Grouping of hantaviruses by small (S) genome segment polymerase chain reaction and amplification of viral RNA from wild-caught rats. A single pair of consensus primers in the polymerase chain reaction (PCR) amplified a conserved region of the small genome segment of twenty hantavirus isolates. Isolates tested included representatives of the four recognized hantaviruses, Hantaan, Seoul, Puumala and Prospect Hill, as well as isolates from Mus musculus (Leakey), Bandicota indica (Thailand 749), and Suncus murinus (Thottapalayam). Viruses from the Nairovirus and Phlebovirus genera yielded negative results. The amplification products were 281-nucleotide pairs (np) in length, with the exception of Thottapalayam, which had an amplification product of approximately 320 np. Products of all isolates were detected by Southern hybridization with a 32P-labeled Hantaan 76-118 amplicon, while an oligonucleotide probe to a conserved region of the amplified fragment failed to detect some isolates of Seoul and Puumala viruses. Restriction endonuclease analysis allowed three groupings: Hantaan-like viruses, Seoul-like viruses, and a diverse group of patterns for the other viruses. Differences were found within the Seoul-like virus group by this method, whereas the Hantaan-like viruses were shown to be similar. RNA extracted from tissues of seropositive and seronegative rats trapped in Baltimore showed the practical application of the test. Hantavirus-specific RNA was detected in 12 (92%) of 13 seropositive rats, but not in seronegative rats. This simple method for detecting and characterizing hantaviruses has potential for epidemiologic studies and for diagnosing human hantavirus infections.


Dendrocyte population in cutaneous and extracutaneous Kaposi's sarcoma. In order to investigate the hypothesis that the spindle cells of Kaposi's sarcoma originate from dendrocytes, we stained 22 cases of cutaneous and extracutaneous Kaposi's sarcoma for the presence of factor XIIIa. We selected eight lesions from the skin, five from the oral mucosa, four from gastrointestinal mucosa, three from lymph nodes, and one each from esophagus and conjunctiva for this study. The majority of cutaneous and lymph node Kaposi's sarcoma lesions had a considerable number of dendrocytes admixed with the spindle tumor cells. Of the five oral mucosal lesions only three showed a focal presence of dendrocytes. No dendrocytes were observed in the lesions biopsied from the gastrointestinal mucosa, esophagus, and conjunctiva. Our findings suggest that the proliferation of dendrocytes seen within the lesions of Kaposi's sarcoma in the skin and lymph nodes probably represents a nonspecific host response in organs where they are normally present, and it is unlikely that they constitute the neoplastic cells of Kaposi's sarcoma.


Multinucleate cell angiohistiocytoma.

Multinucleate cell angiohistiocytoma. We report a case of multinucleate cell angiohistiocytoma occurring in a 50-year-old woman and presenting with a 4-year history of asymptomatic red papules on the face. Histologically, there was a dermal vascular proliferation associated with numerous multinucleate cells. Immunohistochemical studies showed vascular spaces surrounded with mature vascular endothelial cells, and the presence of numerous interstitial factor XIIIa+ cells. Multinucleate cells exhibited intermediate filaments of the vimentin type, but were not stained by endothelial cell or macrophage markers, and were factor XIIIa-. This condition must be addressed in the differential diagnosis of other skin vascular proliferations, such as Kaposi's sarcoma and angiofibromas.


FMRFamide-like immunoreactivity in the brain and pituitary of the goldfish, Carassius auratus. The distribution of FMRFamide-like immunoreactivity was determined in the brain and the pituitary of the goldfish, Carassius auratus. Immunoreactive perikarya were observed in the olfactory nerve, nucleus entopeduncularis, nucleus anterioris periventricularis, nucleus posterioris periventricularis, lateral part of the nucleus lateralis tuberis pars posterioris, midbrain tegmentum, and medulla oblongata. Immunoreactive fibers were widely distributed in the brain, in particular in the ventral telencephalon and the hypothalamus. A few immunoreactive nerve fibers were observed in the pituitary. The findings are discussed in relation to male sexual behavior and the involvement of FMRFamide-like peptide in pituitary functions in the goldfish.


Multiple restriction fragment length polymorphisms in the porcine calcium release channel gene (CRC): assignment to the halothane (HAL) linkage group. Two cDNA probes for the porcine calcium release channel gene (CRC) were used in restriction fragment length polymorphism (RFLP) analysis in an attempt to develop genetic markers linked to the malignant hyperthermia (stress susceptibility) gene (HAL). Three TaqI RFLPs, denoted CRC1-CRC3, each composed of two alleles, were detected. RFLPs were also detected with MspI and PvuII, but the MspI RFLP correlated completely with CRC3 in this material and the PvuII RFLP could not be scored reliably due to a minute size difference between the two allelic fragments. The autosomal codominant inheritance of these RFLP loci was confirmed by family analyses. Significant evidence for genetic linkage between the CRC1/CRC3 loci and the A1BG locus in the HAL linkage group confirmed a previous assignment of the CRC gene to chromosome 6 in the pig.


Restriction fragment length polymorphism analysis of major histocompatibility complex class IV (B-G) genotypes in meat-type chickens. Restriction fragment length polymorphism (RFLP) was used as a molecular genotyping approach to characterize differences in major histocompatibility complex class IV genes in meat-type chickens. A high level of polymorphism was observed following digestion with each of the two restriction endonucleases PvuII and BglII. Examination of DNA from 54 chickens revealed 23 polymorphic fragments. Application of RFLP techniques in the analysis of family groups should make possible the determination of B-G genotypes in the meat type chickens.


Listeriosis in patients with chronic lymphocytic leukemia who were treated with fludarabine and prednisone. To determine whether therapy with fludarabine plus prednisone in patients with chronic lymphocytic leukemia increases the risk for developing listeriosis. Retrospective cohort study based on hospital surveillance data. Referral cancer center. A total of 795 patients with chronic lymphocytic leukemia who received care at The University of Texas M. D. Anderson Cancer Center between 1980 and 1990. Patients were treated with fludarabine alone or fludarabine and prednisone. The listeriosis attack rate was analyzed according to the type of treatment received. Seven of 408 patients in the fludarabine group developed listeriosis (1.7%; 95% Cl, 0.2% to 6%) compared with 0 of 387 patients who received conventional chemotherapy alone (0% to 0.9%; P = 0.015). The 7 patients were among 248 patients who were treated with both fludarabine and prednisone; none of 160 patients treated with fludarabine alone developed listeriosis (P = 0.045 by the Fisher exact test). A dramatic reduction in CD4 counts occurred in patients after fludarabine and prednisone treatment and coincided with the development of listeriosis. The administration of fludarabine plus prednisone is associated with an increased incidence of listeriosis in patients with chronic lymphocytic leukemia. The depletion of CD4 cells may underlie the pathogenesis.


Mitochondrial and nuclear variants in a U.S. black population: origins of a hybrid population. The maternal transmission of mitochondria in higher eukaryotes makes it possible to distinguish between reciprocal matings, since offspring possess the mitochondrial DNA variants received from the mother. This possibility can be extended to hybrid populations, the mitochondrial frequencies reflecting the relative maternal contributions from the parental populations. Nuclear variations reflect the relative genetic contributions of the parental populations, irrespective of parental sex. The U.S. Black population is a hybrid of West African and European populations. The African-European matings that contributed to the present Black population are traditionally considered to have been almost exclusively between African females and European males. We have studied nuclear and mitochondrial variants in a sample of U.S. Blacks, comparing them with published frequencies from African and Caucasian groups. Our results suggest that the mitochondria of present-day American Blacks are derived from Caucasians to an extent similar to nuclear genes. In contrast to traditional beliefs, the contribution from Caucasian females is of the same magnitude as that from Caucasian males.


The proliferative fraction in lymph nodes: a comparison of proliferating cell nuclear antigen morphometry to flow cytometry. A monoclonal antibody to proliferating cell nuclear antigen (PCNA/cyclin) has recently become available. This antibody, as opposed to Ki-67, can be used on formalin-fixed, paraffin embedded tissue sections and allows retrospective comparison of PCNA positivity to percent S + G2 + M of the cell cycle. To compare fresh lymphoma deoxyribonucleic acid (DNA) analysis with PCNA activity on fixed, paraffin-embedded sections, prospective flow cytometric studies of cell cycle analysis were performed on lymph nodes removed from 10 patients for diagnosis. Six patients had T-cell lymphoma, two had B-cell lymphoma, and two were benign. Using the peroxidase-antiperoxidase method, the nuclear positivity of archival lymphoma cases was also examined. To quantify PCNA positivity, a unique morphometric method was employed that utilized digital imaging by high definition television and ELAS (Earth Land Application Software), a geoscience software used extensively for color quantitation of remote sensed data. The immunologic percent PCNA positivity was 26.1 +/- 20 vs. percent S + G2 + M by flow cytometry of 22.4 +/- 10 with a correlation coefficient (r) of 0.55. This r-value compared favorably to data generated for Ki-67 in solid malignant neoplasms. The six more concordant cases had a percent PCNA positivity of 26.5 +/- 10.0 and a percent S + G2 + M of 27.3 +/- 8.6, r = 0.96. Our study is unique in that it compared fresh lymphoma DNA analysis data with paraffin PCNA data. It is our conclusion that immunologic PCNA positivity in paraffin sections correlates with fresh flow cytometric S + G2 + M in lymph nodes, although careful attention must be paid to the area of the node quantified for PCNA.


Pharmacokinetics of cefpodoxime proxetil and interactions with an antacid and an H2 receptor antagonist. Cefpodoxime proxetil is a new oral esterified cephem antibiotic with a broad antibacterial spectrum. The dissolution of cefpodoxime proxetil is pH dependent. The objectives of this study were to characterize the pharmacokinetics of cefpodoxime proxetil in two different oral doses and to examine possible interactions with an antacid, aluminum magnesium hydroxide (Maalox 70), and an H2 receptor antagonist, famotidine. Two studies involving the same 10 healthy volunteers were performed. In the first study, cefpodoxime proxetil was administered in two doses, 0.1 and 0.2 g. In the second study, two interventions were performed in a randomized crossover design. For one intervention, the volunteers were pretreated with 40 mg of famotidine 1 h before 0.2 g of cefpodoxime proxetil was administered. In the second trial, participants were given 10 ml of Maalox 70 2 h and 10 ml of Maalox 70 15 min before they received 0.2 g of cefpodoxime proxetil. Serum and urine concentrations were determined by high-performance liquid chromatography. For the statistical evaluation, these data were tested by using the pharmacokinetics of 0.2 g of cefpodoxime proxetil from the first study. The maximum concentrations were 1.19 +/- 0.32 mg/liter after 0.1 g of cefpodoxime proxetil and 2.54 +/- 0.64 mg/liter after 0.2 g of cefpodoxime proxetil. The elimination half-lives were 149 min for 0.1 g and 172 min for 0.2 g of cefpodoxime proxetil. The total increase in the area under the concentration-time curve (AUC) was dose dependent. Combination with Maalox 70 caused a reduction in the AUC from 14.0 +/- 3.9 to 8.44 +/- 1.85 mg.h/liter. After famotidine, the AUC decreased to 8.36 +/- 2.0 mg . h/liter. Corresponding changes were registered for the maximum concentration of drug in serum, 24-h urine recovery, and the time to maximum concentration of drug serum. Cefpodoxime proxetil was well tolerated without any seriously adverse drug reactions.


