Histoplasma capsulatum modulates the acidification of phagolysosomes.

The phagolysosome is perhaps the most effective antimicrobial site within macrophages due both to its acidity and to its variety of hydrolytic enzymes. Few species of pathogens survive and multiply in these vesicles. However, one strategy for microbial survival would be to induce a higher pH within these organelles, thus interfering with the activity of many lysosomal enzymes. