Stefan Gerber

Tissue engineered fetal skin constructs for paediatric burns

Autologous skin-grafting is the gold standard for treatment of deep second and third degree burns. Available bioengineered skin products also necessitate this two-step surgical procedure. Therefore, we developed fetal skin constructs to improve healing of such degree burns. A bank of fetal skin cells was developed from one organ donation (4 cm2 of skin allowing the preparation of several million three-dimensional skin constructs, 9x12 cm, on native horse collagen). Successive fetal constructs were applied to eight patients at every change of dressing during 1-3 weeks in an outpatient setting. Complete closure was rapid (mean 15.3 days [SD 5.5]) with little hypertrophy of new skin and no retraction seen. This simple technique provided complete treatment without auto-grafting, showing that fetal skin cells might have great potential to treat burns and eventually acute and chronic wounds of other types.

Mycoplasma hominis in mid-trimester amniotic fluid: relation to pregnancy outcome

Abstract Objective: The relationship between detection of Mycoplasma hominis in mid-trimester amniotic fluid and subsequent pregnancy outcome was investigated. Study design: Amniotic fluids from 456 women of European background who underwent a transabdominal amniocentesis at weeks 15–17 of pregnancy were tested for M. hominis by polymerase chain reaction (PCR). The amplicons were hybridized to an internal probe and detected by ELISA. Pregnancy outcomes and clinical data were subsequently obtained. Results: M. hominis were identified in 29 (6.4%) of the amniotic fluids. The rate of preterm labor in women positive for M. hominis (14.3%) was higher than in the negative women (3.3%) (p=0.01). Similarly, a spontaneous preterm birth with intact membranes occurred in 10.7% of the M. hominis-positive women as opposed to only 1.9% of the negative women (p=0.02). The presence of this mycoplasma was not correlated with fetal chromosomal aberrations, intrauterine growth restriction or preeclampsia. Conclusions: Detection of M. hominis in second-trimester amniotic fluids can identify women at increased risk for subsequent preterm labor and delivery.

Wound-healing Gene Family Expression Differences Between Fetal and Foreskin Cells Used for Bioengineered Skin Substitutes

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be implicated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-beta) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-beta superfamily genes analyzed by real-time reverse transcription-polymerase chain reaction, three were found to be significantly different with sixfold up-regulated for TGF-beta2, and 3.8-fold for BMP-6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells.

Evidence of maternal-fetal transmission of Parachlamydia acanthamoebae.

To the Editor: Parachlamydia acanthamoebae is a recently identified agent of pneumonia (1–3) and has been linked to adverse pregnancy outcomes, including human miscarriage and bovine abortion (4,5). Parachlamydial sequences have also been detected in human cervical smears (4) and in guinea pig inclusion conjunctivitis (6). We present direct evidence of maternal–fetal transmission of P. acanthamoebae. n nWe tested 78 amniotic fluid samples from patients who delivered prematurely (defined as spontaneous delivery before 37 weeks of gestation) at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, from 2003 to 2006. DNA was extracted by using the QIAampDNA Mini kit (QIAGEN, Hilden, Germany) and was tested by using a specific Parachlamydia real-time PCR (7). One positive sample (threshold cycle of 34.2) was confirmed by using the 16SigF-Rp2Chlam PCR, which targets a large DNA segment of the 16S rRNA gene (8). Because these 2 PCRs target different DNA segments, the positive PCR results were not due to PCR contamination with amplicons. The sequence exhibited 99.6% similarity with P. acanthamoebae strain Hall’s coccus (1) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF366365,term_id:14030551,term_text:AF366365}}AF366365). n nThe sample was obtained from a 29-year-old woman during her second pregnancy, at 16 weeks of gestation. Amniocentesis was performed because a first-trimester test suggested a Down syndrome risk of 1/100. At the time of amniocentesis, the patient had cough and flu-like symptoms of 3 weeks’ duration, which resolved spontaneously in a few weeks. Cytogenetic analysis showed a normal 46XX karyotype; amniotic fluid culture remained sterile. The pregnancy ended prematurely at 35 weeks and 6 days with the vaginal delivery of a 2,060-g newborn (<5th percentile). The mother and child had an uneventful hospital course. n nThe role of Parachlamydia as the etiologic agent of premature labor and intrauterine growth retardation is likely because 1) all vaginal, placental, and urinary cultures were negative; 2) results of routine serologic tests were negative; and 3) only Parachlamydia was detected in the amniotic fluid. Intrauterine infection caused by Parachlamydia spp. may be chronic and asymptomatic until adverse pregnancy outcomes occur (4). n nThe infection of this pregnant woman might have occurred through zoonotic contact through her work as a butcher in a rural area known for cattle breeding. Of interest, a recent study of 482 healthy Swiss men found 3 who were seropositive for Parachlamydia sp., and all 3 came from the same rural area near Lausanne (within <20-km radius) (9). Moreover, the patient owns 2 guinea pigs, potential vectors of the bacterium (6). Other modes of transmission are possible, e.g., contaminated water (free-living amebae may serve as hosts for Parachlamydia spp. and are widespread in water networks) and ingestion of undercooked meat or contaminated cow milk. n nA plausible pathogenic scenario for this case of possible maternal–fetal transmission of P. acanthamoebae might include bacteremia in the context of a lung infection. This could have resulted in intrauterine infection and intrauterine growth restriction.