Cerebrospinal fluid analysis in human immunodeficiency virus infection. Cerebrospinal fluid (CSF) analytes were evaluated in 59 human immunodeficiency virus (HIV+) individuals to assess neurological involvement. Glucose, total protein, cell counts, p24 antigen, CSF: serum albumin/IgG ratios, and oligoclonal bands were measured. Eighty percent of samples showed abnormalities in one or more analyte. In some patients samples, these abnormalities could mimic those of secondary opportunistic infection when none was present. The presence of oligoclonal banding in CSF (31 percent) and disturbances in CSF: serum albumin/IgG ratio (30 percent) were related to decreases in serum CD4+ lymphocytes. Disturbances in CSF: Serum albumin/IgG ratio were also related to severity of non-neurological HIV disease staging. Cerebrospinal fluid oligoclonal bands were distinct from that found in serum in the same subjects. Since immune complexes between immunoglobulins and enzymes are observed in these same patients, these oligoclonal bands may result in artifactually elevated enzyme results secondary to decreased clearance leading to erroneous clinical decisions. There was no significant relationship between any abnormalities and the presence of neurologic disease as established by a wide variety of other studies. It is important to recognize the limits of CSF interpretation in this patient group.


Pregnancy in aplastic anemia treated with fetal liver and bone marrow hemopoietic cells and antithymocyte globulin. This report describes a patient who had severe refractory anemia and thrombocytopenia during her first pregnancy. She had a remission after delivery but two years later aplastic anemia refractory to steroid and splenectomy developed. After treatment with steroids and antithymocyte globulin the patient had repeated infusions of hemopoietic cells derived from fetal liver and bone marrow, leading to a 7-year remission during which time she had a further successful pregnancy. The patient had a relapse one year later.


Expression of several resistance mechanisms in untreated human kidney and lung carcinomas. Thirty human renal cell carcinomas and 94 non-small cell lung carcinomas of previously untreated patients were analyzed for the presence of P-glycoprotein, glutathione S-transferase-pi and topoisomerase II by means of immunohistochemistry. In the renal cell carcinomas investigated, two resistance markers were seen in 53% and three resistance markers in 36% of the cases. In only three tumors was one resistance mechanism observed. In the 94 non-small cell lung carcinomas 34% had two and 20% three resistance mechanisms, whereas 24% of the tumors revealed only one resistance mechanism. For determining the resistance of the tumors against drugs an in vitro short-term test was used. Only 12% of the sensitive lung tumors had more than one resistance mechanism, whereas 70% of the resistant tumors did. Thus a significant relationship exists between the resistance measured in vitro and the overexpression of P-glycoprotein or glutathione S-transferase-pi and the down-regulation of topoisomerase II.


The advantages of pylorus-preserving pancreatoduodenectomy in malignant disease of the pancreas and periampullary region. The aim of this study was to establish whether the pylorus-preserving pancreatoduodenectomy (PPPD) is a safe and radical procedure in malignant disease of the head of the pancreas and periampullary region, without increased morbidity and mortality rates compared with the standard Whipple's procedure. During the period 1984 to 1990, a Whipple's procedure (n = 44) or PPPD (n = 47) was performed in 91 patient. In-hospital mortality rates were 2% after PPPD and 5% after Whipple's procedure. Median duration of the resection procedure and median blood loss in the PPPD group were 210 minutes and 1800 mL, respectively. After Whipple's procedure, these figures were 255 minutes and 2500 mL, both significantly different (p less than 0.01) as compared with PPPD. No difference was found during follow-up with respect to days of gastric suctioning, start of liquid diet, normal diet, complaints of ulcer disease, postoperative complications, recurrence of disease, and survival. In all patients, curative resection was performed with comparable TNM (tumor, nodes, metastases) staging. The number of tumor-containing duodenal or gastric resection margins did not differ in both groups of patients (two patients after PPPD, two patients after Whipple's procedure). Hospital stay was significantly (p = 0.02) shorter after PPPD; median 14 days, compared with median 18 days after Whipple's procedure. The advantage of the PPPD is that it is an easier and less time-consuming operation, with less blood loss, a shorter hospital stay, and better weight gain (p = 0.02) during follow-up. In conclusion, PPPD is a safe and radical procedure for cancer in the head of the pancreas or periampullary region, with the same survival and appearance of locoregional recurrence and distant metastases as after standard Whipple's resection.


The vertebrate limb: a model system to study the Hox/HOM gene network during development and evolution. The potential of the vertebrate limb as a model system to study developmental mechanisms is particularly well illustrated by the analysis of the Hox gene network. These genes are probably involved in the establishment of patterns encoding positional information. Their functional organisation during both limb and trunk development are very similar and seem to involve the progressive activation in time, along the chromosome, of a battery of genes whose products could differentially instruct those cells where they are expressed. This process may be common to all organisms that develop according to an anterior-posterior morphogenetic progression. The possible linkage of this system to a particular mechanism of segmentation as well as its phylogenetic implications are discussed.


DNA synthesis enzymes and proliferating cell nuclear antigen in normal and neoplastic nerve cells. The activity of nuclear DNA polymerases alpha, beta and delta/epsilon, uracil-DNA glycosylase, thymidine kinase and the presence of Proliferating Cell Nuclear Antigen (PCNA) have been examined in developing rat glial cells, in rat and human glioma, in human neuroblastoma and in differentiated neuroblastoma cell lines in vitro. During glial development the activity of all enzymes tested, except DNA polymerase beta, markedly decreased, suggesting their coordinate regulation in respect to the proliferative state of the cells. Glioma and neuroblastoma cell lines restore the enzymatic activities that were no longer expressed in normal adult cells. Neuroblastoma cell lines induced to differentiate in vitro by retinoic acid showed a decline of the activities of DNA polymerase alpha, DNA polymerase delta/epsilon, uracil-DNA glycosylase and thymidine kinase similar to that observed during in vivo differentiation. We also demonstrate that PCNA is not detectable in glial and neuronal cells at all developmental stages, but can be found in tumor nerve cells. A possible use of enzymatic assays or anti-PCNA antibodies to detect brain tumors is discussed.


A new restriction fragment length polymorphism of the ceruloplasmin gene in rat. Using a cDNA probe of the ceruloplasmin (CP) gene, a new restriction fragment length polymorphism (RFLP) was detected in inbred strains of rat with the restriction enzyme KpnI. The RFLP behaved as a codominant trait on a single autosomal locus. Two alleles of the CP gene, which were tentatively designated as CP-A and CP-B, were almost equally distributed in 10 inbred strains. These indicate that the RFLP of the CP gene is a useful marker locus of the rat. We utilized this CP gene polymorphism for the investigation of the pathogenesis of the aberrant hepatic copper metabolism in LEC mutant rat, since CP has a pivotal role in copper metabolism in the liver. Using backcross progenies originating from LEC and BN rats, we found that the CP gene is not associated with the excess hepatic copper accumulation and the deficiency in serum CP activity, both of which are congenital abnormal phenotypes exhibited in LEC mutant rat.


Amino acid transport in multidrug-resistant Chinese hamster ovary cells. In the process of assessing the effect of anthracycline drugs on cellular membrane function in cultured multidrug resistant (MDR) and its parental cells, experiments were undertaken to investigate the kinetics of neutral amino acid membrane transport (the sodium dependent A and ASC systems). P-glycoprotein, a high molecular weight energy requiring integral membrane protein responsible for actively pumping drugs out of cells, has been shown to be overexpressed in MDR cells. It was our hypothesis that its presence might affect other membrane energy requiring systems such as amino acid transport. On establishing the concentrations of P-glycoprotein by western blotting in the two cell lines to be studied, the kinetics of membrane transport of the neutral amino acids alpha-aminoisobutyric acid (AIB) and serine (SER) were investigated using the CHRC5 multidrug resistant and AUX B1 parental Chinese hamster ovary (CHO) cells. In CHRC5 cells, the amount and rate (Vmax) of accumulated amino acids, was significantly depressed when compared to AUX B1 cells, however, there was no difference in the rates of amino acid efflux between these two cell lines. Using 1,6-diphenyl 1,3,5-hexatriene (DPH) polarization to evaluate the state of membrane fluidity in the two cell lines studied, it was seen that CHRC5 cells showed a slightly lower degree of polarization than that observed in AUX B1 cells. These results suggest, that the P-glycoprotein does not alter amino acid transport directly but may modify the activity or numbers of functional transport carriers.


Synthesis of a 11-deoxyprostanoid in the area of preclavulones: (+-)-8,12-trans-(5Z-14Z)-9-oxo-prosta-5,14-dienoic acid from 2-allyl-2-cyclopenten-1-one. Following our research on the arachidonic acid metabolites and their derivatives with potential biological activity, we describe the synthesis of the (+-)-8,12-trans-(5Z, 14Z)-9-oxo-prosta-5,14-dienoic acid, a 11-deoxyprostanoid correlated to the class of Preclavulones, one of the unusual families of marine eicosanoids from the coral Clavularia Viridis with considerable biological interest.


Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes on in vitro transformation of human mammary epithelial cells. MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-alpha (TGF-alpha) gene in MCF-10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF-10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-10A Ha-ras and MCF-10A TGF-alpha cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-10A TGF-alpha/c-erbB-2 (MCF-10A TE) and 10 MCF-10A Ha-ras/c-erbB-2 (MCF-10A HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-10A TE and MCF-10A HE clones expressed a 10-fold to 40-fold increase in p185 erbB-2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF-alpha compared with MCF-10A cells. Moreover, both MCF-10A TE and MCF-10A HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-10A Ha-ras, MCF-10A c-erbB-2, or MCF-10A TGF-alpha clones. However, neither MCF-10A TE nor MCF-10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erbB-2, and TGF-alpha genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.


