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1.
Arch Orthop Trauma Surg ; 143(7): 4331-4337, 2023 Jul.
Article in English | MEDLINE | ID: mdl-36933071

ABSTRACT

OBJECTIVE: To investigate the clinical outcome of patients that underwent conversion of a medial unicondylar knee arthroplasty (UKA) to a total knee arthroplasty (TKA) and to compare that outcome to patients that underwent primary TKA. It was hypothesized that those groups would significantly differ in terms of knee score outcome and implant survival. METHODS: A retrospective-comparative study was conducted utilizing data from the Federal state's arthroplasty registry. Included were patients from our department that undergone a conversion of a medial UKA to a TKA (UKA-TKA group). The Western Ontario and MacMaster Universities Osteoarthritis Index (WOMAC) from preoperative and 1-year postoperative was used. Moreover, the implant survival was analyzed. RESULTS: In the UKA-TKA group, there were 51 cases (age 67 ± 10, 74% women), and in the TKA group, there were 2247 cases (age 69 ± 9, 66% women). The one-year postoperative WOMAC total score was 33 in the UKA-TKA group und 21 in the TKA group (p < 0.001). Similarly, the WOMAC pain, WOMAC stiffness, and WOMAC function scores were significantly worse in the UKA-TKA. After 5 years, the survival rates were 82% and 95% (p = 0.001). The 10-years prosthesis survival was 74% and 91% in the UKA-TKA and TKA groups, respectively (p < 0.001). CONCLUSIONS: Based on our findings it is concluded that patients who received a TKA after UKA have inferior results than those that directly receive a TKA. This is true for both patient-reported knee outcome and prosthesis survival. Converting UKA to TKA should not be seen as an easy operation, but should rather be done by surgeons with considerable experience in both primary and revision knee arthroplasty.


Subject(s)
Arthroplasty, Replacement, Knee , Osteoarthritis, Knee , Humans , Female , Middle Aged , Aged , Male , Arthroplasty, Replacement, Knee/adverse effects , Prosthesis Failure , Retrospective Studies , Treatment Outcome , Reoperation , Knee Joint/surgery
2.
Phys Rev E Stat Nonlin Soft Matter Phys ; 78(5 Pt 2): 055203, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19113178

ABSTRACT

It is shown that the pulses which develop in the NO+H2 reaction on an alkali promoted Rh(110) surface reaction can transport alkali metal. This leads to the accumulation of a substantial alkali-metal concentration in the collision area of pulse trains. Realistic simulations revealed that the effect is due to the strong energetic interactions of the alkali metal with coadsorbates, i.e., the attractive interaction with coadsorbed oxygen and the effectively repulsive interaction with coadsorbed nitrogen.

3.
Anal Bioanal Chem ; 391(5): 1759-72, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18425643

ABSTRACT

This paper introduces the electrically detected displacement assay (EDDA), a electrical biosensor detection principle for applications in medical and clinical diagnosis, and compares the method to currently available microarray technologies in this field. The sensor can be integrated into automated systems of routine diagnosis, but may also be used as a sensor that is directly applied to the polymerase chain reaction (PCR) reaction vessel to detect unlabeled target amplicons within a few minutes. Major aspects of sensor assembly like immobilization procedure, accessibility of the capture probes, and prevention from nonspecific target adsorption, that are a prerequisite for a robust and reliable performance of the sensor, are demonstrated. Additionally, exemplary results from a human papillomavirus assay are presented.


Subject(s)
Biological Assay/methods , Biosensing Techniques/methods , Molecular Diagnostic Techniques/methods , Nucleic Acids/analysis , Papillomaviridae/isolation & purification , Papillomavirus Infections/diagnosis , Base Sequence , Electricity , Genotype , Humans , Microarray Analysis/methods , Molecular Sequence Data , Nucleic Acids/genetics , Oligonucleotide Probes/genetics , Papillomaviridae/genetics , Polymerase Chain Reaction/methods , Sensitivity and Specificity
4.
Nucleosides Nucleotides Nucleic Acids ; 26(8-9): 1167-72, 2007.
Article in English | MEDLINE | ID: mdl-18058558

ABSTRACT

Oligonucleotide-loaded nanoparticles, which are of interest for biomedical application, up to now, could not be prepared by in-situ synthesis, due to difficulty of handling in automated synthesizers. To overcome this problem, we have introduced the "support-on-support" concept. It is based on the reversible anchoring of nanoparticles to the surface of microparticles. These composite beads easily can be used for automated synthesis, being released after completion of chain elongations. As examples, dextran-coated magnetite nanoparticles were attached to polystyrene microparticles through (1) a gelatine or (2) a silica layer. Release involved dissolution of the bonding layer by (1) proteases or (2) alkali.


Subject(s)
Nanoparticles , Oligodeoxyribonucleotides/chemical synthesis , Dextrans , Ferrosoferric Oxide , Metal Nanoparticles , Nanotechnology , Polyhydroxyethyl Methacrylate , Polystyrenes , Spectrophotometry, Ultraviolet
5.
J Physiol Pharmacol ; 57 Suppl 4: 119-29, 2006 Sep.
Article in English | MEDLINE | ID: mdl-17072038

ABSTRACT

The objective of this investigation was to assess the association between the presence of sleep disordered breathing (SDB) and daytime sleepiness, body mass index, hospitalisation, and survival. To this end, a prospective longitudinal study was conducted in the elderly population consisting of 80 patients of either sex over the age of 65 years admitted to a city hospital in Germany without any history of SDB. All patients met the following exclusion criteria: age <65 yr, heart failure, and chronic obstructive lung disease. Baseline anthropometric and cardiorespiratory (one-night portable polygraphic recording) data, and a standardized sleep and sleepiness-questionnaires (Epworth Sleepiness Scale, ESS) were obtained in 1999. A second screening was conducted in 2003. Thirty one women and 34 men completed the follow-up after 3 years. These patients were divided into two subgroups: (i) no clinically relevant SDB and (ii) SDB (apnea-hypopnea index, AHI, >or=5 plus excessive day time sleepiness, ESS, >9). Six men and 3 women fulfilled the criteria of SDB. Thirty three percent of patients with SDB and 20% of patients without SDB died during the follow-up period. Duration of hospital stay was 35 days for the SDB patients and 20 days for patients without it. Body weight and sleepiness did not change significant over the 3-year period between the two cohorts. We conclude that the presence of SDB was associated with a 1.5-fold higher mortality and longer hospital stay in elderly patients over a period of 3 years even in persons without previous history of SDB. Daytime sleepiness was a better predictor than AHI or BMI for death.


Subject(s)
Fatigue/epidemiology , Sleep Apnea Syndromes/epidemiology , Aged , Body Mass Index , Fatigue/etiology , Female , Hospitalization/statistics & numerical data , Humans , Longitudinal Studies , Male , Polysomnography , Surveys and Questionnaires
6.
Int J Oral Maxillofac Surg ; 35(9): 861-4, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16697147

ABSTRACT

This case demonstrates the successful aesthetic and functional reconstruction of a complex facial gun-shot injury with extended bone defects and soft tissue destructions using a 3-step procedure. Initially, a reconstruction plate was inserted, later a fibula transplant enabled the basic reconstruction and finally was distructed in a 3rd session. The rationale behind the sequencing of surgical sessions was the extended bony defect and soft-tissue destruction. The main problem in this type of wound is hypoxia or anoxia of the receptor bed for the transplant. A microvascular anastomosized bone transplant is necessary for sufficient oxygen tension in the recipient site. The anatomical dimensional disproportion of the transplanted free fibula graft and the shape of the mandible were corrected prior to the insertion of dental implants by means of vertical distraction.


Subject(s)
Facial Injuries/surgery , Fibula/transplantation , Mandibular Injuries/surgery , Osteogenesis, Distraction/methods , Wounds, Gunshot/surgery , Bone Plates , Fibula/blood supply , Humans , Male , Middle Aged , Suicide, Attempted
7.
J Physiol Pharmacol ; 56 Suppl 4: 85-91, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16204780

ABSTRACT

The prevalence of obstructive sleep apnea syndrome in patients up to the age of 60 is known to be two times higher in men then in women. Hormonal changes during menopause might underlie changes in this relationship in the elderly. This study was designed to detect differences in the type and frequency of sleep-disordered breathing between women and men over the age of 65 years, having the same body mass index. The study was conducted using a matched-pair approach consisting of a sample population of 40 pairs of patients over the age of 65. All patients met the following exclusion criteria: age below 65, heart failure, chronic obstructive lung disease. Polygraphy was conducted by means of a portable recorder. All measured indices were higher in men than in women. The apnea index was 2.8 +/-4.1 in men and 0.6 +/-1.4 in women. The apnea/hypopnea index was 10.2 +/-11.4 and 4.8 +/-3.9, respectively. These differences were significant (P<0.05). Significant differences also were observed when central (men 8.1 +/-13.1, women 3.1 +/-8.2), mixed (men 5.1 +/-11.4, women 0.4 +/-1.3), and obstructive (women men 8.6 +/-20.1, 1.0 +/-4.3) apnea indices were compared. In conclusion, the study demonstrates that elderly patients showed gender-dependent differences in the type and frequency of sleep-related breathing disorders. Men suffered from all kinds of apnea more frequently than women.


Subject(s)
Aging , Respiration , Sleep Apnea, Obstructive/physiopathology , Sleep , Aged , Female , Humans , Male , Matched-Pair Analysis , Polysomnography , Severity of Illness Index , Sex Distribution , Sex Factors , Surveys and Questionnaires
8.
Transplant Proc ; 37(1): 472-6, 2005.
Article in English | MEDLINE | ID: mdl-15808680

ABSTRACT

INTRODUCTION: In a discordant orthotopic xenotransplantation model (pig-to-baboon) donor pigs expressing human decay accelerating factor (hDAF) as a regulator of complement activity were used to prevent hyperacute xenograft rejection (HXR). We investigated a modified immunosuppressive therapy consisting of ERL080 (Novartis Pharma AG, Base, Switzerland), cyclosporin A (Neoral), steroids, and a cyclophosphamide (CyP) induction protocol with several reduced doses to prevent acute vascular rejection (AVR). METHODS: Donor hearts were harvested from hDAF-transgenic pigs (18.8 +/- 2.6 kg, Imutran Ltd., a Novartis Pharma AG Company). Four adult baboons (25.6 +/- 2.7 kg) with high titers of xenoreactive antibodies (XAb) served as recipients. Serological and hemodynamic parameters were measured. Finally, myocardial tissue was sampled for histological and immunohistochemical examinations. RESULTS: In the first baboon, an acute graft failure occurred after 1 hour due to preservation injury. The second succumbed after 11.1 day due to an acute renal failure. The third died after 13.1 days of an ileus. The fourth baboon had continuously excellent cardiac function (mean echocardiographic ejection fraction, 69.2%), but succumbed on day 20 due to anemia. Corrected mean xenograft survival (excluding the first baboon because of a technical failure) was 14.6 +/- 2.6 days. XAb decreased after day 3 to constantly low levels (<1:64 titer) after CyP induction. White blood cell count decreased from 10.3 +/- 0.8 to 0.9 +/- 0.3 G/L after day 3. Macroscopically and histologically no typical signs of HXR or severe AVR could be detected. CONCLUSIONS: These results confirm that hDAF transgen blocks HXR in this life-supporting model. AVR was prevented by using a modified quadruple immunosuppressive drug combination (Neoral, ERL080, steroids, and several small single doses of CyP). An optimum "fine-tuning" of immunosuppression is required to achieve the best risk-benefit ratio.


Subject(s)
CD55 Antigens/genetics , Graft Survival/physiology , Heart Transplantation/physiology , Sertoli Cells/transplantation , Transplantation, Heterologous/physiology , Animals , Animals, Genetically Modified , Antibodies, Heterophile/blood , Heart Transplantation/methods , Hemodynamics , Humans , Male , Papio , Rats , Rats, Sprague-Dawley , Swine , Time Factors , Transplantation, Heterologous/methods
9.
Transplant Proc ; 37(1): 483-6, 2005.
Article in English | MEDLINE | ID: mdl-15808683

ABSTRACT

INTRODUCTION: Hyperacute xenograft rejection (HXR) and acute vascular rejection (AVR) after xenotransplantation are triggered by xenoreactive antibodies (XAb) and an activated complement cascade. In a heterotopic (abdominal) xenotransplantation model we combined immunoadsorption (IA, Ig-Therasorb column) and a quadruple immunosuppressive drug therapy in recipient baboons with donor pig hearts transgenic for human decay accelerating factor (hDAF). METHODS: According to XAb titers between 6 and 14 cycles of IA were performed preoperatively in 4 recipient baboons (18.6 +/- 2.5 kg). Hearts of hDAF-transgenic donor pigs (6.1 +/- 1.1 kg, Imutran Ltd., a Novartis Pharma AG Company, Basel, Switzerland) were heterotopically transplanted using the abdominal technique in baboons. Immunosuppression consisted of cyclophosphamide (CyP) induction therapy, ERL080 (Novartis Pharma AG), cyclosporin A (CyA, Neoral), and steroids. Blood levels of mycophenolate, CyA, immunoglobulins (Ig), anti-pig-antibodies, complement factors, and cardiac enzymes were determined. Abdominal electrocardiography (ECG), echocardiography, and palpation were used for monitoring of the pig hearts. Myocardial tissue specimens were examined using immunohistochemistry, light microscope (LM), and electron microscope (EM). RESULTS: Ten cycles of IA alone removed 78% of XAb and accordingly IgM, IgG, IgA, complement C3, and C4. None of the xenografts was hyperacutely rejected, but xenograft failure occurred after 5.0 +/- 1.3 days (range, 2.4-8.0 days) because of an AVR associated with a rapid XAb increase within 24 hours. White blood cell count (10.3 +/- 2.2 G/L) showed a maximum of 13.1 +/- 2.1 (day 1) and constant levels (1.4 +/- 0.3-2.1 +/- 1.3 G/L) between day 3 and 6. Histology (LM/EM) showed massive hemorrhage, necrosis, and vascular thrombi as signs of AVR. CONCLUSION: Although HXR was prevented by using IA and hDAF-transgenic donor hearts, AVR was not avoided due to insufficient immunosuppressive regimen used and a missed postoperative IA treatment as a result of an inefficient control of XAb production.


Subject(s)
CD55 Antigens/genetics , Heart Transplantation/immunology , Adrenal Cortex Hormones/therapeutic use , Animals , Animals, Genetically Modified , Antibodies, Heterophile/blood , Humans , Immunosorbent Techniques , Immunosuppressive Agents/therapeutic use , Mycophenolic Acid/therapeutic use , Papio , Swine , Transplantation, Heterologous
10.
Peptides ; 25(5): 819-25, 2004 May.
Article in English | MEDLINE | ID: mdl-15177877

ABSTRACT

The central cornea of 10 cadavers and 33 patients suffering from keratoconus, herpetic keratitis, Fuchs' dystrophy and pterygium were analysed focusing on the expression of TFF peptides by means of reverse transcription polymerase chain reaction and immunohistochemistry. TFF1 and TFF3 transcripts were detected in healthy corneae as well as in pterygia. Only TFF3 mRNA was transcribed in keratoconus, Fuchs' dystrophy and herpetic keratitis. Immunohistochemistry revealed absence of all three TFF peptides in healthy corneae but production of TFF3 in each of the diseased corneae. In pterygia both TFF1 and TFF3 synthesis was detectable in goblet cells. The absence of TFF peptide production in the healthy cornea indicates that TFF3 secretion is induced in different corneal diseases by yet unknown stimuli. Here TFF3 synthesis can be interpreted as a protection mechanism, because all corneal diseases analysed are characterized by progressive tissue destruction. TFF1 and TFF3 production by goblet cells in pterygia is comparable to the healthy conjunctiva suggesting that TFF peptides do not play a significant role in the pathogenesis of pterygia.


