Antibiotic Cycling Reverts Extensive Drug Resistance in Burkholderia multivorans.

Antibiotic collateral sensitivity, in which acquired resistance to one drug leads to decreased resistance to a different drug, occurs in Burkholderia multivorans. Here, we observed that treatment of extensively drug-resistant variants evolved from a cystic fibrosis (CF) sputum sample isolate with either meropenem or sulfamethoxazole-trimethoprim, depending on past resistance phenotypes, resulted in increased sensitivity to five different classes of antibiotics. We further identified mutations, including putative resistance-nodulation-division efflux pump regulators and uncharacterized pumps, that may be involved in this phenotype in B. multivorans.

X-ray photoelectron and scanning Auger electron spectroscopy study of electrodeposited ZnCr coatings on steel.

Zn-Cr alloyed coatings electrochemically deposited are of high interest for leading steel manufacturing companies because of their novel properties and high corrosion resistance compared with conventional Zn coatings on steel. For tuning and optimizing the properties of the electrodeposited Zn-Cr coatings, a broad range of the deposition conditions must be studied. For this reason, two different types of material were investigated in this study, one with a low electrolyte temperature and one with an elevated electrolyte pH, compared with the standard values. Because different corrosion performance and delamination behaviour of the layers were observed for the two types, advanced surface analysis was conducted to understand the origin of this behaviour and to discover differences in the formation of the coatings. The topmost surface, the shallow subsurface region, and the whole bulk down to the coating-steel interface surface were analysed in detail by X-ray photoelectron spectroscopy (XPS) and high-resolution scanning Auger electron spectroscopy to determine the elemental and the chemical composition. For better understanding of the resulting layer structure, multiple reference samples and materials were measured and their Auger and XPS spectra were fitted to the experimental data. The results showed that one coating type is composed of metallic Zn and Cr, with oxide residing only on the surface and interface, whereas the other type contains significant amounts of Zn and Cr oxides throughout the whole coating thickness.

Non-pegylated liposomal doxorubicin and docetaxel in metastatic breast cancer: final results of a phase II trial.

BACKGROUND: Non-pegylated liposomal doxorubicin (NPLD) has demonstrated equivalent antitumor activity to conventional doxorubicin and a significantly lower risk of cardiotoxicity when given as single agent or in combination with cyclophosphamide, but there is limited experience with the combination of NPLD and taxanes. This phase II study was performed to evaluate the efficacy and safety of the NPLD and docetaxel in patients with metastatic breast cancer. PATIENTS AND METHODS: A total of 51 patients were treated with NPLD (60 mg/m(2)) and docetaxel (75 mg/m(2)) in 3-weeks intervals for up to eight cycles. RESULTS: The overall response rate was 50% and 78% of patients derived a clinical benefit. Median time to progression and overall survival were 10.0 months (95% CI, 6.9-13.1 months) and 25 months (95% CI, 22.1-29.8 months), respectively. Median duration of response was 12.0 months (95% CI 7.1-16.9). The treatment was generally well tolerated and associated with toxicities that were consistent with the known side-effects of the individual agents and of anthracycline/taxane combinations. There were no symptomatic cardiac averse events and mild asymptomatic LVEF changes were reported in five patients. CONCLUSIONS: The combination of NPLD and docetaxel is well tolerated and has high antitumour activity in MBC patients.

[Placenta praevia percreta--a serious complication after previous cesarean section]. / Plazenta praevia percreta--eine schwere Komplikation nach vorausgegangener Sectio caesarea.

A 36-year-old G4P1 presented at 20 weeks gestation with vaginal bleeding. Her obstetrical history was significant for two first-trimester spontaneous abortions requiring curettage and a cesarean section. On admission placenta praevia was suspected by ultrasound. A placenta percreta was suspected by ultrasound follow-up at 30 weeks. At 33 weeks she underwent cesarean section because of serious vaginal bleeding. The profound blood loss with consecutive coagulopathy required an emergency hysterectomy and multiple blood transfusions. Placenta percreta is a rare but dramatic complication after previous cesarean section. This should be kept in mind as the rate of elective cesarean sections is rising continuously.

