Urethral atresia in a neonate with alveolar capillary dysplasia and pulmonary venous misalignment.

Urethral atresia and alveolar capillary dysplasia (ACD) are rare congenital malformations. Urethral atresia is associated with severe pulmonary hypoplasia secondary to oligohydramnios. ACD is associated with pulmonary venous misalignment, results in severe pulmonary hypertension, and is uniformly fatal. We present a case of urethral atresia with successful, early placement of vesicoamniotic shunting, with resolution of the oligohydramnios, in which the neonate rapidly progressed to respiratory failure and death. Postmortem examination confirmed urethral atresia and diagnosed ACD. Given the surprisingly high mortality rate after vesicoamniotic shunting in patients with urethral atresia, we question whether there might be a possible link to ACD.

Fournier's disease.

Fournier's gangrene is an aggressive synergistic fasciitis of the perineum. The disease can no longer be considered to be idiopathic; in most cases a urologic, colorectal, or cutaneous source can be identified. Despite antibiotics and aggressive debridement, the mortality rate remains high, particularly in the elderly, in patients with renal failure, and in patients with extensive disease. The presentation is highly variable, necessitating a high index of suspicion. High-risk patients include diabetics, alcoholics, and debilitated and immunosuppressed individuals. As the AIDS population increases, the incidence of Fournier's gangrene may increase as well. In questionable cases, imaging modalities should be performed to allow early diagnosis and to reduce missed diagnoses. Broad-spectrum antibiotics and aggressive debridement remain the hallmarks of treatment. Hyperbaric oxygen therapy and improved local wound care may decrease the extent of tissue destruction. Reconstructive techniques afford better cosmetic results. With early recognition, prompt treatment, improved wound care, and reconstructive efforts, the mortality rates and cosmetic results should continue to improve.

GABAA alpha 2 mRNA levels are decreased following induction of spontaneous epileptiform discharges in hippocampal-entorhinal cortical slices.

Exposure of hippocampal slices to Mg2+ free media (0 Mg) has been shown to trigger full production of stimulus-induced seizure activity after restoration of physiological conditions [1]. In the present study employing hippocampal entorhinal cortical slices (HEC), spontaneous epileptiform discharges (SEDs) were induced using 0 Mg treatment following the return of the slices to physiological conditions. To evaluate the effect of sustained epileptiform activity on gene expression in this HEC slice preparation, changes in mRNA levels of the GABAA alpha 1 and alpha 2 and beta CaM Kinase II subunits were measured using in situ hybridization. HEC slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence or absence of Mg2+ for 3 h, then placed in oxygenated ACSF containing Mg2+ for up to 3 h. Control slices were maintained in Mg2+ containing ACSF for up to 6 h. Recurrent SEDs were observed in 0 Mg pre-treated slices while no epileptiform discharges were seen in control slices. Following induction of SEDs by 0 Mg pre-treatment, a significant decrease in mRNA encoding GABAA alpha 2 was found in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus for up to 3 h after treatment. Levels of mRNA for GABAA alpha 1 and beta CaM Kinase II were not affected. The results document a decrease in GABAA alpha 2 gene expression following the induction of SEDs in the HEC slice preparation and suggest that rapid changes in neuronal gene expression may contribute to long lasting excitability changes associated with the induction of epilepsy.

Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans.

The success of adenoviral vectors for gene therapy of lung disease in cystic fibrosis (CF) depends on efficient transfer of the complementary DNA encoding the correct version of the cystic fibrosis transmembrane regulator (CFTR) to the affected columnar epithelial cells lining the airways of the lung. Pre-clinical studies in vitro suggest that low doses of adenovirus vectors carrying this CFTR cDNA can correct defective Cl- transport in cultured human CF airway epithelia. Here we use mice carrying the disrupted CF gene to test the efficacy of this transfer system in vivo. We find that even repeated high doses can only partially (50%) correct the CF defect in Cl- transport in vivo and do not correct the Na+ transport defect at all. We investigated this discrepancy between the in vivo and in vitro transfer efficiency using CF mouse and human samples, and found that it reflects a difference in the susceptibility to adenovirus-5 transduction of the epithelial cell types dosed in vivo (columnar) and in vitro (basal-cell-like). These studies indicate that more efficient adenoviral gene-transfer vectors and/or refinement of dosing strategies are needed for therapy of CF lung disease.