Subject(s)
Cornea/metabolism , Fuchs' Endothelial Dystrophy/metabolism , Mucins/metabolism , Muscle Proteins/metabolism , Proteins/metabolism , Pterygium/metabolism , Cornea/pathology , Fuchs' Endothelial Dystrophy/pathology , Goblet Cells/metabolism , Goblet Cells/pathology , Humans , Immunohistochemistry , Keratitis, Herpetic/metabolism , Keratitis, Herpetic/pathology , Keratoconus/metabolism , Keratoconus/pathology , Peptides , Pterygium/pathology , Trefoil Factor-1 , Trefoil Factor-3 , Tumor Suppressor Proteins
11.
Phys Rev Lett ; 92(19): 198305, 2004 May 14.
Article in English | MEDLINE | ID: mdl-15169457

ABSTRACT

Promoters are adsorbed mobile species which do not directly participate in a catalytic surface reaction, but can influence its rate. Often, they are characterized by strong attractive interactions with one of the reactants. We show that these conditions lead to a Turing instability of the uniform state and to the formation of reaction-induced periodic concentration patterns. Experimentally such patterns are observed in catalytic water formation on a Rh(110) surface in the presence of coadsorbed potassium.

12.
Ophthalmologe ; 101(1): 19-24, 2004 Jan.
Article in German | MEDLINE | ID: mdl-14872263

ABSTRACT

The epithelial lining of the lacrimal sac and the nasolacrimal duct consists of pseudo-stratified, columnar epithelia rich in goblet cells. Major secretory products of the epithelial cells are mucins together with TFF peptides. Expression and distribution of several mucins and TFF peptides in the human efferent tear ducts was investigated by means of reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. mRNAs for all the mucins investigated, MUC1, MUC2, MUC4, MUC5AC, MUC5B and MUC7, were detected in healthy human lacrimal sacs and nasolacrimal ducts. Both MUC5AC and MUC5B were detected in goblet cells forming intraepithelial mucous glands. MUC7 together with TFF3 occurred only in columnar epithelial cells of the efferent tear duct system. The mucin diversity of the efferent tear ducts could enhance tear transport and antimicrobial defense. The absence of some mucins in non-functioning although patent segments of the lacrimal passage, suggests that mucins ease tear flow through the efferent tear ducts because these conditions are associated with epiphora. Disorders in the balance of single mucins could be of importance with regard to dacryostenosis, dacryocystitis and dacryolith formation.


Subject(s)
Epithelium/metabolism , Lacrimal Apparatus/metabolism , Mucins/metabolism , Tears/metabolism , Biological Transport, Active/physiology , Humans , Muscle Proteins/metabolism , Nasolacrimal Duct/metabolism , Peptides , Reverse Transcriptase Polymerase Chain Reaction/methods , Trefoil Factor-3
13.
Curr Pharm Biotechnol ; 4(6): 379-95, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14683432

ABSTRACT

The interactions of nucleic acids technology and the technology of arrayed nucleic acids are described, showing the interdependence of nucleic acids chemistry, surface chemistry, (micro-) technology and the requirements of bio-medical applications. The methods and problems of the production of large numbers of oligonucleotides as well as the methods of arraying oligonucleotides are highlighted. The basic approaches, in-situ synthesis and postsynthetic immobilization, are described with a special emphasis on the postsynthetic immobilization of ready-made oligonucleotides on support materials. Techniques for the detection of nucleic acids interactions on arrays are outlined in brief.