Selective impairment of long-term but not short-term conditional fear by the N-methyl-D-aspartate antagonist APV. Previous research has indicated that the competitive N-methyl-D-aspartate (NMDA) antagonist APV (DL-2-amino-5-phosphonovalerate) prevents the Pavlovian conditioning of fear to contextual stimuli when tested 24 hr, but not immediately, after training. The present study investigated this differential time-dependent effect of APV on fear conditioning. Rats were given either APV or saline and presented with 3 footshocks in a distinctive chamber. Promptly after the shock, rats that had received APV exhibited a species-typical fear response-freezing. However, the freezing lasted for only a short period of time (less than 3 min) compared with that of controls. An immediate-shock procedure showed that freezing was entirely a conditional response to the chamber. In addition, the results of a savings test suggest that APV impairs storage rather than retrieval processes. These results indicate that there are two temporally distinct associative fear processes, a short-term NMDA-independent conditional fear and a long-term NMDA-dependent conditional fear.


Delayed-nonmatching-to-sample performance is impaired by extensive, but not by limited, lesions of the thalamus in the rat. Two experiments were conducted to determine whether lesions affecting limited areas of the thalamus can impair the performance of rats on a spatial delayed-nonmatching-to-sample (DNMTS) task trained before surgery. In Experiment 1, DNMTS was not affected by lesions produced by injecting 5 microliters of 1 mM N-methyl-D-aspartate into either the midline thalamus (n = 16) or bilaterally 1.0 mm from the midline (n = 16). In experiment 2, radio-frequency lesions were made 1.0 mm lateral to the midline at 3 anterior-posterior locations that destroyed the full rostral-caudal extent of the lateral internal medullary lamina (L-IML; n = 8), or at single anterior-posterior locations that destroyed either the anterior (n = 8) or posterior (n = 8) portions of the L-IML site. Although complete L-IML lesions disrupted DNMTS performance to an extent comparable to that of another study (Mair & Lacourse, 1992), lesions that were restricted to either the anterior or posterior portion of the L-IML site had no significant effect on this task.


Equivalent recognition of HIV proteins, Env, Gag and Pol, by CD4+ and CD8+ cytotoxic T-lymphocytes. Cytotoxic T-lymphocytes (CTL) appear to be an important defense mechanism against HIV infection. This study proposes to examine the major histocompatibility complex (MHC)-restricted HIV-1 Env-, Gag- and Pol-specific CTL activities in HIV-infected asymptomatic patients. CD4+ and CD8+ CTL were examined to establish whether the same HIV-1 protein (Env, Gag or Pol) was recognized by both CD4+ and CD8+ CTL with MHC antigen restriction. Peripheral blood mononuclear cells, CD4+ and CD8+ T-cells from 17 HIV-infected asymptomatic patients and 10 HIV-seronegative individuals were examined for HIV-1 Env-, Gag- and Pol-specific MHC-restricted cytotoxicity using autologous and heterologous B-lymphoblastoid cell lines infected with vaccinia recombinant expressing HIV-1 Env, Gag and Pol proteins as targets. CD4+ and CD8+ CTL specific for the HIV-1 Env, Gag and Pol were demonstrated in the peripheral blood. DR4 and DQw2 were possible sites of MHC class II restriction of CD4+ CTL. Possible MHC class I restriction sites of CD8+ CTL included A2 and B8 for Env, A1 and A2 for Gag, and A2 and B8 for Pol antigen. These observations should help to define more precisely the nature and elements of protective immunity and to evaluate AIDS vaccine strategies.


Biphasic rate of CD4+ cell count decline during progression to AIDS correlates with HIV-1 phenotype. To determine the kinetics of decline of CD4+ lymphocytes in HIV-1-infected asymptomatic homosexual men. CD4+ lymphocytes were enumerated in a cohort of 187 HIV-1-infected initially asymptomatic homosexual men seen at 3-month intervals over 5 years. During follow-up, 45 men progressed to AIDS (excluding cases presenting with Kaposi's sarcoma). Correlation between rate of CD4+ cell decline and presence of a particular HIV-1 biological phenotype was analysed in 43 participants. CD4+ cell counts declined slowly and continuously in HIV-1-seropositive men who remained asymptomatic during follow-up. A biphasic CD4+ cell count decline was observed in the group who developed AIDS: the decline was slow and steady (5.6 x 10(6)/l per month, similar to that observed in the asymptomatic group) until 18 months before AIDS diagnosis, but became three to five times faster thereafter. Rapid CD4+ cell decline was significantly related to syncytium-inducing, fast-replicating HIV-1 isolates; during the period of slow and steady CD4+ cell count decline, non-syncytium-inducing isolates were predominant. At an average of 18 months preceding AIDS diagnosis, a three to fivefold increase in the rate of loss of CD4+ lymphocytes occurs, and may be related to the appearance of a more virulent HIV-1 phenotype.


Clinical and laboratory predictors of survival in Gambian patients with symptomatic HIV-1 or HIV-2 infection. To determine which clinical and immunological features of patients with symptomatic HIV-1 and HIV-2 infection best predict survival in The Gambia. All patients presenting to two hospitals in The Gambia between January 1987 and June 1990 with symptoms or signs suggesting chronic HIV infection were tested for HIV-1 and HIV-2 antibodies. Eighteen HIV-1 and 31 HIV-2-infected patients were recruited to the study, investigated intensively on admission and followed up until the end of 1990. Presenting clinical features, such as Karnofsky score, diagnosis of AIDS according to World Health Organization Bangui or Centers for Disease Control criteria and number of associated infections, together with five immunological measurements, as well as type of HIV infection, were related to length of survival using proportional hazard models fitted to Kaplan-Meier plots of survival times. Karnofsky score and diagnosis of AIDS were the best clinical predictors of survival. Type of HIV infection or number of associated infections did not predict outcome. The most powerful laboratory predictors were log(e) serum neopterin level, CD4 cell count and log(e) serum beta 2-microglobulin (beta 2M) level. The estimated median survival times (90% confidence interval) of the HIV-1 and HIV-2-infected were six (4-11) and 13 (9-20) months, respectively. These survival times do not differ significantly. The Karnofsky score and measurements of serum neopterin or beta 2M, which are easier and cheaper to perform than CD4 counts, may prove to be useful guides to prognosis for HIV infection in Africa.


Vapreotide, a somatostatin analogue, in cryptosporidiosis and other AIDS-related diarrhoeal diseases. To evaluate the efficacy and tolerance of vapreotide, a new somatostatin analogue, in the treatment of refractory AIDS-related diarrhoea. An open, non-comparative pilot trial. The trial was conducted in 10 medical centres in France. Thirty-four AIDS patients with chronic diarrhoea unresponsive to conventional antidiarrhoeal therapy were enrolled. Cryptosporidiosis was diagnosed in 21 out of 30 evaluable patients. Mean number of stools prior to therapy was 10.1 +/- 4.9 per day (range, 3-20 stools per day). After initial baseline studies, patients received subcutaneous vapreotide at escalating doses of 400 (23 patients) or 500 micrograms (seven patients), between two and six times daily. Efficacy was assessed after 14 days of therapy, when it was found to be effective. Responders were offered the opportunity to continue receiving therapy. Four patients demonstrated a complete response and 12 a partial response with greater than 50% reduction in daily stool emission. Fourteen patients did not respond to doses up to 2400 micrograms/day. Patients with conditions other than cryptosporidiosis had a significantly higher probability of response (P = 0.013), as did those with milder diarrhoea (less than 10 stools per day). Median duration of response was 1.5 months (range, 0.5-5 months); relapse occurred in five out of eight responders despite maintenance therapy. Toxicity was minimal. We conclude that AIDS patients with diarrhoea not caused by Cryptosporidium may benefit from vapreotide therapy.


Application of the World Health Organization system for HIV infection in a cohort of homosexual men in developing a prognostically meaningful staging system. Validation of a modified version of the recently proposed World Health Organization (WHO) staging system for HIV infection and disease in a cohort of homosexual men. Five hundred and thirty HIV-positive men followed for a median of 51 months (range, 1-98 months) were eligible for analysis. Subjects were classified into stages at their first seropositive visit and at all subsequent visits. As of 1 April 1991, 136 subjects (26%) had progressed to stage IV of the modified WHO system on the basis of their CD4 lymphocyte counts, and 78 subjects (15%) had died. Kaplan-Meier estimates for progression to stage IV from stages I, II and III were 52.8 +/- 7.5% over 6.6 years, 58.1 +/- 7.1% over 5.9 years and 66.5 +/- 9.7% over 5.7 years (log-rank P = 0.0001). Estimated median times to stage IV were 6.4, 5.3 and 3.8 years from stages I, II and III, respectively. Estimated median times to death were 10.9, 8.2, 6.3 and 1.7 years from stages I to IV, respectively. Results remained unchanged when CD4 lymphocyte count was replaced with lymphocyte count in the laboratory axis of the staging system. The proposed staging scheme, based on the WHO system, provides a prognostically meaningful classification for HIV infection in a cohort of homosexual men. Furthermore, the use of absolute lymphocyte count as a valid alternative for CD4 lymphocyte count has implications for the applicability of this system in many parts of the world where diagnostic resources are limited.


Adverse effects of systemic opioid analgesics. Adverse effects of opioids are multiple. They are most often receptor-mediated and inseparable from their desired effects. The most severe mishaps with opioids are related to their respiratory depressant effect, which is widely influenced by factors such as pain, previous opioid experience and awareness. Other relevant central nervous system effects of opioids include cough suppression, nausea and vomiting, rigidity, pruritus and miosis. The cardiovascular adverse effects of opioids are mainly related to histamine release and differ widely between agonists and agonist-antagonists. Gastrointestinal effects such as constipation, reflux and spasms of the bile duct are well described. Adverse effects on endocrine, immunological and haematological functions are possible, while allergic reactions are extremely rare. The adverse effects of long term use are overestimated. Systemic toxicity is negligible and development of tolerance is minimal while treating pain. In the clinical setting of pain control, addiction and withdrawal do not pose significant problems. Nevertheless, the possible effects of opioids on the unborn child should always be considered. Overall, opioids show a good record of safety. Their use should not be unduly limited by unfounded fears of adverse effects, but these effects should be avoided by anticipation and prevention.