Hyperabsorption of Na+ and raised Ca(2+)-mediated Cl- secretion in nasal epithelia of CF mice.

We investigated the effect of homozygous genetic disruption of the murine cystic fibrosis transmembrane regulator (CFTR) gene on regulation of the rates of Na+ absorption and Cl- secretion by nasal epithelia in cystic fibrosis (CF) mice. The basal in vivo nasal potential difference (PD; -28.8 +/- 1.8 mV, n = 10) and amiloride-sensitive PD (delta 13.8 +/- 1.0 mV, n = 10) were raised in CF mice compared with controls [-7.8 +/- 0.8 mV, n = 14 (basal); delta 4.5 +/- 0.7 mV, n = 14 (amiloride)], consistent with raised Na+ transport. In vitro studies of freshly excised nasal epithelia confirmed that CF epithelia exhibited a greater basal equivalent short-circuit current (Ieq; 63.5 +/- 12 microA/cm2, n = 15) vs. control (30.2 +/- 7.2 microA/cm2, n = 16) and amiloride-sensitive Ieq (delta 46.2 +/- 12.5 microA/cm2) vs. control (delta 11.3 +/- 4.5 microA/cm2). Tissue from normal mice failed to secrete Cl- in response to ionomycin (delta Ieq: -1.2 +/- 1.9 microA/cm2, n = 18), whereas CF murine tissue responded with a large rise in Ieq (delta 55.1 +/- 19.1 microA/cm2, n = 13). We conclude that CF murine nasal epithelia exhibit Na+ hyperabsorption, providing strong evidence for a regulatory link between CFTR and Na+ channel activity in airway epithelia. We speculate that upregulation of the Ca(2+)-mediated Cl- secretory pathway buffers the severity of airway disease in the CF mouse.

Kindling produces long-lasting and selective changes in gene expression of hippocampal neurons.

To test the hypothesis that repeated subconvulsive stimulations required to induce kindling can permanently alter gene expression of hippocampal neurons, we used Northern and in situ hybridization analyses to measure steady-state mRNA levels encoding several phenotypic proteins. mRNA encoding a membrane-bound protein, ligatin, was significantly reduced in kindled brains relative to naive and sham control animals. This change in gene expression persisted for over 4 months after kindling, was associated with a decrease in ligatin protein expression, was not produced by single or multiple seizures that did not induce the kindling phenomena, and was blocked by MK801. These results provide direct evidence that kindling can cause persistent changes in the expression of specific genes in hippocampal neurons and suggest that N-methyl-D-aspartate receptor-activated changes in gene expression may be a basic molecular mechanism underlying some of the long-lasting plasticity changes seen in kindling or models of long-term memory.

Cell cycle and proliferation dynamics of adult rat oligodendrocytes.

Adult oligodendrocytes (OLGs) have been shown to be mitotically responsive when co-cultured with dorsal root ganglion. We have investigated the population dynamics of the OLG proliferative response, including the maximum percent of cells which will proliferate and the cell cycle duration time of the adult rat OLG. Adult OLGs were stimulated with pituitary extract and either continuously labeled or pulse labeled with bromodeoxyuridine (BrdU). The labeled cells were stained with a fluorescent anti-BrdU monoclonal antibody, and fluorescence measurements were made with a multiparameter flow cytometer. A maximum number of 53% of the adult OLGs entered the cell division cycle after 9 days. The cell division cycle duration time (Tc) was determined to be approximately 35.4 hr, and the durations of G1, S, and G2M found to be 12.1, 8.5, and 5.8 hr, respectively. A scenario was generated for adult OLG proliferation in which 2.2% of the initial population is stimulated to enter the cell division cycle in the first 24 hr. This initial population divides and continues in the cell division cycle. Another 2.2% of the initial population enters the cell division cycle during the second 24 hr period, divides, and continues in the cell division cycle. This entry of 2.2% of the cells into the proliferative phase continues until 53% of the initial population has left a quiescent state and entered the cell cycle.

Mitotic potential of adult rat oligodendrocytes in culture.