Impact of three-dimensionally measured endometrial volume on the pregnancy rate after intrauterine insemination.

OBJECTIVE: The success of artificial reproductive techniques not only depends on the quality of oocytes and spermatozoa but also on the receptivity of the endometrium. The aim of this study was to assess the role of endometrial volume measurement by three-dimensional ultrasound in predicting the pregnancy rate (PR) in women receiving controlled ovarian hyperstimulation followed by intrauterine insemination. MATERIAL AND METHODS: 104 patients having intrauterine insemination (IUI) were included in this prospective trial. Ovarian hyperstimulation was performed with gonadotropins in 73 % of patients and with clomiphene citrate (CC) in 27 % of patients. Endometrial thickness, pattern and three-dimensional volume were measured immediately before insemination. RESULTS: In 104 IUI cycles a total of 14 clinical pregnancies were recorded (PR=13.5 %). The endometrial volume was 3.5 ml and was not significantly different in pregnant (4.0 +/- 1.5 ml) from non-pregnant women (3.4 +/- 1.9 ml). In the subgroup of women with an endometrial volume > or = 2 ml and trilaminar endometrium the pregnancy rate was 22 %, significantly higher than that in women without these two criteria (PR 6 %, p < 0.05). The negative predictive value of an endometrial volume < 2 ml for a clinical pregnancy after IUI was 96 %. Endometrial volume and thickness were significantly higher after ovarian hyperstimulation with gonadotropins (3.7 ml and 11.0 mm) than with CC (2.8 ml and 9.5 mm; p < 0.05). CONCLUSIONS: An endometrial volume < 2 ml at the day of insemination is associated with a poor likelihood of pregnancy. Endometrial volume measured by 3D ultrasound is a new objective parameter to predict endometrial receptivity.

[Expression of the molecules HLA-G and HLA-E modulates cytokine production of monocyte generated dendritic cells]. / Die Expression der Moleküle HLA-G und HLA-E moduliert die Zytokinproduktion monozytengenerierter dendritischer Zellen.

OBJECTIVE: Preferential secretion of Th1-like cytokine is mainly a property of monocyte derived dendritic cells (DC). Since normal early pregnancy is characterized by a shift towards a Th2-like cytokine pattern, it may be assumed that cytokine secretion by DC during early pregnancy could be modulated by the non-classical HLA molecules G and E present on invasive trophoblast. MATERIAL AND METHODS: DC were cultivated from monocytes isolated from peripheral blood mononuclear cells. DC were cocultured with K-562 leukemia cells lacking the class I and II HLA antigens transfected with either HLA-G or HLA-E or ultratransfected cells (controls) and the concentrations of IL-8, IL-10, IL-12p70, IL-18 and TNF-alpha were measured in the supernatants by ELISA. RESULTS: Coculture with ultratransfected cells resulted in a significant increase of the production of IL-8 and TNF-alpha by mature and immature DC and of IL-10 by immature DC (p < 0.01). When cocultured with HLA-G and HLA-E transfected K-562 cells, the secretion of IL-8 by immature and mature DC and that of IL-10 and TNF-alpha by immature DC was significantly (p < 0.01) decreased. The contact with HLA-G and HLA-E transfected cells had no effect on the production of IL-12p70 and IL-18 by DC. CONCLUSIONS: These results show that DC react with an increased cytokine release upon contact with cells lacking HLA class I and II antigens. The suppressive effect of HLA-G and HLA-E on the secretion of TNF-alpha (Th1 cytokine), IL-10 (Th2 cytokine) and IL-8 (chemokine) by immature DC could be interpreted as further evidence for the central immunotolerance role of HLA-G and HLA-E during early pregnancy.

[First experiences with human blastocyst culture after IVF/ICSI under the conditions of the German embryo protection law]. / Erste Erfahrungen mit der Blastozystenkultur nach IVF oder ICSI unter den Bedingungen des Deutschen Embryonenschutzgesetzes.