Oligonucleotide hybridization used to detect short non-contiguous sequences. We describe the use of an oligonucleotide construction as a hybridization probe to detect short noncontiguous regions of sequence identity. Oligonucleotides complementary to various portions of the conserved heptamer and nonamer sequences flanking immunoglobulin variable region genes at the 3' end were used in this model system. We show that short oligonucleotides alone (7 bp or 9 bp) cannot be used as hybridization probes, but a construction containing both conserved sequences linked by a bridge will hybridize. The bridge is formed by degenerate bases (any base potentially at each position) and serves to maintain the spacing originally present between heptamer and nonamer. We show that such a bridging oligonucleotide probe can be used for hybridization analysis both on Southern blots and in bacterial screening.


Immunotoxic effects of MPT-IP containing 60% methylparathion in mice. The effects of a single large and repeated small doses of MPT-IP (the industrial product used to produce Wofatox EC 50) containing 60% methylparathion, on the humoral and cellular immunoreactivity of CFLP mice were investigated. Administration of a single LD50/2 dose 3 d prior to immunization caused a 40% increase in the number of splenic PFC on the 5th day but no significant increase in serum antibody titre on the 7th day after immunization. Treatment for 4 weeks with an LD50/40 dose resulted in a 100% increase in splenic PFC, also not associated with a change in serum antibody titre. Under the same conditions and LD50/20 dose had no effect on these parameters. Neither the single large nor the repeated small doses had any effect on the intensity or time course of a DTH reaction. The results show that MPT-IP has an immunotoxic potential in mice under certain experimental conditions.


Assessment of pentachlorophenol exposure in humans using the clearance concept. 1. Pentachlorophenol (PeCP), a widely-used wood preservative, is a ubiquitous compound which has been found to be carcinogenic in mice. The objective of this study is to assess the average net daily intake of PeCP in cohorts of individuals who are: (1) not specifically exposed to PeCP, (2) residents of homes made of PeCP-treated logs and (3) occupationally exposed to PeCP. 2. The average net daily intake was calculated using a basic pharmacokinetic principle, the clearance (CL) concept: net daily intake equals CL (in 1 d-1) times the average steady-state concentration of PeCP in plasma (Css). Css values reported in the literature were used for the calculations. 3. Because the two definitive studies on PeCP toxicokinetics in humans have given conflicting results, kinetic information from human exposure to PeCP was reviewed. Plasma clearance was estimated from retrospective analysis of urine and plasma concentrations measured in people after long-term exposure to PeCP. An overall clearance of 0.425 l d-1 was obtained. 4. In groups of individuals who are not specifically exposed to PeCP, net daily intake estimated in eight countries varied from 5 micrograms (Nigeria) to 37 micrograms (The Netherlands). Net intake was between 51 micrograms d-1 and 157 micrograms d-1 in residents of homes made of PeCP-treated logs. In individuals occupationally exposed to PeCP, net daily intake varied widely (from 35 micrograms to about 24,000 micrograms) depending on the type of work.


Dangerous mixture of household detergents in an old-style toilet: a case report with simulation experiments of the working environment and warning of potential hazard relevant to the general environment. A housewife cleaned toilet porcelain connected directly to a sewage storage tank with a mixture of cleaning agents; sodium hypochlorite (NaOCl) and hydrochloric acid (HCl) solutions. She complained of insomnia on the night after cleaning and suffered from severe metabolic acidosis with extremely low blood pH, PCO2 and bicarbonate values. She recovered from the acidosis after bicarbonate transfusion, plasmapheresis and plasma exchange. Permanent blindness ensued, however, from the third day after the event. These clinical symptoms suggested that the toxic substances responsible were chloramine and methyl chloride. Their generation was confirmed by in-vitro experiments, mixing NaOCl, HCl and pooled urine from normal people. In the simulation, the methyl chloride level far exceeded (100,000 ppm) the maximal allowable concentration recommended (ca 400 ppm) by the American Conference of Governmental Industrial Hygienists (ACGIH). Chloramine's toxic actions were confirmed using purified enzyme assay, and the inhibition of carbonic anhydrase and aldehyde dehydrogenase and the enhancement of superoxide dismutase activity were confirmed in neutral pH. The patient's clinical symptoms suggested that insomnia and permanent blindness seemed to be partly ascribable to chronic repetitive exposure to methyl chloride; catching a cold, drug intake and alcohol intake, in addition, precipitated the patient's visual loss. The possibility of this kind of intoxication with such a mixture of agents may lie latent in any situation where sewage or garbage are exposed to the open air.


Elevation of lead in human blood from its controlled ingestion in beer. 1. Nine volunteers ingested high levels of lead in beer over a 28-d period. The increase in blood lead varied by a factor of about two. There was a similar two-fold variability in the whole-body uptake (mean 14%) of a single oral dose of the short-lived tracer 203Pb. 2. The average elevations led to estimates of the potential increment from consumption of alcoholic beverages which accord broadly with epidemiological observation and which, if relevant to intakes of lead in table wine, raise the possibility of considerably elevated levels in the blood of avid consumers. 3. Rate constants inferred for removal of stable lead from blood were lower than reported following intake of the tracer, reflecting feedback of lead from other compartments.


1H-NMR spectroscopy as a means of monitoring nephrotoxicity as exemplified by studies with cephaloridine. 1. Male albino rats were dosed intravenously with either 0.9% saline or cephaloridine in saline at doses of 650, 750 or 950 mg kg-1 d-1 for 7 d. 2. Urine analysis on day 3, after two doses of cephaloridine showed dose-related increases in glucose, total protein, N-acetyl beta-D-glucosaminidase, gamma-glutamyltranspeptidase, alkaline phosphatase and lactate dehydrogenase. 1H-NMR spectroscopy showed corresponding disturbed profiles of products of intermediary metabolism indicative of a disruption of renal function. 3. By day 6, after five doses of cephaloridine, analysis by both 1H-NMR and conventional methods showed that all indices had returned to normal. 1H-NMR was demonstrated to provide useful complementary information to conventional techniques on the time course of the onset of the nephrotoxicity and the recovery phase, and was at least as sensitive as conventional urine analysis.


Aldrin and dieldrin residues in human fat, milk and blood serum collected from Delhi. 1. Aldrin and dieldrin residues were monitored in the fat, breast milk and blood serum from female residents of Delhi. 2. The average aldrin and dieldrin contents were 0.048 and 0.099 ppb in adipose tissue, 0.003 and 0.060 ppb in breast milk and 0.004 and 0.002 ppb in blood serum, respectively. 3. The older donors contained higher levels of aldrin and dieldrin in their adipose tissue. 4. Primagravidae contained more of these chemicals in their breast milk. 5. A positive correlation was observed between the aldrin concentration in adipose tissue and breast milk, and that in adipose tissue and blood serum. Similarly, a significant correlation was found between dieldrin in adipose tissue and blood serum. 6. The levels of aldrin and dieldrin were low in samples from residents of Delhi when compared to those in developed countries.


Fatal cyanide poisoning from cassava-based meal. Three patients admitted to the Accident and Emergency Unit of Lagos University Teaching Hospital (LUTH) after eating a cassava based meal 'Gari' died shortly after admission. The patients vomited and complained of abdominal pain immediately after the meal. They were unconscious with renal failure and died of cardiopulmonary arrest. The cyanide levels in the blood and urine averaged 1.12 and 0.54 mg l-1, respectively. Cassava contains cyanogenic glycosides which slowly release cyanide and this may have been responsible for the death of these patients. There is an urgent need to establish maximum tolerable levels of cyanide in 'Gari' and other cassava food products.


Toxicological findings after fatal fenfluramine self-poisoning. A fatal case involving the suicidal ingestion of fenfluramine is presented. The drug was quantified using gas chromatography. The blood concentration of fenfluramine was 7.46 mg l-1. Chronic use of fenfluramine was clearly demonstrated by the presence of drug in hair at 14.1 ng mg-1.


An acute mercuric mercury poisoning: chemical speciation of hair mercury shows a peak of inorganic mercury value. A woman ingested a dose of sublimate (approximately 0.9 g) in an attempted suicide. She survived and recovered in response to a combination of therapies including chelate (BAL) therapy, plasma exchange, haemodialysis and peritoneal dialysis. Serum inorganic mercury concentration, urinary inorganic mercury excretion and hair inorganic and organic mercury and selenium concentrations, along the length from the scalp to the distal part, were measured. Longitudinal analysis of hair, revealed a peak in inorganic mercury corresponding to the time of mercury ingestion. Organic mercury and selenium in the hair had different patterns of longitudinal variation from that of inorganic mercury. The biological half-life (23.5 d) of serum inorganic mercury levels was in good agreement with values previously reported in the literature.


Cobalamin deficiency and the pathogenesis of nervous system disease. Neuropathy commonly complicates cobalamin (Cb1) deficiency in humans, monkeys, fruit bats, and pigs. The neuropathy is characterized by demyelination of the posterolateral columns of the spinal cord (subacute combined degeneration). The lesion was thought to arise primarily from impairment of the adenosylcobalamin-dependent methylmalonyl CoA mutase reaction, leading to the formation of abnormal odd-chain and branched-chain fatty acids and their incorporation into myelin with resultant demyelination. Data from recently developed animal models of the Cb1 neuropathy induced by exposure to nitrous oxide do not substantiate this hypothesis, but rather identify impairment of the methylcobalamin-dependent methionine synthetase reaction as the more important basic defect. The key evidence for this hypothesis is the ability of methionine to delay the onset of Cb1 neuropathy in experimental Cb1 deficiency. In the Cb1-deficient pig, adenosylhomocysteine accumulates in neural tissue, presumably owing to the inability to recycle homocysteine via the defective methionine synthetase reaction. Accumulation of adenosylhomocysteine results in a fall in the adenosylmethionine:adenosylhomocysteine methylation ratio, and this change is believed to cause defective methylation and demyelination in the nervous system. However, in the Cb1 neuropathy in the fruit bat, adenosylhomocysteine does not accumulate in the nervous system, the methylation ratio does not change, and no defect can be demonstrated in the methylation of myelin lipid or basic protein. Although a central role for methionine in the pathogenesis of the Cb1 neuropathy has been established, defective methylation attendant upon impairment of the methionine synthetase reaction may not be the universal defect underlying the Cb1 neuropathy. This would suggest that the methionine effect could be mediated via its role in formate metabolism or polyamine synthesis, or by some as yet unidentified pathway.