Subject(s)
DNA Probes/chemistry , Oligonucleotide Array Sequence Analysis/instrumentation , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotides/analysis , Oligonucleotides/chemistry , Sequence Analysis, DNA/instrumentation , Sequence Analysis, DNA/methods , Adsorption , Biotechnology/methods , DNA Probes/chemical synthesis , Equipment Design , Nucleic Acid Hybridization/methods , Surface Properties
14.
Planta ; 213(6): 927-36, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11722129

ABSTRACT

Water conductance of the cuticular membrane (CM) of sweet cherry (Prunus avium L. cv. Sam) fruit during stages II and III (31-78 days after full bloom, DAFB) was investigated by gravimetrically monitoring water loss through segments of the exocarp. Segments were mounted in stainless-steel diffusion cells, filled with 0.5 ml of deionized water and incubated for 8 h at 25 +/- 2 degrees C over dry silica. Conductance was calculated by dividing the amount of water transpired per unit surface area and time by the difference in water vapor concentration across the segment (23.07 g m(-3) at 25 degrees C). Fruit mass and fruit surface area increased 4.9- and 2.8-fold between 31 and 78 DAFB, respectively. However, CM mass per unit area decreased from 3.9 to 1.5 g m(-2) and percentage of total wax content remained constant at about 31%. Stomatal density decreased from 0.8 to 0.2 mm(-2) (31-78 DAFB). Total conductance of the CM on the fruit cheek (gtot.) remained constant during stage II of development (approx. 1.38 x 10(-4) m s(-1) from 31 to 37 DAFB), increased to 1.73 x 10(-4) m s(-1) during early stage III of fruit growth (43-64 DAFB) then decreased to 0.95 x 10(-4) m s(-1) at maturity (78 DAFB). Partitioning gtot. into cuticular (gcut.) and stomatal conductance (gsto.) revealed that the relative contribution of gcut. to gtot. increased linearly from 30% to 87% of gtot. between 31 and 78 DAFB. respectively. On a whole-fruit basis, g,tot. and gcut. consistently increased up to 64 DAFB, and decreased thereafter. A significant negative linear relationship was obtained between gcut. and CM thickness, but not between the permeability coefficient (p) and CM thickness. Further, p was positively related to strain rate, suggesting that strain associated with expansion of the fruit surface increased p.


Subject(s)
Fruit/metabolism , Prunus/metabolism , Water/metabolism , Algorithms , Biological Transport , Fruit/growth & development , Models, Biological , Permeability , Plant Epidermis/chemistry , Plant Epidermis/growth & development , Plant Epidermis/metabolism , Plant Transpiration , Prunus/growth & development , Waxes/analysis , Waxes/isolation & purification
15.
Blood ; 97(9): 2798-807, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11313274

ABSTRACT

Constitutively activated nuclear factor (NF)-kappaB is observed in a variety of neoplastic diseases and is a hallmark of the malignant Hodgkin and Reed-Sternberg cells (H/RS) in Hodgkin lymphoma. Given the distinctive role of constitutive NF-kappaB for H/RS cell viability, NF-kappaB-dependent target genes were searched for by using adenoviral expression of the super-repressor IkappaBDeltaN. A surprisingly small but characteristic set of genes, including the cell-cycle regulatory protein cyclin D2, the antiapoptotic proteins Bfl-1/A1, c-IAP2, TRAF1, and Bcl-x(L), and the cell surface receptors CD86 and CD40 were identified. Thus, constitutive NF-kappaB activity maintains expression of a network of genes, which are known for frequent, marker-like expression in primary or cultured H/RS cells. Intriguingly, CD40, which is able to activate CD86 or Bcl-x(L) via NF-kappaB, is itself transcriptionally regulated by NF-kappaB through a promoter proximal binding site. NF-kappaB inhibition resulted in massive spontaneous and p53-independent apoptosis, which could be rescued by ectopic expression of Bcl-x(L), underscoring its dominant role in survival of H/RS cells. Hence, NF-kappaB controls a signaling network in H/RS cells, which promotes tumor cell growth and confers resistance to apoptosis.


Subject(s)
Antigens, CD/genetics , Apoptosis/genetics , CD40 Antigens/genetics , Membrane Glycoproteins/genetics , NF-kappa B/genetics , Reed-Sternberg Cells/pathology , Reed-Sternberg Cells/physiology , Antigens, CD/metabolism , B7-2 Antigen , CD40 Antigens/metabolism , Gene Expression Regulation, Neoplastic , Hodgkin Disease/genetics , Hodgkin Disease/pathology , Humans , Membrane Glycoproteins/metabolism , NF-kappa B/biosynthesis
16.
Cell Tissue Res ; 303(1): 109-15, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11235998

ABSTRACT

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of mucin-producing cells and are thought to influence the rheological properties of mucous gels. Here, the localization of these peptides in the human uterus was investigated. An analysis of TFF-peptides mRNA by the polymerase chain reaction revealed TFF3 mainly in the endocervix and smaller amounts in the endometrium. TFF1 and TFF2 mRNA was detectable occasionally in the endocervix and very rarely in the endometrium. Western blot analysis detected only TFF3 in tissue extracts of the endocervix and as a constituent of human cervical mucus. Immunofluorescence localized TFF3 in the surface epithelium of the endocervix and in gland-like structures of the cervical epithelium.


Subject(s)
Endometrium/chemistry , Endometrium/metabolism , Growth Substances/analysis , Growth Substances/genetics , Muscle Proteins , Neuropeptides , Peptides/analysis , Peptides/genetics , Blotting, Western , Cervix Uteri/chemistry , Cervix Uteri/metabolism , Female , Fluorescent Antibody Technique , Gene Expression/physiology , Humans , Mucins/metabolism , Oligonucleotide Probes , Pregnancy , Proteins/analysis , Proteins/genetics , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Trefoil Factor-2 , Trefoil Factor-3
17.
J Biotechnol ; 86(3): 203-24, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11257532

ABSTRACT

We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper Földes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project.


Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Sequence Analysis, DNA/methods , Base Sequence , DNA/analysis , Molecular Sequence Data , Polymerase Chain Reaction/methods , Rhodamines/chemistry , Spectrometry, Fluorescence/methods , Taq Polymerase/chemistry , Templates, Genetic
18.
J Biotechnol ; 86(3): 281-8, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11257537

ABSTRACT

Different kinds of particles were investigated for their potential use as supports for exonucleolytic sequence analysis. Composite beads composed of an unreactive polystyrene "core" and a "shell" of functionalized silica nanoparticles were found to best fulfill the various prerequisites. The biotin/streptavidin system was used for attachment of DNA to composite beads of 6 microm diameter. Applying M13 ssDNA in extremely high dilution (approximately 1 molecule versus 100 beads) with internal fluorescent labels, only a small fraction of beads was found to be associated with fluorescent entities, which likely correspond to a very small number of bound DNA molecules per particle. For better selection and transfer of DNA-containing beads into microstructures for exonuclease degradation the loading experiments were repeated with composite beads of 2.3 microm diameter. In this case a covalent bond was formed between carboxylate-functionalized beads and amino-terminated oligonucleotides, which were detected through external labelling with fluorescent nanoparticles interacting with biotinylated segments of the complementary strand.


Subject(s)
Polymers/chemistry , Biotin/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Fluorescent Dyes/chemistry , Microspheres , Sequence Analysis, DNA , Streptavidin/chemistry
19.
Transpl Int ; 13 Suppl 1: S494-503, 2000.
Article in English | MEDLINE | ID: mdl-11112061

