication of oriP plasmids is directly dependent on the ability of the polypeptides to bind to the EBNA 1 consensus binding sequence.

Construction and characterization of viable deletion mutants of simian virus 40 lacking sequences near the 3' end of the early region.

Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized. These mutants lack 15 to 60 base pairs between map positions 0.198 and 0.218, near the 3' end of the early region of SV40 and extend further into the body of the A gene, encoding the large T antigen, than previously described deletion mutants. These mutants were isolated after transfection of monkey kidney CV-1p cells with full-sized linear DNA prepared by partial digestion of form I SV40 DNA with restriction endonucleases HinfI or MboII, followed by removal of approximately 25 base pairs of DNA from the 5' termini using lambda-5'-exonuclease and purification of the DNA in agarose gels. Based on camparisons of the DNA sequence of SV40 and polyoma virus, these mutations map in the 19% of the SV40 A gene that shares no homology with the A gene of polyoma virus. The mutations exist in two different genetic backgrounds: the original set of mutants (dl2401 through dl2405) was prepared, using as a parent SV40 mutant dl862, which has a deletion at the single HpaII site (0.725 map unit). A second set (dl2491 through dl2495) contains the same deletions in a wild-type SV40 (strain SV-S) background. Relative to wild-type SV40, the original mutants showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental dl862, although mutant progeny yields were usually lower than yields of dl862, suggesting a possible interaction between the two deletions. The second set of mutants had growth properties and progeny yields similar to those of wild-type SV40; however, Southern blotting experiments indicated that viral DNA replication proceeds at a slightly reduced rate. All of the mutants transformed mouse NIH/3T3 cells and mouse embryo fibroblasts at the same frequency as wild-type SV40. Mutants dl2402, dl2492, and dl2405 consistently produced denser and larger foci in both types of cells. All mutants directed the synthesis of shortened large T antigens. Adenovirus helper function was retained by all mutants.

[Clinical experience with Spersadex comp. gtt]. / Klinické zkousení prípravku Spersadex comp. gtt.

The preparation Spersadex comp. gtt. was tested on three groups of patients (60 patients, 109 eyes) in order to assess its effectiveness in indications outlined by the manufacturer. The preparation has a very favourable antimicrobial, and antiphlogistic action. It proved very effective also in the treatment of chlamydial infections and in the postoperative treatment the combination of the antiphlogistic effect and the antibiotic action proved very useful, though tested only in small groups of patients. Spersadex comp. gtt. is a very effective preparation, well tolerated by patients and it supplements in a useful way the range of eye drops by a new antibiotic-corticoid combination.

Increased opsonization of a prtH-defective mutant of Porphyromonas gingivalis W83 is caused by reduced degradation of complement-derived opsonins.

Periodontitis is a disease of the supporting structures of the teeth that is caused by bacteria whose common ecologic niche is the gingival crevice or the periodontal pocket. Tissue destruction occurs in spite of both local and systemic immune responses against such bacteria. Porphyromonas gingivalis is considered to be an important pathogen in some forms of human periodontitis and is particularly interesting because of its multiplicity of virulence factors. We have previously observed that phagocytosis-resistant invasive strains of P. gingivalis proteolytically degrade C3 and IgG and accumulate less C3-derived opsonins during complement activation. We recently have cloned the prtH gene from P. gingivalis W83 that encodes a 97-kDa active protease, which has the capacity to degrade purified C3 protein. By using this cloned gene we created an allelic exchange mutant of P. gingivalis W83, designated V2296, in which the prtH gene was inactivated. This mutant was previously shown to be less virulent than its parent strain W83 in a mouse model of bacterial invasiveness. In the present study we have assessed the relative capacity of V2296 and W83 to be opsonized by complement and to be taken up by PMNs. The data demonstrate that V2296, in comparison with its parent strain W83, is less able to degrade C3 and that it accumulates significantly greater numbers of molecules of C3-derived opsonins on the bacterial surface in the form of C3b and iC3b during complement activation. Furthermore, opsonized V2296 is taken up in much higher numbers by human PMNs than W83, suggesting that the prtH gene product may be important in evasion of host defense mechanisms.

Adrenal vasoactive intestinal peptide participates in neonatal corticosteroid production in the rat.

Neonatal rats (3-14 days old) exhibit a period of adrenal hyporesponsiveness characterized by blunted corticosterone (B) responses to stress and reduced adrenal sensitivity to adrenocorticotropic hormone (ACTH). Several adrenomedullary peptidergic systems like vasoactive intestinal peptide (VIP) are postulated to influence cortical function. VIP is known to stimulate corticosterone secretion in vitro and to be released from the adrenal medulla following splanchnic nerve stimulation. Here, we tested whether 1) accelerated sympathetic innervation of the adrenal gland by daily L-thyroxine (T4) treatment modified the ontogeny of adrenal VIP and 2) an increase in VIP synthesis could prematurely increase adrenal sensitivity and corticosteroid output during neonatal life. Immunohistochemical VIP staining revealed a different ontogenetic pattern between adrenal regions from days 2-18 and different sensitivities to T4 treatment. Capsular staining was most abundant at all ages and increased with T4 treatment, whereas medullary staining was seen by day 18 and was not affected by T4. Throughout development, VIP receptors were detected mostly in the capsular region, but not in the adrenal cortex. Although receptor levels were not modified by T4 injections, T4 significantly enhanced VIP mRNA levels in the whole adrenal at all ages. In vivo administration of VIP (0.1-2.0 mg/kg body wt ip) to 9- to 12-day-old neonates increased pituitary ACTH, adrenal B, and aldosterone secretion significantly. Corticotropin-releasing factor immunoneutralization before VIP injection diminished VIP-induced ACTH release but still produced small but significant B and aldosterone secretion. Our results show that 1) VIP innervation of the adrenal capsule is present soon after birth and is increased by sympathetic activity whereas VIP appears only much later in the medulla and does not coincide with the onset of splanchnic innervation and 2) exogenous VIP stimulates ACTH, B, and aldosterone release during development and the effect of VIP on steroidogenic secretion is occurring through ACTH secretion, but also, at least in part, directly at the level of the adrenal gland.

Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment.

We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.

Mutational analysis of Epstein-Barr virus nuclear antigen 1 (EBNA 1).

We have constructed a set of nonsense mutants in the EBNA 1 gene of Epstein-Barr virus by inserting a synthetic oligonucleotide, which has translational termination codons in all three reading frames, at various positions in a cloned copy of the EBNA 1 gene. The EBNA 1 proteins encoded by these mutants and three deletion mutants were analyzed using several functional assays. It was determined that there are two separable phosphorylation domains in the carboxy half of the molecule. The carboxy half of the molecule was also found to contain a region between the unique Sac I and Sac II sites that is required for transactivation of the EBNA 1-specific enhancer element found within ori P. The mutants also served to identify a 248 bp region that affects the pattern of intranuclear localization of the protein. Correlations between the functional domains established by these studies and other properties of EBNA 1 are discussed.