The proliferation of adult oligodendrocytes (OLGs) was examined in response to membrane bound and soluble mitogens. OLGs were isolated according to Vick, et al., J Neurosci Res 25:524-534, 1990, and co-cultured with dorsal root ganglia (DRGs). Less than 5% of the total cells incorporated a 48 hr pulse of 3H thymidine during the first 4 days of co-culture. From day 4 to day 6 there was a dramatic increase in proliferation which reached a plateau at 40% and gradually decreased to 25% from days 10 to 20 of co-culture. Axolemma-enriched fractions (AEF) were weak mitogens for OLGs (less than 5% proliferation after 7 days of stimulation), however, heparin extracts of AEF were five-fold more mitogenic than the AEF from which they were derived. Basic and acidic fibroblast growth factor were effective mitogens for the adult OLGs (labelling indices of 28% and 12%, respectively) provided that the cells were treated for 7 days with the growth factor and that the cells had been in culture for at least 14 days. Other soluble growth factors (IL-2 and PDGF) gave no mitotic response. We conclude that adult OLGs are mitotically responsive to mitogens provided that (1) the adult OLG has been cultured for sufficient time (14 days, acidic or basic fibroblast growth factor); (2) the axonal mitogen is allowed to interact with the OLG for a sufficient time (neuritic mitogen); and (3) the axonal mitogen is presented to the OLG in an activated form (AEF-heparin extract).

Role of adult oligodendrocytes in remyelination after neural injury.

Traumatic neural injury is often accompanied by demyelination. The factors that determine the ability of the CNS to remyelinate are examined as well as the origin of the cells responsible for the remyelination. Evidence is presented that suggests that both a precursor-type oligodendrocyte as well as an oligodendrocyte that previously formed a myelin sheath are able to remyelinate in the CNS. A key determinate of the success of remyelination is the ability for either cell type to proliferate before remyelination. We have developed a method to isolate the oligodendrocyte from the mature CNS and studied the mitotic potential of these adult oligodendrocytes in vitro. In contrast to neonatal oligodendrocytes, the adult oligodendrocytes respond weakly to soluble and particulate mitogens. However, when cocultured with neurites, the adult oligodendrocytes demonstrate a more vigorous mitotic response, which may be related to the ability of the neurite to upregulate receptors which transduce the mitotic signal. Since we have identified fibroblast growth factor as a mitogen associated with the axonal plasma membrane that stimulates neonatal oligodendrocytes to divide, we have examined the redistribution of membrane-associated fibroblast growth factor in an in vitro model of neuronal injury. After neuritic injury, fibroblast growth factor containing membrane vesicles were redistributed to the surface of cocultured oligodendrocytes. After invasion of macrophages to a site of neural injury, enzymes secreted by macrophages can release extracellular stores of fibroblast growth factor into the vicinity. This burst of mitotic potential may preferentially stimulate astrocyte rather than oligodendrocyte division, leading to glial scarring and a subsequent failure of remyelination. Other factors to be considered in the potential for remyelination after injury are astrocyte-derived factors that inhibit myelination and the proteins in oligodendrocytes that prevent axonal regrowth indirectly influencing remyelination potential. Thus, provided the oligodendrocyte can gain access to relevant mitogens either in the axonal plasma membrane or in a soluble form and undergo a wave of proliferation, there is good potential for remyelination after neural injury. However, if axonal regrowth is inhibited and astrocytes preferentially are stimulated to divide and form a glial scar, the prognosis for remyelination is poor.

Isolation, culture, and characterization of adult rat oligodendrocytes.

Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSA). The pellet was resuspended and placed in an untreated 6-well culture dish overnight to allow the astrocytes to attach. The non-adherent cells were replated on poly-l-lysine-treated coverslips. Approximately 8.25 x 10(5) cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2-5 days in vitro (DIV), the oligodendrocytes extended 6-10 thick processes. A membrane sheath between these processes was immunostainable with either anti-galactocerebroside (GC), anti-O4, anti-myelin basic protein (MBP), or anti-2'3' cyclic nucleotide 3' phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up to 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 A with up to 11 concentric layers of membrane. Immunologically, the adult oligodendrocytes are GC+, O4+, MBP+, CNPase+, and GFAP-. The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.