CD4-binding of gp130 micelles isolated from SIVagmTYO-7. The binding and binding inhibition of the SIVagmTYO-7 external glycoprotein gp130 in micellar form to the CD4 molecule on human Molt-4 clone 8 cells was investigated. The best binding of gp130 to Molt-4 clone 8 cells occurred at pH 5.5 to 6.5 at 37 degrees C after 4 h or at room temperature after 10 h. The dissociation constant of this reaction was 0.2-0.4 nM, with both soluble CD4 or CD4 on Molt-4 clone 8 cells. This value is close to 0.15 nM determined for the antihuman CD4 monoclonal antibody 30F16H5. After partial deglycosylation of gp130, a 90 kD product arose which still bound to CD4. Fully deglycosylated gp130 of 60 kD was still immunoprecipitable, but had lost the CD4 binding activity. Lens culinaris agglutinin was able to inhibit the gp130-CD4 interaction very efficiently, while the agglutinin of Phaseolus vulgaris was half as efficient and Canavalia ensiformis was inefficient. CD4 binding of gp130 micelles was also inhibited with several anti CD4 monoclonal antibodies directed against the OKT4a epitope as well as with soluble recombinant CD4.


Somatostatin binding in normal and malignant human gastrointestinal mucosa. Somatostatin is a regulatory peptide implicated in the control of cellular proliferation in epithelial tissues and this regulation may occur directly via membrane bound receptor activation. The aim of this study was to investigate somatostatin binding in human gastrointestinal cancer and normal mucosa. Plasma membranes were prepared from specimens of tumour and normal mucosa from 51 patients undergoing surgical resection for malignancy (28 gastric, 23 colorectal). Using a competitive displacement assay, specific 125I-tyrosine-11-somatostatin-14 binding to plasma membranes was assessed and and characterised in terms of receptor affinity (Kd) and maximum binding capacity (Bmax) as determined by Scatchard analysis. Specific low affinity (Kd = 166 nM), high capacity (Bmax = 1.2 pmol mg-1 protein) somatostatin binding was demonstrated in 22 of the gastric cancers and 17 of the colorectal cancers (Kd = 140 nM, Bmax = 1.8 pmol mg-1 protein). Similar affinity and binding capacity was demonstrable in normal mucosal samples. High affinity receptors for somatostatin were expressed by one gastric carcinoma (Kd = 0.9 nM; Bmax = 0.23 pmol mg-1 protein). Thus, low affinity, high capacity binding is a common feature of gastrointestinal tumours and normal mucosa, and high affinity receptors may occasionally be demonstrated. The functional significance of these low affinity binding sites requires elucidation to determine whether long-acting somatostatin analogues may have therapeutic benefit in gastrointestinal malignancy.


Innervation of mucosal immune cells in the gastrointestinal tract. There is mounting evidence for interactions between the immune and peripheral nervous systems. Many regulatory molecules are candidate mediators for communication between inflammatory cells and nerves; however, the extent of targeting of neurotransmitters (and interleukins) as intersystem messengers has been somewhat overlooked. Lymphoid tissues are well supplied by nerves and the gastrointestinal lamina propria, which is populated by a variety of immune cell types, is densely innervated. One-half to two-thirds of mast cells are closely apposed to nerves in the intestinal mucosa, in both rodents and humans, and nerve stimulation has been reported to cause mast cell activation. Although less extensively studied, both eosinophils and plasma cells in the gastrointestinal mucosa are also positioned for interaction with nerves, and intra-epithelial leukocytes may be subject to diffusible neurally-derived mediators. Peyer's patches are relatively sparsely innervated but appear to express neuropeptide receptors in inflammatory conditions. Although the nerves in the mucosa have traditionally been thought of as a static component, recent experiments suggest that these may undergo extensive remodelling during nematode-induced inflammation. Such data suggest a dynamic interplay between the immune and nervous systems during inflammatory episodes in the gut, although considerable work is still needed to determine the importance of neuro-immune interactions in gastrointestinal homeostasis and inflammation.


Regulation of mucosal immune responses by T lymphocytes: the effect of chronic CD4+ T cell deficiency on IgA synthesis. Mechanisms of immune defence at the mucosal surface has been elucidated by recent advances in molecular and cellular immunology. IgA is undoubtedly the most important defense factor in the mucosal immune system. It has been shown that T cells are essential for the induction and regulation of IgA synthesis. In T cell regulation of IgA synthesis, various cytokines (e.g., TGF-beta, IL-2, IL-5, and IL-6) which are secreted by CD4+ T cells, play important roles for the induction and regulation of IgA isotype switching and terminal differentiation of sIgA+ B cells to become IgA producing cells. The chronic treatment of mice with anti-CD4 mAb induced a market deficiency of CD4+ T cells in both mucosal and systemic tissues. IgA plasma cells were significantly reduced in treated mice when compared with normal mice (greater than 80% reduction), while the numbers of sIgA+ B cells in IgA inductive sites (e.g., PP) remained normal. CD4+ Th cells are a critical element for the induction of appropriate IgA responses in mucosal associated tissues. Elucidation of the precise cellular and molecular network for the regulation of mucosal immune defense system is important and useful for the consideration of prevention of infectious diseases. In this regard, the effective and sophisticated mucosal administration of vaccines using the concept of the common mucosal immune system should induce effective immune responses which prevent the pathogen from entering the host through large surface areas of mucosal membranes. This goal cannot be achieved without a more complete understanding of regulatory T cells and cytokines for mucosal immune responses.


Low-frequency restriction fragment analysis of Frankia strains (Actinomycetales). Low-frequency restriction fragment analysis of more than 100 strains of the genus Frankia showed that restriction enzyme DraI (recognition site, TTT'AAA) gave rise to large DNA fragments (200 to 1,500 kb), which, when they were subjected to cluster analysis, reflected the host plants from which the strains were isolated. Our results support the conclusions of Lalonde and his colleagues (M. Lalonde, L. Simon, J. Bousquet, and A. Seguin, p. 671-680, in H. Bothe, F. J. de Bruijn, and W. E. Newton, ed., Nitrogen Fixation: Hundred Years After, 1988; P. Normand, P. Simonet, and R. Bardin, Mol. Gen. Genet. 213:238-246, 1988) and Fernandez et al. (M. P. Fernandez, H. Meugnier, P. A. D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989) that various biochemical and genomic analyses can give rise to groupings of Frankia strains that are consistent with the host plants from which the strains are isolated and that the resulting groups may form a basis for defining Frankia species.


Differentiation of the subspecies of Campylobacter fetus by genomic sizing. Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.


Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen. A ribosomal DNA restriction analysis of 17 strains belonging to the Saccharomyces cerevisiae complex revealed two major groups, one of which corresponded to Saccharomyces bayanus. Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S. bayanus should be reinstated as a separate taxon.


Some technical parameters influencing the precision and accuracy of fragment size determination for RFLP's. A comparison was carried out between 3 computer-assisted systems for the estimation of DNA fragment length: (1) the "Digitab" system (developed at our institute), (2) the FBI analysis system (FBI Quantico, USA) and (3) the BioImage system (Waters/Millipore, USA). Both the FBI system and the BioImage system were found to be much more precise than the manual Digitab system. In an additional experiment, a possible influence of the field strength on the "accuracy" of fragment size measurement was checked. Lowering the field strength to approx. 1V/cm led to a statistically significant increase in the measured fragment size.


Forensic identification of urine samples. VNTR polymorphisms were investigated for the possible individualisation of human urine samples with reference to doping cases in sport. Investigations were carried out with the RFLP single locus system YNH24/Hinf I and the PCR-VNTR systems Apo B and COL2A1 (AMPFLPs) as well as SE 33 and TC 11 (STRs). Urine samples were tested using 3 different volumes (10 ml, 1 ml and 0.1 ml) after 2 days and 2-5 weeks storage at 4 degrees C. Positive results were obtained for STR systems in all cases and with the smallest volume of urine tested (0.1 ml). The AMPFLP systems gave positive results in 4 out of 8 (Apo B) and 5 out of 8 (COL2A1) samples and YNH24 was successful in 1 out of 3 samples. Negative results were obtained for the AMPFLP systems and YNH24 after longer storage periods whereas the STRs were positive.


Sodium nitroprusside alters dark voltage and light responses in isolated retinal rods during whole-cell recording. Dark voltage and light responses of isolated retinal rods of Rana esculenta were investigated by employing the whole-cell patch-clamp technique. When the recording pipette was filled with a medium devoid of nucleotides, a spontaneous hyperpolarization of the dark voltage partly due to a diffusional loss of cGMP and its precursor GTP and a retardation in the recovery of the light responses was observed. The larger part of the retardation of the light responses was prevented by 1 mM ATP. Addition of GTP attenuated the hyperpolarization, but did not abolish it completely. When the nitric-oxide-releasing substance sodium nitroprusside plus GTP was applied, the tendency of hyperpolarization disappeared and a stable dark voltage or even a slight depolarization was measured during the whole-cell recording period. Similar results were also obtained when GTP was given in combination with either EGTA or IBMX which are both known to interfere with the cGMP regulating enzymes in retinal rods. In addition to its effects on the dark voltage, an acceleration of the recovery phase of the light responses by sodium nitroprusside was also observed. Our observations strongly suggest that sodium nitroprusside activates guanylate cyclase in photoreceptors, as it does in other tissues, but we cannot exclude with certainty an effect on the phosphodiesterase.


Circulating intercellular adhesion molecule-1 in melanoma patients: induction by interleukin-2 therapy. Intercellular adhesion molecule-1 (ICAM-1, CD54), a molecule bound to the cell surface membrane, mediates various cell-cell interactions in inflammation and immunosurveillance. By means of a new specific enzyme-linked immunosorbent assay (ELISA) for soluble ICAM-1, free circulating ICAM-1 was measured in serum from five healthy volunteers, 10 melanoma patients at different stages of their disease, and eight patients receiving high-dose interleukin-2 (IL-2) for metastatic melanoma. No correlation between the concentration of circulating ICAM-1 and the tumor burden could be detected. In melanoma patients receiving high-dose IL-2, we observed an increase of circulating ICAM-1 of up to 200%, compared to the concentration prior to therapy, ranging between 4 and 13 ng/ml. The increase in circulating ICAM-1 was associated with the induction of tumor necrosis factor-alpha and interferon-gamma.