Screening for infectious diseases

IntroductionFetal brain injury is an essential cause of lifelong morbidity. Infection appears as a cause of brain damage. Apart from chorioamnionitis, screening for infectious diseases must be considered in pregnancies with a risk of congenital infection or cases with abnormal cerebral ultrasound findings.DiscussionCongenital infections include most of the major components of the TORCH complex: toxoplasmosis, rubella, cytomegalovirus, herpes, and varicella. Seronegative mothers can develop primary infection, which carries a risk of vertical transmission. The timing of the infection is a critical point, because fetal damage often depends on the gestational age at which acute maternal infection took place and occurs more likely in the first half of pregnancy. Antenatal ultrasound can detect brain abnormalities, like hydrocephalus, periventricular leukomalacia, calcifications or hemorrhage. Maternal serologic tests must be performed to look for an infectious etiology; the most frequent agents are the components of the TORCH complex. But additional serology must include parvovirus B19, HIV, and coxsackieviruses.

The morcellator knife: a new laparoscopic instrument for supracervical hysterectomy and morcellation

Background Cutting the cervix, morcellation, and extraction of the uterus and myomata remain major problems in endoscopic surgery. We developed an efficient, safe, reusable, and inexpensive instrument to cut the cervix and morcellate the uterus and myomata: the morcellator knife. Instrument The morcellator knife is a classic lancet with an interchangeable blade, transformed into an endoscopic instrument that can be inserted easily through a 10-mm-diameter trocar. The blade has an automatic retraction system and is set in the standby position, ensuring security. Cutting the cervix and uterine or myoma fragmentation are easy. The mass to be cut is held between two grasping forceps for easy cutting with the blade, under permanent visual control. After morcellation, extraction of the masses is performed through a posterior culdotomy. Experience We have used this morcellator knife in 54 subtotal hysterectomies and 16 myomectomies. There were no complications during the procedures. Morcellation lasted 3–14 minutes and involved the use of an average of two to three blades. Conclusion The morcellator knife is a simple, safe, reusable, and inexpensive instrument with a low maintenance cost.

Human muscular fetal cells: a potential cell source for muscular therapies

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30xa0years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5xa0years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4xa0days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

Colposcopic evaluation after a repeat atypical squamous cells of undetermined significance (ASCUS) smear

Objectives: Management of patients with atypical squamous cells of undetermined significance (ASCUS) remains controversial. We chose to repeat the Pap smear after four months. If ASCUS persisted in this second test, the patient was advised to undergo colposcopy. Our objective is to determine the clinical significance and the prediction of neoplasia among these patients through a colposcopic examination. Methods: Of 29 827 patients who had a Pap smear, ASCUS were detected in 1387 (5%) and persisted in the repeat smear of 225 (16%). Colposcopy and an additional Pap smear were performed on 186 patients. Results: Out of 186 colposcopic evaluations, 91 (49%) were normal and the patients had a negative Pap smear. Colposcopy was abnormal in 95/186 patients (51%) ( Table 1 ). Histology of the directed biopsies revealed 38 (21%) low‐grade squamous intraepithelial lesions (LSIL) and 17 (9%) high‐grade squamous intraepithelial lesions (HSIL). Forty patients (21%) with normal biopsies had ASCUS for the third time in the Pap smear. Conclusions: Colposcopic evaluation after a repeated Pap smear with ASCUS is an appropriate cost‐effective management. Finding 30% of LSIL or HSIL justifies this additional investigation.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Mechanical ventilation (MV) is life‐saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non‐surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation‐free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8u2009hr of MV with 60% oxygen (O2), 24u2009hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O2 (LPSu2009+u2009MVu2009+u2009O2). Lungs were recovered either directly following (T:0u2009hr) or 48u2009hr after MV (T:48u2009hr). Histologically, signs of ventilator‐induced lung injury (VILI) were observed in LPSu2009+u2009MVu2009+u2009O2 lungs at T:0u2009hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48u2009hr. At T:0u2009hr, LPSu2009+u2009MVu2009+u2009O2 increased gene expression of pro‐inflammatory MIP‐2. In parallel anti‐inflammatory IL‐1Ra gene expression was increased in LPS and LPSu2009+u2009MVu2009+u2009O2 groups. At T:48u2009hr, pro‐ and anti‐inflammatory genes had returned to their basal expression. MMP‐2 gene expression was decreased in LPS and LPSu2009+u2009MVu2009+u2009O2 groups at T:0u2009hr, but no longer at T:48u2009hr. MMP‐9 gene expression levels were unchanged directly after MV. However, at T:48u2009hr, gene and protein expression increased in LPSu2009+u2009MVu2009+u2009O2 group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation‐free period in newborn rats and may help to further understand the time‐course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure‐related genes. Pediatr Pulmonol. 2012; 47:1204–1214.

Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction

OBJECTIVEnInsulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth.nnnSTUDY DESIGNnAmniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR.nnnRESULTSnAmniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups.nnnCONCLUSIONSnHigh IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.