OBJECTIVE: Due to the improvements in human embryo culture in the recent years, it is now possible to transfer embryos five days after oocyte retrieval and IVF or ICSI at the blastocyst stage with favorable implantation rates. In Germany it is illegal to cultivate more than 3 embryos, therefore the selection has to be done at the pronuclear stage. There we report our experiences of human blastocyst culture in a routine IVF/ICSI programme under the conditions of the German Embryo Protection Law. MATERIALS AND METHODS: The data of 100 couples undergoing the IVF-ICSI programme at the University Clinic of Würzburg were analysed prospectively. 14-18 hours after insemination or micro-injection two or three zygotes with the best pronuclear development were selected for further cultivation. Fertilized oocytes were cultured in sequential media and were then transferred into the uterus 5 days after oocyte recovery. The blastocysts were graded from 1-8. RESULTS: In 100 cycles a total of 859 oocytes were collected, of whom 663 were fertilized and reached the pronuclear stage (median fertilization rate 88.9 %). 251 zygotes were selected at the PN stage. 51 % of the selected zygotes achieved the blastocyst stage after 5 days (grade 1-5), 28 % were morulae (grade 6-7) and 21 % of the embryos arrested in their development (grade 8). The clinical pregnancy rate was 26 %. Women who conceived had a significant better development of blastocysts on day 5 (grade 4 versus grade 6, P < 0.01) than those not achieving pregnancy. CONCLUSIONS: In summary, under the current legal conditions in Germany, blastocyst culture cannot improve pregnancy rates as the rate of arrested embryos of over 20 % limits the chances of implantation.

The use of a detailed zygote score after IVF/ICSI to obtain good quality blastocysts: the German experience.

BACKGROUND: Due to improvements in embryo culture, it is now possible to transfer embryos 5 days after oocyte retrieval and IVF/ICSI at the blastocyst stage, giving a better synchronization with the female reproductive tract. In Germany it is illegal to culture more than three embryos. Therefore, there is need for a sufficient selection at the pronuclear (PN) stage to select the best zygotes and exclude those of poor quality. METHODS: A prospective trial was conducted in 168 IVF and ICSI cycles including the size, number and alignment of pronuclei and nucleoli, cytoplasmic halo effect, the presence of vacuoles and granularity of ooplasm. Based on the above criteria, the best zygotes were selected (score <15) for embryo transfer on day 5. Blastocysts were classified in eight grades based on the cleavage speed. RESULTS: A total of 1450 oocytes were collected, of which 1119 reached the pronuclear stage. Of the zygotes (n = 424) selected at the PN stage, 46% achieved the blastocyst stage after 5 days (grade 1-5), 26% the morula stage (grade 6-7) and 28% were arrested (grade 8). The mean zygote score showed a significant positive correlation with the mean blastocyst quality in ICSI, but not in IVF cycles. A cut-off of 15 was calculated for ICSI cycles giving the best discrimination with blastocyst grades (6 versus 7) and number of arrested embryos (23 versus 45%) below and above this cut-off. A total of 33 clinical pregnancies was achieved (20%). Women conceiving had a significantly better mean blastocyst development than those not conceiving. Strong cytoplasmic vacuolization and an extreme or no halo effect had a negative effect on blastocyst development. CONCLUSIONS: The data show that PN stage morphology is related to blastocyst development, but the rate of arrested embryos of almost 30% limits the chance of conception under the conditions of the German Embryo Protection Law.

Th1- and Th2-like cytokine production by first trimester decidual large granular lymphocytes is influenced by HLA-G and HLA-E.