ABSTRACT

In xenotransplantation long ischemic time of grafts is supposed to have a marked influence on hyperacute rejection (HXR). We investigated the influence of different cold ischemic times on HXR of ex vivo "working pig hearts" perfused with human blood. Xenoreactive natural antibodies (XNAb) as a trigger of HXR were eliminated by Ig-Therasorb immunoadsorption (IA). Explanted Landrace pig hearts of group G1 and group G3 (with additional IA) underwent 4 h of cold ischemia prior to xenoperfusion. Control groups G2 and G4 (with IA) were kept ischemic for only 46.6 +/- 15.8 and 51.2 +/- 4.2 min, respectively. Ischemic time prolonged the perfusion time in our working heart model (G1: 356 +/- 46.1 min; G2: 125 +/- 31 min; P < 0.05). IA had no additional impact on perfusion time but was effective by itself. The heart weight increased fourfold more in G2 as compared to the other groups. IA without ischemia significantly improved cardiac output in G4 (G3: 198.8 +/- 15.4 mL/min; G4: 338.5 +/- 16.0 mL/min). Coronary flow in G2 was significantly lower than in G1 (G1: 157.9 +/- 9.15 mL/min; G2: 59.4 +/- 20.1 mL/min). Histological signs of HXR (light and electron microscopy) could be found in G2 in contrast to the other groups. Parameters of serological damage showed a minimum in G4 and the maximum in G2. In G1 XNAb were nearly equally eliminated immediately after the start of xenoperfusion as in IA groups G4 and G3. Four hours of ischemic time showed beneficial effects in preventing HXR, possibly caused by changes of the endothelial cell surface (for example, glycosylation or loss of alpha1-3Gal epitopes with a hapten effect).


Subject(s)
Graft Rejection/immunology , Heart Transplantation/immunology , Heart , Myocardial Ischemia , Myocardial Reperfusion , Transplantation, Heterologous/immunology , Acute Disease , Animals , Antibodies, Heterophile/isolation & purification , Cardiac Output , Cell Membrane/physiology , Endothelium, Vascular/physiology , Heart/physiology , Heart Arrest, Induced , Humans , Immunosorbent Techniques , In Vitro Techniques , Models, Cardiovascular , Myocardium/cytology , Myocardium/pathology , Organ Preservation , Swine
20.
Transpl Int ; 13 Suppl 1: S508-17, 2000.
Article in English | MEDLINE | ID: mdl-11112063

ABSTRACT

To prevent hyperacute xenograft rejection (HXR) caused by preformed natural antibodies (XNAb) after orthotopic heart xenotransplantation (oXHTx) of landrace pig hearts into baboons, we used immunoadsorption of immunoglobulins IgG, IgM and IgA and complement with the reusable Ig-Therasorb column. In addition to functional data, tissue was sampled for histological, immunohistochemical and electron microscopical analysis. We performed three oXHTx of landrace pig hearts to baboons using extracorporeal circulation (ECC) connected to the immunoadsorption unit. Intraoperative treatment consisted of four cycles of immunoabsorption (IA). One oXHTx of a baboon without IA served as a control. A mismatch of donor and recipient heart size was prevented by selecting a 30-40% lower body weight of donor pigs than recipients. Four cycles of IA removed more than 80% of IgG, IgM and IgA, 86% of antipig antibodies and 66% of complement factors C3 and C4 from plasma. The graft of the control animal failed after 29 min. Orthotopic xenotransplantation with IA was selectively terminated after 100 min, 11 h and 21 h, respectively without any histological signs of HXR in light and electron microscopy. After weaning off from ECC these donor xenografts showed sufficient function with normal ECG and excellent cardiac output in echocardiography and invasive measurement (1.93 +/- 0.035 l/min). The myocardium of the control xenograft demonstrated more deposits of Ig and complement components (C3, C4) than in the IA group. Baboons survive HXR after orthotopic pig heart xenotransplantation due to antibody depletion by reusable Ig-Therasorb column treatment. Long-term survival in an orthotopic baboon xenotransplantation model after IA, especially in combination with transgenic pig organs, could be a reliable preclinical trial for future clinical xenotransplantation programs.


Subject(s)
Graft Rejection/prevention & control , Heart Transplantation/immunology , Immunosuppression Therapy/methods , Transplantation, Heterologous/immunology , Acute Disease , Animals , Antibodies/blood , Complement System Proteins/isolation & purification , Extracorporeal Circulation/instrumentation , Extracorporeal Circulation/methods , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Immunosorbent Techniques , Papio , Swine
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