Ex vivo culture of CD34+/Lin-/DR- cells in stroma-derived soluble factors, interleukin-3, and macrophage inflammatory protein-1alpha maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1alpha (MIP-1alpha). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin-/DR- cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin-/DR- cells assayed in SNC cultures supplemented with IL-3 and MIP-1alpha. When CD34+/Lin-/DR- progeny from the SNC culture were plated sequentially into "NK cell progenitor switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3- NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1alpha directly to "NK cell differentiation" conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33- cells present after SNC cultures with IL-3 and MIP-1alpha, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33- population similar to fresh sorted CD34+/Lin-/DR- cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1alpha, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.     

Dynamics of antigenemia and coproantigens during a human Fasciola hepatica outbreak

In the present study the dynamics of antigenemia and coproantigens were studied in patients with Fasciola hepatica infection during an outbreak occurring in La Palma, Pinar del Río, in the West Province of Cuba. Stool and serum samples were collected from 67 patients and 40 healthy subjects. Stool samples were studied by a simple gravity sedimentation technique and an ES78 sandwich enzyme-linked immunosorbent assay (ELISA) for observation of eggs and detection of parasite coproantigens, respectively. Serum samples were also studied by the ES78 sandwich ELISA and an indirect ELISA to detect circulating antigens and antibodies, respectively. At the beginning of the study, 8 of 67 patients had patent infections and 59 had prepatent infections, which was determined by the recent consumption of lettuce contaminated with metacercariae of F. hepatica, the presence of clinical symptoms, and the absence of Fasciola eggs in their stools. Patients with prepatent infections were monitored by all techniques until patency. Circulating antigens were not detected in patients with patent infections. However, coproantigens were clearly detected in all patients with patent infections. On the other hand, 28.8% of patients with prepatent infections tested positive for circulating antigens and 81.4% tested positive for coproantigens in the first stool sample studied. Only two other coproantigen determinations were necessary to diagnose 93.2% of the patients. While circulating antigen levels diminished in all patients during the infection, coproantigen levels increased. The present study demonstrates that the ES78 sandwich ELISA is a better tool than parasitological examination for diagnosis of active early infection, since by the combination of the circulating-antigen detection assay and the coproantigen detection assay 91% of patients were able to be diagnosed at the beginning of the study. In contrast, a coprologic analysis repeated over several weeks was necessary to diagnose 100% of the patients.     

Congestive heart failure in the community: a study of all incident cases in Olmsted County, Minnesota, in 1991

BACKGROUND: Data are limited regarding the classification and prognosis of patients with congestive heart failure (CHF) in the community. METHODS AND RESULTS: Using the resources of the Rochester Epidemiology Project, we evaluated all patients receiving a first diagnosis of CHF in Olmsted County, Minnesota, in 1991 (n=216). Among these patients, 88% were >/=65 years and 49% were >/=80 years of age. The prognosis of patients with a new diagnosis of CHF was poor; survival was 86+/-2% at 3 months, 76+/-3% at 1 year, and 35+/-3% at 5 years. Of the 216 patients, 137 (63%) had an assessment of ejection fraction. In these patients, systolic function was preserved (ejection fraction >/=50%) in 59 (43%) and reduced (ejection fraction <50%) in 78 (57%). Survival adjusted for age, sex, NYHA class, and coronary artery disease was not significantly different between patients with preserved and those with reduced systolic function (relative risk, 0.80; P=0.369). ACE inhibitors were used in only 44% of the total population with CHF. CONCLUSIONS: The present study reports the clinical characteristics and natural history of CHF as it presents in the community in the vasodilator era. CHF is a disease of the "very elderly," frequently occurs in the setting of normal ejection fraction, and has a poor prognosis, regardless of the level of systolic function. Diagnostic and therapeutic methods are underused in the community.     

Study of dosimetry consistency for kilovoltage x-ray beams

In this paper, the consistency of kilovoltage (tube potentials between 40 and 300 kV) x-ray beam dosimetry using the "in-air" method and the in-phantom measurement has been studied. The procedures for the measurement of the central-axis depth-dose curve, which serve as a link between the dose at the reference depth to the dose elsewhere in a phantom, were examined. The uncertainties on the measured dose distributions were analyzed with the emphasis on the surface dose measurement. The Monte Carlo method was used to calculate the perturbation correction factors for a photon diode and a NACP plane-parallel ionization chamber at different depths in a water phantom irradiated by 100-300 kV (2.43 mm Al-3.67 mm Cu half-value layer) x-ray beams. The depth-dose curves measured with these two detectors, after correcting for the perturbation effect (up to 15% corrections), agreed with each other to within 1.5%. Comparisons of the doses at the phantom surface and at 2 cm depth in water for photon beams of 100-300 kV tube potential obtained using the "backscatter" method and those using the "in-phantom" measurement have shown that the "in-air" method can be equally applied to this energy range if the depth-dose curve can be measured accurately. To this end, measured depth ionization curves require depth-dependent correction factors.     