During normal early pregnancy, a particular immune environment in the decidua and the expression of non-classical HLA-G and HLA-E molecules on the invading trophoblast are assumed to be essential for the tolerance of the fetus. To assess whether HLA-G and HLA-E influence the cytokine production of their putative target cells [large granular lymphocytes (LGL)], we analysed the concentrations of tumour necrosis factor (TNF-alpha), interferon (IFN)-gamma, interleukin (IL)-10, IL-13 and granulocyte-macrophage colony stimulating factor (GM-CSF) in supernatants of isolated first trimester LGL co-cultured with HLA-G or HLA-E transfected K-562 leukaemia cells lacking the classical HLA class I and II molecules. In comparison with that observed with untransfected K-562 cells, co-culture of LGL with HLA-G-expressing cells significantly reduced the concentration of all cytokines investigated (TNF-alpha, IL-10 and GM-CSF, P < 0.01; IFN-gamma and IL-13, P < 0.05). In contrast, co-culture of LGL with HLA-E-expressing cells significantly (P < 0.01) decreased only IL-10 production, although a strong tendency towards reduced IL-13 levels was also observed. In the co-culture system presented, membrane-bound HLA-G and, to a lesser extent, HLA-E expression affected cytokine release by decidual LGL in a manner not consistent with the Th1/Th2 paradigm. In conclusion, our data are indicative of a general immune-suppressive effect of HLA-G on LGL activity.

Octreotide LAR treatment throughout pregnancy in an acromegalic woman.

We report a 24-year-old woman with active acromegaly despite pituitary surgery and irradiation who received continuous octreotide LAR treatment for the control of GH excess throughout her pregnancy. The patient delivered a healthy girl following an uneventful pregnancy. Despite a substantial materno-fetal transfer of octreotide, postnatal development was normal with length parameters around the 50th percentile at 3 months of age. In almost all previously described cases (n = 13) octreotide was stopped after the diagnosis of pregnancy was established. No side-effects of mother or fetus have been reported. Octreotide treatment in pregnancy seems to be feasible and safe. Due to the still-limited number of reported cases, the potential benefits of octreotide treatment should be weighed carefully against its possible risks.

Concentrations of copper thought to be toxic to Escherichia coli can induce the viable but nonculturable condition.

We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of Escherichia coli to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.

The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection.

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.

[Immunotherapy for prevention of abortion and for improving implantation in extracorporeal fertilization]. / Immuntherapie zur Abortprophylaxe und zur Verbesserung der Implantation bei der extrakorporalen Befruchtung.

In women with a history of recurrent in-vitro fertilization failure (at least three transfers of good quality embryos), there is an increased prevalence of organo-specific and not organo-specific autoantibodies, similar to women with a history of recurrent miscarriage. It is still unknown whether these autoantibodies are causally related to the failure of implantation or merely reflect an underlying systemic disorder. Several therapeutic intervention for the improvement of implantation after in-vitro fertilization and embryo transfer have been tried, including prednisolone (10 mg/d), heparin and aspirin (80-100 mg/d) alone or in combination with immunoglobulins and active immunotherapy using partner's leukocytes. At present, these interventions in combination with IVF should be regarded as experimental, as controlled, randomized and prospective trials in a large number of couples are still lacking.

Semen sample collection in medium enhances the implantation rate following ICSI in patients with severe oligoasthenoteratozoospermia.

Ejaculation in medium increases the proportion of antibody-free spermatozoa in semen samples containing anti-sperm antibodies and thereby enhances the fertilization rate in vitro. The aim of this study was to investigate whether this technique is also beneficial in semen samples with severe oligoasthenoteratozoospermia (OAT) where bacteria and detritus are often present. A prospective randomized controlled trial was carried out to study the results of sperm preparation and fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) for OAT. Of the 114 couples (one cycle per couple) studied between 1998 and 2000, 55 men were randomized to have semen collection into sterile dry pots (group A) and the remaining 59 had samples collected into 20 ml HEPES buffered Ham's F-10 medium with 10% human serum albumin (group B). In group B the ejaculates were incubated for 30 min and mixed gently. The samples were then processed by mild centrifugation and washing followed by a mini-swim-up technique. The ejaculates in group A were prepared by the swim-up procedure only. The overall fertilization rate was 71.8% and was similar in groups A (fertilization rate = 66.7%) and B (fertilization rate = 64.3%). In group A, 10/55 clinical pregnancies were recorded (pregnancy rate 18%), with an implantation rate (IR) of 6.9% per embryo. In group B, 16 of 59 patients conceived leading to significantly higher implantation (9.9%, P < 0.001) and clinical pregnancy rates (27%, P < 0.001). It is postulated that the addition of medium before liquefaction could inhibit the binding of bacteria and detritus to the sperm surface and may diminish DNA damage caused by reactive oxygen species, leading to improved efficiency of fertilization. The results demonstrate that the addition of HEPES buffered Ham's F-10 medium to sample collection pots significantly improves the pregnancy rate after ICSI in patients with severe OAT.

Assessment of endocrine status in patients undergoing in-vitro fertilization treatment. Is it necessary?

Endocrine evaluation as a prerequisite for every patient undergoing routine in-vitro fertilization (IVF) and embryo transfer for tubal or male factor infertility is still a matter of debate. The aim of this study was to determine if a full endocrine work-up, including the measurement of androgens, gonadotropins, prolactin and TSH, is conclusive for the subsequent success in IVF/ET. 71 infertile women without known endocrinopathies (e.g., polycystic ovarian disease), who were scheduled to enter the IVF/ET program were studied under strictly standardized conditions during the follicular phase of a natural cycle. Fasting serum concentrations of follicle stimulating hormone, luteinizing hormone, oestradiol, progesterone, prolactin, testosterone (T), dehydroepiandrosterone, 17-OH-progesterone, androstenedione and thyroid stimulating hormone (TSH) were measured using commercially available radioimmunoassays. Ovarian stimulation was performed by a long gonadotrophin-releasing hormone agonist/human menopausal gonadotrophin protocol. The overall clinical pregnancy rate was 15.5% in the first started IVF cycle. While patients who conceived in the first treatment cycle had significantly lower T levels (368 +/- 49 pg/ml) than those who did not (518 +/- 27 pg/ml, p=0.042, Kruskal & Wallis H-test), but the percentage of women with elevated T concentrations was not different. Similarly, TSH concentrations were significantly higher in women with a clinical pregnancy (1.9 +/- 0.2 mU/ml) than in non-pregnant women (1.4 +/- 0.3 mU/ml, p=0.046), but levels were still within the normal range. There were no further significant differences in hormone levels between pregnant and non-pregnant patients. These results do not suggest the measurement of a full hormonal profile in all infertile women before IVF/ET in non-endocrine infertility, taking into account the low likelihood to identify endocrinological disturbances, the considerable cost of endocrine testing and the paucity of therapeutic consequences.

[Effects of prepartum infusion solutions on glucose and bilirubin metabolism of mother and child in the prepartum and postpartum period]. / Auswirkungen von präpartal verabreichten Infusionslösungen auf den Zucker- und Bilirubinstoffwechsel von Mutter und Kind peri- und postpartal.

BACKGROUND: Administration of glucose 5% infusion is regularly used in obstetrics. The purpose of the study was to investigate the effects of glucose as compared to xylose and electrolyte solutions on parameters of maternal and fetal glucose and bilirubin metabolism during labour and after delivery. PATIENTS AND METHODS: 53 pregnant women (> or = 37 weeks of gestation, uncomplicated pregnancies) were randomised by entering the delivery ward. Under labour either glucose 5%, xylose 5% or electrolyte infusions were administered. Maternal serum glucose, serum osmolarity, insulin and glucagon were analysed before administration and 20 minutes after delivery. In the newborn blood osmolarity and serum glucose levels were analysed in the umbilical cord directly after birth and in capillary blood samplings 2 hours after birth. RESULTS: Maternal blood glucose levels 20 min. post partum were significantly different (p < 0.05). Maternal insulin and glucagon concentrations 20 min. pp showed similar trends with glucose levels but were not significantly different. Glucose levels in the umbilical cord were significantly higher in the glucose than in the electrolyte group, but not higher than in the Xylit group. In contrast, the glucose-levels in the newborns after 2 h were significantly higher in the glucose group compared with both other groups. No significant differences were observed in bilirubin levels. Osmolarity in the umbilical cord between groups differed significantly. CONCLUSIONS AND DISCUSSION: In conclusion, the administration of different solutions showed a distinct influence on the maternal and neonatal glucose metabolism. A significant impact on the bilirubin levels could not be shown in this study.