Mediated Na(+)-independent transport of L-glutamate and L-cystine in 1- and 2-cell mouse conceptuses. L-Glutamate and L-cystine appeared to compete for transport via a mediated Na(+)-independent transport process in 1- and 2-cell conceptuses. Not only did these substances competitively inhibit each others' uptake by conceptuses, but their Ki values for inhibition were about equal to their Km values for transport in 1-cell conceptuses. Moreover, the transport process interacted strongly with L-amino acids that had 3-6 atoms in a chain between their negatively charged groups, whereas it interacted weakly or not at all with amino acids that did not have these characteristics or that were N-methylated. Transport of anionic amino acids was not altered greatly by pH in the range 4.5-8.0, but transport of L-cystine was much faster at higher pH values. The slower cystine transport at lower pH values was due primarily to protonation of its second amino group. A small increase in the degree of deprotonation of cystine's carboxyl groups also probably contributed slightly to its faster transport at higher pH values. By all of these criteria, the transport process in conceptuses appears to be a form of amino acid transport system xc-. System xc- activity was several-fold higher in 1- than in 2-cell conceptuses. Similarly, L-glutamate uptake by unfertilized eggs was relatively rapid, whereas it was much slower in immature, fully-grown oocytes. System xc- activity in 1-cell conceptuses also appeared to increase in response to the oxidative stress of culture, whereas no such increase was observed for 2-cell conceptuses. We suggest that this transient increase in the activity of system xc- activity during development of 2-cell conceptuses from immature, fully-grown oocytes could help protect unfertilized and fertilized eggs from oxidative stresses in situ.


Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II. The CO2 hydration activities of cloned human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) and variants with Lys, Glu, Gln or Ala replacing His at sequence position 64 have been measured in a variety of different buffers in the pH range 6-9. The variants with Lys-64, Gln-64 and Ala-64 showed non-Michaelis-Menten behavior under some conditions, apparent substrate inhibition being prominent near pH 9. However, asymptotic Michaelis-Menten parameters could be estimated for the limit of low substrate concentrations. All variants show distinct buffer specificities, and imidazole derivatives, Ches and phosphate buffers yield higher kcat values that Bicine, Taps and Mops buffers under otherwise similar conditions. These results are interpreted in terms of different pathways for a rate-limiting proton transfer. In unmodified enzyme, the very high catalytic activity depends on His-64 functioning as an efficient proton transfer group, but this pathway is not available in the variants with Gln-64 and Ala-64. Imidazoles, Ches and phosphate are thought to participate in a metal center-to-buffer proton transfer pathway, whereas Bicine, Taps, Mops and Mes appear to lack this capacity, so that the rate-limiting proton transfer occurs in a metal center-to-bulk water pathway for these variants. The Lys-64 and Glu-64 variants give significantly higher kcat values in Taps, Mops and Mes buffers than the Ala-64 and Gln-64 variants. The pH dependencies of these kcat values are compatible with the hypothesis that Lys-64 and Glu-64 can function as proton transfer groups. Thus, at pH near 9, Lys-64 appears to be only 5-times less efficient than His-64, while Glu-64 is inefficient. At pH 6, Lys-64 is an inefficient proton transfer group, but Glu-64 is only 2-3-times less efficient than His-64. The data indicate that Lys-64 and Glu-64 have pKa values near 8 and below 6, respectively.


Expression of haptoglobin receptors in human hepatoma cells. The uptake of radio-labeled hemoglobin-haptoglobin complex (Hb-Hp) by human hepatoma PLC/PRF/5 and HepG2 cells was investigated in an attempt to characterize the uptake process and intracellular transport. Human hepatoma cells took up Hb-Hp in a receptor-mediated manner. Scatchard analysis of binding revealed that PLC/PRF/5 and HepG2 cells exhibited about 21,000 and 63,000 haptoglobin receptors/cell, with a dissociation constant (Kd) of 8.0 and 17 nM, respectively. Human hepatocytes in primary culture also expressed about 84,000 receptors/cells, with a Kd of 7.4 nM. The hemoglobin-haptoglobin complex was internalized and subsequently the internalized Hb-Hp was slowly degraded in the cells. Preincubation of the cells with Hb-Hp resulted in a decrease in binding of the radioactive Hb-Hp to the cell surface, and was accompanied with an accumulation of intracellular receptors. The uptake of Hb-Hp by the cells was not inhibited by 100 microM chloroquine or by 10 mM methylamine, but was inhibited by 50 microM monodansylcadaverine. Hemoglobin-heme taken up by the cells induced microsomal heme oxygenase. Thus, human hepatoma PLC/PRF/5 and HepG2 cells can take up Hb-Hp by haptoglobin receptor-mediated endocytosis and Hb-Hp probably causes translocation of the haptoglobin receptors from the cell surface to the cell interior where they can be degraded. The internalized heme-moiety of hemoglobin can regulate the expression of heme oxygenase.


Influence of wearing masks on the density of airborne bacteria in the vicinity of the surgical wound. To find out if the wearing of masks influenced the number of colony forming units (CFU) of bacteria in the vicinity of the wound, 14 operations on the thyroid gland were divided in 30 min periods during which personnel were randomly allocated to wear or not to wear masks. There was at least one period with, and one without masks during each operation. Air was sampled 20 cm from the wound using a Sartorius membrane filter sampler. There were no significant differences between the groups in either numbers of CFU or species of pathogenic bacteria found.


Effect of suture technique on early healing of intestinal anastomoses in rats. To see if the site and tension of sutures had any influence on early breaking strength in intestinal anastomoses. 161 Male Wistar rats. First experiment: after laparotomy and division of the ileum an inverted end to end anastomosis was made in a single layer with 14 interrupted sutures of 7/0 polypropylene. All sutures were inserted 3 mm from the cut edges. The rats were randomly allocated to one of four groups according to the size of the loop of suture used: tight (n = 38), 0.4 mm (n = 22), 0.9 mm (n = 35), or 2.0 mm (n = 24). Second experiment: the method used was the same except that sutures were placed 1.5 mm from the cut edges and the rats were randomly allocated to one of two groups according to the size of loop of suture--tight (n = 20), or 0.9 mm (n = 22). Breaking strength measured in a tensiometer (both experiments); and myeloperoxidase activity/g tissue, hydroxyproline content and solubility/biopsy specimen (first experiment only). Breaking strength decreased proportionally the tighter the sutures were tied, and the differences were significantly greater when the sutures were placed closer to the cut edges. Anastomotic myeloperoxidase activity, hydroxyproline content, and soluble: total hydroxyproline ratio were similar in all groups. Tight sutures may promote proteolytic tissue degradation by reducing the local availability of circulating proteinase inhibitors.


Stab wounds to the pericardium and heart: an analysis of 85 consecutive patients. To audit all stab wounds of the pericardium and heart treated by a policy of immediate exploration over a period of 17 years, and identify any factors predictive of outcome. Retrospective study. Urban referral centre. 85 consecutive patients with stab wounds of pericardium and heart. Immediate exploration. Mortality and morbidity. Eleven patients died (13%), 10 of them on the operating table. No powerful predictor of outcome was identified, but measurable blood pressure on admission (p = 0.04) and a low cardiac trauma index score (p = 0.07) seemed to be associated with survival. Some unrecordable factors--for example, the skill of the team on duty--may have had a considerable influence on outcome.


Catheter-related complications of continuous ambulatory peritoneal dialysis. To assess the effectiveness of continuous ambulatory peritoneal dialysis (CAPD) with particular reference to morbidity. Open study. Two city general hospitals. 104 Adults and 11 children with end stage renal failure. Morbidity. There were 29 complications (25%), the most common being obstruction of the tube (n = 8, 7%), and migration of the tube (n = 7, 6%). Others were peritonitis (n = 5), haemorrhage (n = 4), infection at the exit site (n = 3), and leakage of fluid (n = 2). All were readily treatable. Fixing the catheter in two places may prevent its migration. The complication rate of CAPD is acceptable, and in children with end stage renal failure it is a suitable alternative to haemodialysis while they are waiting for renal transplantation.


Assessment of the physiological importance of iliac artery stenosis by laser Doppler flowmetry in pigs. To test the hypothesis that a curve with two peak values (double hump) recorded by laser Doppler flowmetry over the skin of the lower limb during postocclusive hyperaemia reflects pathological vascular resistance in the aortoiliac segment. Open study. Six Norwegian Landrace pigs. Arterial stenoses were induced in the external iliac arteries. Presence of double humped laser Doppler curves, relative decrease in laser Doppler flux between the two peaks, and time taken to reach peak hyperaemic flux. Double humped curves were seen only when arterial stenoses were present. The relative decrease in laser Doppler flux between the two peaks, and the time to reach peak hyperaemic flux were related to the blood pressure gradient (mmHg) at the stenosis (r = 0.88 and 0.83, p less than 0.0001). The laser Doppler curve pattern can be explained by similar dynamic changes in arterial blood pressure distal to the tourniquet during hyperaemia. These results confirm the hypothesis, and suggest that laser Doppler flowmetry recordings of postocclusive hyperaemia may be a non-invasive way of assessing the condition of the iliac artery.


Association between haematoma after inguinal hernia repair and site of heparin injection. To see if subcutaneous heparin prophylaxis against deep vein thrombosis and pulmonary embolism given into the abdominal wall caused more haematomas after repair of inguinal hernia than the same dose given into the shoulder. Random control trial. District hospital. 101 consecutive patients admitted for elective inguinal hernia repair. Four injections of sodium heparin 5,000 IU given either into the abdominal wall or the shoulder, the first two hours, before, and the last 24 hours after operation. Incidence of haematoma after operation. There was no significant difference in the incidence of haematoma between the groups. Haematoma formation was associated with a fall in systolic blood pressure of more than 25% (p = 0.055), which in turn was significantly associated with age over 60 years (p less than 0.0003). Injection of heparin subcutaneously into the abdominal wall does not lead to more wound haematomas than injection into the shoulder. Haematoma formation seems to be associated with a drop in systolic blood pressure of 25% or more, and thus requires further investigation.