Variations in adductor spasmodic dysphonia: acoustic evidence

The development of a validated primate model of progressive parkinsonism is a critical step in the evaluation of drugs that might halt or slow progression of Parkinson's disease. In this pilot study, we gradually exposed 14 cynomolgus monkeys to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), at a weekly dose of 0.5 mg/kg s.c. for 10 weeks, to determine their probability of not reaching a predetermined endpoint on a disability scale by Kaplan-Meier analysis. Four other MPTP-exposed animals were coadministered the potent free radical scavenger 7-hydroxy-1-[4-(3-methoxyphenyl)-1-piperazinyl]acetylamino-2,2,4,6- tetramethylindan (OPC-14117) as a single oral daily dose of 0.6 g/kg, starting 2 weeks before MPTP initiation. The risk of reaching endpoint by week 10 was 79% and mean time before reaching endpoint was 6 weeks. Global motor activity, recorded in a subset of animals using a portable activity monitor, declined following the first MPTP dose and never recovered. Several cerebrospinal fluid indices of central monoamine metabolism collected by suboccipital puncture at 0, 5, and 10 weeks, including HVA, DOPAC, and tetrahydrobiopterin but not MHPG, were found to be "trait" markers for MPTP exposure, whereas CSF DOPAC and tetrahydrobiopterin constituted potential "state" markers for reaching endpoint. The antioxidant OPC-14117 did not protect against MPTP-induced parkinsonism. Further attempts to validate this incremental model of neurotoxin-induced parkinsonism as a predictor of patient responses to putative neuroprotective agents appear warranted.     

Comparison of 51Cr-EDTA with 99m-Tc-DTPA slope clearance for estimation of glomerular filtration rate using the one compartment model

AIM: Of this study is to determine the relationship between 51Cr-EDTA and 99mTc-DTPA slope clearance applying the "one-compartment model". METHODS: The "one-compartment model" was chosen to calculate and to compare the glomerular filtration rates of 25 patients with normal and pathological creatinin values after injection of 51Cr-EDTA and 99mTc-DTPA simultaneously. RESULTS: The two clearance values correlated well (r = 0.996), and the 99mTc-DTPA clearance was systematically higher (28%). The 99mTc-DTPA was calculated and compared after taking three plasma samples. Taking two samples, only minor differences were seen and the correlation was high (r = 0.992). CONCLUSION: The results of this study encouraged us to adopt the use of 99mTc-DTPA instead of 51Cr-EDTA in determining the glomerular filtration applying the "one-compartment model" in slope with two plasma samples.     

Ventral subiculum regulates hypothalamo-pituitary-adrenocortical and behavioural responses to cognitive stressors

The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined. This structure shows important differences from the lower resolution structure deposited in 1982. The crystal used to determine the new structure was of a different form, having spacegroup P2(1). The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction. The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts. Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides. The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase. However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal. An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge. The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure. Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.     

Estimating risk associated with care in alternative settings: deterioration among children hospitalized

The incidence of ductal carcinoma in situ (DCIS) has increased with the widespread use of screening mammography. DCIS is often suspected when clustered microcalcifications are evidenced on routinely performed mammography. High quality mammographies are required and should be completed with magnification views. Mammographic--pathologic correlations are described according to the new classifications as well as unusual forms of presentation on mammography. Early contrast enhancement in DCIS on dynamic MRI is reported and seems to be related with angiogenesis. A wire localization procedure of non-palpable lesions has to be performed and per-operative specimen radiography is mandatory. Stereotaxic large core needle biopsy is a valuable alternative to surgical biopsy but a multidisciplinary team approach is necessary and follow-up is recommended if no excisional biopsy is done. Quality in the management of DCIS depends on the coherence of the "multidisciplinary team".     

Oxyanion-mediated inhibition of serine proteases

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].