Viable but nonculturable uropathogenic bacteria are present in the mouse urinary tract following urinary tract infection and antibiotic therapy.

Involvement of the viable but nonculturable (VBNC) condition in recurrent urinary tract infections (UTIs) was investigated. VBNC bacteria are those which are alive but do not give rise to visible growth under nonselective growth conditions. Urine, bladder, and kidney samples collected over a 2-month period from BALB/c mice inoculated with the uropathogenic Escherichia coli strain J96 were examined to determine the level of culturable and viable bacteria. Urine from uninoculated mice was found to contain more viable than culturable bacteria. Inoculated mice had a transient increase in the level of culturable forms of the uropathogen in their urine, followed by a decrease to background levels; they also had multiple log higher levels of viable cells than culturable cells. The culturable pathogenic bacteria in mice that were inoculated and received antibiotic treatment dropped to undetectable levels within 1 week. At 2 out of 12 subsequent time points spanning an additional 65 days, culturable forms of the inoculated pathogenic bacteria were recovered. Polymerase chain reaction (PCR) analysis confirmed that DNA from the inoculated bacteria was present in a sample that yielded no culturable bacteria. These data indicate that the inoculated uropathogenic E. coli was not eliminated by antibiotic therapy, and suggest that these bacteria may escape detection by current standard culturability assays because they are VBNC.

Constrained profile retrieval applied to the observation mode of the michelson interferometer for passive atmospheric sounding.

To investigate the atmosphere of Earth and to detect changes in its environment, the Environmental Satellite will be launched by the European Space Agency in a polar orbit in October 2001. One of its payload instruments is a Fourier spectrometer, the Michelson Interferometer for Passive Atmospheric Sounding, designed to measure the spectral thermal emission of molecules in the atmosphere in a limb-viewing mode. The goal of this experiment is to derive operationally vertical profiles of pressure and temperature as well as of trace gases O(3), H(2)O, CH(4), N(2)O, NO(2), and HNO(3) from spectra on a global scale. A major topic in the analysis of the computational methodology for obtaining the profiles is how available a priori knowledge can be used and how this a priori knowledge affects corresponding results. Retrieval methods were compared and it was shown that an optimal estimation formalism can be used in a highly flexible way for this kind of data analysis. Beyond this, diagnostic tools, such as estimated standard deviation, vertical resolution, or degrees of freedom, have been used to characterize the results. Optimized regularization parameters have been determined, and a great effect from the choice of regularization and discretization on the results was demonstrated. In particular, we show that the optimal estimation formalism can be used to emulate purely smoothing constraints.

[Fetal complications after failed pregnancy termination in the first trimester]. / Fetale Komplikationen nach misslungenem Schwangerschaftsabbruch im ersten Trimester.

BACKGROUND: Two different methods are in use for induction of abortion: either dilation and curettage (D & C) or medical termination with antigestagens and/or prostaglandins. The risk of continuing pregnancy after D & C ranges from 0.023 to 1% and is inversely correlated with gestational age. METHODS AND RESULTS: Several authors reported fetal anomalies (limb and oro-facial defects) in such cases. For the medical induction of abortion a combination of Mifepristone and Misoprostole is the treatment of choice in order to reduce the risk of ongoing pregnancy. Using single drug therapy carries an increased risk of failure and of subsequent fetal anomaly. There seems to be an increased incidence of limb defects and Möbius' syndrome in fetuses born after failed termination of pregnancy, but normal fetal development is entirely possible. CONCLUSION/DISCUSSION: The patients concerned must be informed in detail of the risks and probable consequences which must be included in the counselling concept of the treating gynaecologist.

Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles.

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g. trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.