Hypercalcaemia and pancreatic ultrastructure in cats. To study the effects of local and systemic infusions of calcium on the ultrastructure of the pancreas in cats. Controlled study. Three groups of four cats each had local infusions (into the splenic artery) of calcium gluconate 0.6 mmol/kg.hour or potassium chloride 1.1 mmol/kg.hour, or sodium chloride 0.9%, for three hours. Two groups of eight cats each had systemic infusions (into the jugular vein) of either calcium gluconate 0.6 mmol/kg.hour or sodium chloride 0.9%, for twelve hours. In the group that was given calcium, the infusion rate was reduced after three hours to 0.3 mmol/kg.hour to maintain the hypercalcaemic state for a further nine hours. Local infusion of calcium caused destruction of acinar cells with hydropic degeneration of nuclei, discharge of cell organelles into the interstitial spaces, and extravasation of red blood cells but no apparent damage to the capillaries. There were no ultrastructural changes of any importance in the groups that received potassium or sodium chloride. Systemic infusion of calcium resulted in a 1.8 fold increase in the ionised calcium concentration in the serum, progressive signs of overstimulation of the Golgi apparatus with hypertrophy, fusion of condensing vacuoles, and disruption of the acinar cell polarization. This was followed by clumping of nuclear chromatin and destruction of acinar cells. Acute pancreatitis in cats can result from stimulation and destruction of acinar cells by hypercalcaemia.


Peroperative endotoxin concentrations in portal and peripheral venous blood in patients undergoing right hemicolectomy for carcinoma. To find out the incidence of systemic and portal endotoxaemia in a homogeneous group of patients with colonic cancer undergoing a standard operation, and to compare them with a control group with benign disease. Open study. Department of Surgery, Stockholm Söderhospital. 15 of 17 consecutive patients admitted for right hemicolectomy for cancer, and four control subjects (one with angiodysplasia, and three with ulcerative colitis) who were to undergo right hemicolectomy and proctocolectomy. After mobilisation of the colon, 10 ml samples of blood were taken simultaneously from a mesocolic vein, and from an antecubital vein, for assay of endotoxin. Raised concentrations of endotoxin were found in the portal blood of 4 of the 15 patients with cancer, and in two of the four with benign disease. One of the four with cancer also had a raised value in peripheral blood. There was no correlation between increased endotoxin concentration and tumour size, Dukes' stage, or development of infective complications. Pronounced concentrations of endotoxin are found only rarely in patients undergoing right hemicolectomy for cancer. The most likely explanation is translocation of endotoxin through mucosa damaged by an ulcerating tumour.


Postoperative follow-up of patients with colorectal carcinoma by colonoscopy. To find out if colonoscopy is of use in the follow-up of patients who have been operated on for colorectal carcinoma. Retrospective study. Department of diagnostic radiology, university hospital. 390 consecutive patients operated on for colorectal carcinoma during the 10-year period 1981 to 1990. Number of recurrences, synchronous of metachronous tumours, and number and size of adenomas found on colonoscopy. Neoplastic lesions were found in 175 (45%) of the 390 patients studied. There were 14 anastomotic recurrences and 12 new primary carcinomas. At operation for recurrent tumours Dukes' A or B lesions were found in half of the 14 patients who had no symptoms, and a quarter of the 12 who had had symptoms. Those with recurrent carcinoma were younger than those without. Adenomas 1 cm in diameter or more were found in 44 patients and 104 had adenomas less than 1 cm. In addition one carcinoid was found. Colonoscopy gave a high yield of neoplastic lesions when used to follow-up patients after resection of colorectal carcinoma, particularly at six months, and resulted in half the recurrent carcinomas being diagnosed before the patients had symptoms. We recommend its use for follow-up of high risk patients, but further studies are needed to establish the optimum time intervals.


Digital gangrene after accidental intra-arterial injection of phenytoin (epanutin). A case is described in which inadvertent intra-arterial injection of phenytoin led to digital gangrene, with arteriography showing occlusion of the digital arteries, necessitating amputation of fingers. The high alkalinity of phenytoin is strongly irritant to vessel walls. Intravenous injection of phenytoin should be given only with great care and in emergencies, preferably avoiding the cubital fossa.


Decrease in arterial ketone body ratio indicating graft dysfunction after liver transplantation. In 3 cases of living related liver transplantation, arterial ketone body ratio (AKBR) showed secondary decrease in the early postoperative period, indicating the graft dysfunction more rapidly and sensitively than other liver function tests. Significance of AKBR for monitoring the graft function in postoperative management after liver transplantation is discussed.


Primary benign ulcer of the gall bladder. It is sometimes impossible to come to a final diagnosis in patients with dyspepsia and upper abdominal pain in spite of extensive investigation. Such patients are usually given vague diagnoses like "non-ulcer dyspepsia" and they represent an important diagnostic challenge.


Time course of transmitter release calculated from simulations of a calcium diffusion model. A three-dimensional presynaptic calcium diffusion model developed to account for characteristics of transmitter release was modified to provide for binding of calcium to a receptor and subsequent triggering of exocytosis. When low affinity (20 microM) and rapid kinetics were assumed for the calcium receptor triggering exocytosis, and stimulus parameters were selected to match those of experiments, the simulations predicted a virtual invariance of the time course of transmitter release to paired stimulation, stimulation with pulses of different amplitude, and stimulation in different calcium solutions. The large temperature sensitivity of experimental release time course was explained by a temperature sensitivity of the model's final rate limiting exocytotic process. Inclusion of calcium tail currents and a saturable buffer with finite binding kinetics resulted in high peak calcium transients near release sites, exceeding 100 microM. Models with a single class of calcium binding site to the secretory trigger molecule failed to produce sufficient synaptic facilitation under this condition. When at least one calcium ion binds to a different site having higher affinity and slow kinetics, facilitation again reaches levels similar to those seen experimentally. It is possible that the neurosecretory trigger molecule reacts with calcium at more than one class of binding site.


Nonsurgical treatment of urinary incontinence. Genuine stress urinary incontinence can be treated by surgical or nonsurgical methods. Conservative treatments include pelvic muscle exercises, hormonal and nonhormonal pharmacologic therapy, and functional electrical stimulation with vaginal or anal electrodes. All of these methods improve or cure stress incontinence in a significant proportion of selected women, with less cost and morbidity. Patients with genuine stress incontinence generally should have a trial of conservative therapy before corrective surgery is offered. Behavioral and pharmacologic methods, alone and in combination, are used for women with detrusor instability. Behavioral regimens, including bladder retraining and biofeedback, are particularly effective for urge and stress incontinence, but are dependent on compliance and motivation of both patient and caregiver. Drug therapy is effective, but with potential morbidity. As with genuine stress incontinence, surgical methods should only be employed for patients with detrusor instability who do not respond to nonsurgical treatment.


Gestational trophoblastic diseases: recent advances in the understanding of cytogenetics, histopathology, and natural history. Our understanding of gestational trophoblastic diseases has progressed considerably in recent years in terms of our knowledge of their cytogenetic origin, histopathology, and natural history. Advances in molecular biology have been applied to the study of gestational trophoblastic diseases, providing new insights into their pathogenesis. Molecular biologic studies have now clearly demonstrated that complete molar pregnancies result from fertilization of an anuclear, "empty" ovum. Improved understanding of the cytogenetics and biology of gestational trophoblastic diseases may well contribute to further advances in patient care.


Alzheimer's disease: relationship of cognition and behavior to neurochemistry. Alzheimer's disease is characterized by loss of cells and synapses in specific neural systems. The development of more effective therapies will depend on understanding the relationships between this pathology and the cognitive and behavioral impairments. In this review, focusing primarily on work in our laboratory, we will examine both classic and neuropeptide neurotransmitter systems and will discuss conceptual and methodological problems in relating clinical and biological measures.


Circulating levels and liver tissue distribution of intercellular adhesion molecule-1 during beta-interferon therapy of hepatitis C virus-associated chronic active liver disease. Intercellular adhesion molecule-1, an immunoglobulin supergene family member, is known to account for important steps in cell activation and the immune response. By a non-isotopic slot-dot immunoblotting assay, we measured circulating levels of intercellular adhesion molecule-1 in 26 patients with hepatitis C virus-associated chronic active liver disease before and after beta-interferon therapy, in 6 patients with non-A, non-B acute self-limiting hepatitis and in 13 healthy subjects. Circulating intercellular adhesion molecule-1 was found in 10 of 13 (77%) normal controls at low concentrations which were not statistically different from those measured in patients with hepatitis C virus-associated chronic active liver disease responsive to beta-interferon, whereas significantly higher levels were found in unresponsive patients. Higher serum intercellular adhesion molecule-1 levels were found in 4 of 10 (40%) beta-interferon-responsive patients compared with 13 of 16 (18%) unresponsive patients. Intercellular adhesion molecule-1 levels persisted after discontinuation of beta-interferon treatment and did not correlate with hepatocytolysis (as indicated by alanine aminotransferase serum activity) either in chronic active liver disease or acute hepatitis. However, a good correlation was found between intercellular adhesion molecule-1 and its expression on liver cells, thus emphasizing that induced circulating levels may reflect the state of activation at the sites of the inflammatory process. These data strongly support the view that intercellular adhesion molecule-1 plays an important role in liver cell damage in hepatitis C virus-associated acute and chronic liver disease, and that its circulating levels may be a good prognostic parameter of responsiveness to beta-interferon therapy.


Suspected chemoreceptors in coelenterates and ctenophores. Chemoreceptors in coelenterates and ctenophores have not been identified with certainty. Among prospective chemoreceptive cells are the sensory nerve cells, the cnidocyst-bearing cnidocytes, and the epitheliomuscular cells that are likely to be involved in feeding or aggression. Both behaviors are mediated by coordinated chemical and mechanical reception. This is reflected in the close apposition of putative chemo- and mechanoreceptors. Among the structures that have been designated as likely chemo- and/or mechanoreceptors are stereocilia, kinocilia, and/or microvilli which are universally present on all the putative chemoreceptor complexes, while gland cells and mucous secretions are prevalent. Evidence that the actin-containing stereocilia are chemically modulated mechanoreceptors is presented for several forms.


Enzymatic and chemical demethylenation of (methylenedioxy)amphetamine and (methylenedioxy)methamphetamine by rat brain microsomes. The metabolism of (methylenedioxy)amphetamine (MDA) and (methylenedioxy)methamphetamine (MDMA) was examined in microsomal preparations from rat brains. The products generated from MDA and MDMA were identified as dihydroxyamphetamine (DHA) and dihydroxymethamphetamine (DHMA), respectively. The demethylenation reaction required NADPH and was strongly inhibited by CO/O2 (4:1 v/v), suggesting that the formation of DHA and DHMA is mediated by cytochrome P450. The conversion was inhibited by desipramine, imipramine, and methimazole, whereas SKF-525A and alpha-naphthoflavone had little effect. Lineweaver-Burk plots of MDA and MDMA demethylenation were biphasic in both cases, indicating that multiple isozymes may participate in the oxidation. The microsomal preparation showed no significant stereoselectivity in the demethylenation of either MDA or MDMA. Catechol formation differed with the incubation buffer and was 2.6 times greater when phosphate rather than HEPES buffer was used. This difference disappeared, however, when desferrioxamine B methanesulfonate (desferal) and hydroxyl radical (.OH) scavenging agents were added to either buffer. The demethylenation was also sensitive to catalase and was stimulated by the addition of ferric ion and EDTA to the microsomal incubation mixture. These results indicate that the demethylenation of MDA and MDMA by rat brain microsomes has a cytochrome P450-mediated component as well as a chemical component involving .OH.