Dicroceliasis (lancet fluke disease) in an HIV seropositive man.

Dicroceliasis is an unusual zoonotic trematode infection caused by the lancet liver fluke, Dicrocoelium dendriticum. Grazing herbivores (usually sheep or cattle) are the definitive hosts. The life cycle proceeds through two intermediate hosts: the land snail and the field ant. Human infection is acquired by consuming the field ant. This case report describes a human immunodeficiency virus-seropositive patient who presumably acquired this parasite from bottled water contaminated with ants. A brief discussion of the parasitology, pathology, clinical findings and treatment is presented.

Biological, immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland.

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (micrograms) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with 125I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50-60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.

The treatment of sarcoidosis by levamisole.

Five patients with persistent or progressive pulmonary shadowing due to sarcoidosis were treated with 150 mg levamisole daily and one patient with 150 mg twice weekly. Only the latter patient completed a 12-week course without unwanted side-effects. One of the remaining five patients on full dose completed the course but all experienced symptoms (nausea, malaise, influenza-like syndrome or arthralgia and skin rash) severe enough to cause five to stop the drug. Haematology and biochemistry, however, remained normal, with the exception of transient rise in transaminases in one patient. Radiology, pulmonary function and numbers of circulating T-lymphocytes (E-rosettes) were unchanged, but three patients developed increased intensity of delayed hypersensitivity (DH) skin tests using PPD, Candida and Trichophyton antigens; two of these patients also developed increased in vitro lymphocyte stimulation by mitogen and PPD antigen and the other developed a 'serum sickness' syndrome with evidence of circulating immune complexes.

The contribution of renal and extrarenal mechanisms to hypokalemia induced by glucagon.

Other investigators have shown that infusion of glucagon causes the concentration of potassium, [K+], in the arterial plasma to increase rapidly, then to decrease to less than the beginning value. In studies on anesthetized dogs, we found that the magnitude of the initial, rapid rise of [K+] was increased by nephrectomy but not affected by pancreatectomy. The subsequent decline of [K+] and the persistent hypokalemia were not affected significantly by nephrectomy. Plasma [K+] decreased in the nephrectomized-pancreatectomized dogs, as it did in the nephrectomized and the control groups, but the effect was temporary, and [K+] began to increase again, even though the infusion of glucagon continued; after the infusion was ended, plasma [K+] became significantly higher than the beginning value. These data suggest that the hypokalemia caused by infusion of glucagon initally depends on extrarenal factors other than insulin, and, later, depends on insulin.

The effects of increased blood viscosity on pulmonary vascular resistance.

The isolated left lower lobes of 15 dogs' lungs were perfused by means of a roller pump with blood at hematocrit values ranging from 31 to 80 per cent. Pressure-flow curves were constructed at blood flow rates from one half to three times the normal flow for the left lower lobe at each hematocrit level. The perfusion pressure was normalized with reference to the normal hematocrit(38 to 48 per cent) and normal blood flow for the left lower lobe (20 ml. per kilogram per minute). From these normalized pressure-flow curves, normalized resistance-flow curves were constructed at different mean hematocrit levels. Regression lines were drawn relating normalized pulmonary vascular resistance to hematocrit at different rates of pulmonary blood flow which might be found in patients with congenital heart disease. It was found that pulmonary vascular resistance rose in an exponential fashion as the hematocrit was increased, and that the blood viscosity determined both the shape of the resistance-flow curve and magnitude of the increase in resistance to pulmonary blood flow, especially when the pulmonary blood flow was less than normal and the hematocrit was greater than 54 per cent. The family of regression lines relating pulmonary vascular resistance to hematocrit at different flow rates may be used clinically in patients with congenital heart disease and polycythemia to determine if an elevated pulmonary vascular resistance is due to increased blood viscosity or obstructive pulmonary vascular disease. It is concluded that an increased blood viscosity due to polycythemia significantly alters the pulmonary hemodynamics of patients with congenital heart disease with either increased or decreased pulmonary blood flow. Increased blood viscosity may play an important part in the early initiation and development of pulmonary arteriosclerosis in patients with transposition of the great arteries.