Familial primary hyperparathyroidism: study of the pedigree in three generations. The familial occurrence of primary hyperparathyroidism in which the proband is a 55-year-old man is reported. His 58-year-old sister and 40-year-old brother had undergone partial parathyroidectomy, and histological examination revealed hyperplasia in both cases. Their father and a daughter of the proband had a history of nephrolithiasis. The three siblings showed high levels of plasma parathyroid hormone (even the two postoperative cases). All of them had a history of nephrolithiasis and peptic ulcers. In the proband, image studies did not reveal any abnormality in the neck region. At present, the three cases do not exhibit any abnormalities in the pancreas or the pituitary by imaging studies and endocrine tests.


Physician failure to record alcohol use history when prescribing benzodiazepines. The purpose of this pilot study was (1) to determine the proportion of patients in an ambulatory medical clinic who have an alcohol history recorded when prescribed benzodiazepines, and (2) to assess the adequacy of the alcohol history when obtained. Medical records of 35 outpatients who obtained prescriptions for benzodiazepines at a large inner-city teaching hospital medical clinic were audited. In none of the records was there evidence that the physician had sufficient knowledge of the patient's alcohol use to safely prescribe a benzodiazepine. In 57% of the records, no information about alcohol use was recorded. In the remaining 15 medical records, the information recorded was limited. The implications of prescribing benzodiazepines without knowledge of drinking status are discussed.


Narcotic use in the hospital: reasonably safe? To determine the causes and frequency of overdoses associated with the administration of opioid analgesics in hospitalized patients. Case series. Two acute care teaching hospitals. Eighty-one hospitalized patients who received naloxone for a clinically suspected narcotic overdose. Three investigators reviewed each patient who received naloxone during a 12-month period. The patients were judged to have a narcotic overdose if caregivers documented an immediate improvement in mental status, respiratory rate, or blood pressure after naloxone administration. The number and causes of narcotic overdoses were determined. The frequency of morphine and meperidine overdoses was calculated. The number of incidents reported using incident or adverse drug reaction reports or the appropriate International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) code. In the 22 overdoses that occurred, 14 (64 percent) were caused by medication prescribing, compounding, or administration errors and potentially were preventable. The remaining eight patients experienced an overdose despite receiving appropriate amounts of opioids. The frequency of overdoses was 0.4 and 0.2 percent of total patients receiving morphine or meperidine, respectively, at the two hospitals. Nonreporting of these narcotic overdoses was frequent. In one hospital, 1 incident report and 3 adverse drug reactions were reported for 17 overdoses. At the second hospital, 1 incident report and 1 adverse drug reaction were reported for 6 overdoses. None of the patient charts included an ICD-9-CM code that documented the problem. The causes of overdoses are not limited to prescribing and administration errors. Some patients, despite proper execution of appropriate orders, develop a narcotic overdose. Caregivers must be aware of this problem and monitor patients for a decrease in mental status and respiratory rate. In addition, we conclude that an important number of hospitalized patients develop an overdose even though the frequency is low related to the number of patients receiving narcotics.


Propafenone-induced liver injury.

Propafenone-induced liver injury. To describe propafenone-induced liver injury. Retrospective case report. Referred care in a large tertiary care center. Laboratory tests were performed at the auxiliary site and the tertiary care center. A 71-year-old woman with atrial fibrillation developed elevations of greater than two times the upper limit of normal in alkaline phosphatase (ALK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyltransferase (GGT) after initiation of propafenone therapy. Studies included serial measurements of ALK, ALT, AST, and GGT. The patient developed elevations of greater than two times the upper limit of normal in ALK, ALT, and AST, one month after initiating propafenone therapy. The propafenone dose was decreased from 900 to 675 mg/d and, ten days later, the ALK, ALT, and AST were decreased slightly, but still above the upper limit of normal. One month later, serum transaminases had returned to baseline, but propafenone therapy was discontinued because of recurrent atrial fibrillation, persistent elevation in ALK, and elevation in GGT. Two months after discontinuing propafenone, serum aminotransaminase and ALK concentrations had normalized and GGT had decreased and remained only slightly elevated. The occurrence of liver injury secondary to propafenone therapy is rare. Reported cases appear to be secondary to hepatocellular injury, cholestasis, or a combination of the two. In this case, the pattern demonstrated by elevations in liver enzymes may be classified as acute cholestatic liver injury. Because the reported incidence is 0.1-0.2 percent and there are no known fatalities secondary to propafenone liver injury, routine monitoring of liver function tests in all patients receiving propafenone cannot be recommended at this time. Baseline liver function tests prior to initiating propafenone therapy with follow-up laboratory studies one month later are recommended in patients with known liver dysfunction. If elevations are noted, a reduction in dose may result in lower liver enzyme concentrations, although discontinuation of therapy may be required in some cases.


Hematopoietic progenitors cells in cord blood. Human umbilical cord blood was evaluated as an alternative to bone marrow as a source of stem cells for hematopoietic transplantation. In order to define an optimal collection procedure, we have studied the parameters that influence the collection, handling and storage of cord blood. We have attempted to correlate the quality of the samples with obstetrical and neonatal parameters. Using culture techniques we have studied the long term viability of the cells. Cell separation was also investigated. Our results suggest that most of the sample collected could be suitable for transplantation, in term of progenitors. However the observed variability between samples suggest that an efficient control of the quality of the samples is important.


Human cord blood: a source of transplantable stem cells? In a preliminary study we have shown that optimally collected human umbilical cord (HUC) blood cells grow significantly better in long term cultures (LTC) than normal adult marrow cells (NBM) p = 0.0007. The LTC findings are supported by the observation in clonogenic assay that a similar number of GM-CFC colonies can be grown from HUC blood and NBM mononuclear cells (MNC). Also there is a trend towards a higher proportion of primitive erythroid (BFU-E) and primitive megakaryocyte colonies (MK-CFC containing greater than 20 cells) in HUC blood compared with NBM. We suggest that further work on the feasibility of a HLA typed, cryopreserved HUC blood bank as a source of unrelated haemopoietic 'stem' cells for clinical transplantation is indicated.


Clinical applications of stem cell transfusion from cord blood and rationale for cord blood banking. Umbilical cord blood collected and cryopreserved at birth contains enough hematopoietic progenitor stem cells for engraftment. HLA identical sibling cord blood transplant has been performed for the first time, in a child with Fanconi anemia. Three years latter, this child is alive with a complete donor type bone marrow. Since this first attempt, several other patients with other diseases have been transplanted successfully. Cord blood banking is a safe and easy procedure. Due to the high proliferative capacity of neonatal hematopoietic progenitors and to the relative immunological functional immaturity of neonatal lymphocytes cord blood cells could be used for matched unrelated or partially mismatched transplants.


Fetal liver transplants.

Fetal liver transplants. Transplants of hematopoietic stem cells derived from fetal liver during the second trimester of pregnancy can restore hematopoiesis in animals and humans with bone marrow failure. These cells also have a reduced likelihood of causing graft-versus-host disease. Because fetal liver derived hematopoietic stem cells are relatively pure and considerable proliferative potential, they may be reasonable targets for studies of gene modification. Other possible uses of fetal liver derived stem cells are also considered as are results of fetal liver transplants in animals and humans. These data are compared to alternative sources of hematopoietic stem cells including bone marrow and umbilical cord and adult blood.


In utero transplantation of stem cells in humans: immunological aspects and clinical follow-up of patients. Four human fetuses were treated by transplantation of human fetal liver stem cells. Two of them had severe immunodeficiency disease and the two other ones had thalassemia major. Three of these in utero transplants were followed by engraftment. The three patients are now born: the first one is now very healthy thanks to the reconstitution of cell-mediated immunity associated with this transplant, and he lives normally at home; the two other ones, who have been more recently treated, have a significant improvement of their condition and they also live normally at home. This procedure, for the first time used in humans, has therefore demonstrated its feasibility and its efficacy: during early fetal development, foreign cells engraft readily and may result in cure or significant correction of a large variety of inherited diseases.


T cell repertoire and tolerance after fetal stem cell transplantation. We studied the T cell repertoire and the mechanism of tolerance in two patients with severe combined immunodeficiency transplanted with HLA mismatched fetal liver stem cells. They are 17 and 5 years old now, healthy, and show normal immunoresponses to recall antigens. Their T cells are of donor origin, whereas monocytes and B cells remained of the host. The NK cells have different sources since in one patient they derive from the donor and in the other one from the host. Despite the HLA mismatch between donor and host cells, no acute or chronic graft-versus-host disease was observed. In vitro experiments with PBMC showed specific nonresponsiveness for the HLA antigens expressed by the host cells. However, an extensive clonal analysis showed that CD4+ and CD8+ host-reactive T cell clones recognizing class II and class I HLA molecules of the host, respectively, were present in the peripheral blood of both patients. Limiting dilution experiments indicated that the frequency of CD8+ host-reactive cells was in the same range as that observed for alloreactive T cells. In contrast, no donor reactive CD8+ T cells could be isolated. Host-reactive CD4+ and CD8+ T cell clones were normal in their capacity to produce IL-2, IFN-gamma, GM-CSF and IL-5, but they failed completely to synthesize IL-4. In addition, CD4+ T cell clones from patient RV secreted very high levels of IL-10. Interestingly, exogenous IL-10 was able to inhibit the proliferative responses of the CD4+ host-reactive T cell clones. Our data demonstrate that host-reactive cells are not deleted from the donor T cell repertoire following allogenic fetal liver stem cell transplantation. Therefore, in vivo tolerance between the host and the donor is maintained by a peripheral autoregulatory mechanism in which cytokines may play a role.


Clinical and biological aspects of human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells. Presented is a short review of the current status fo the clinical and biological aspects of using human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells.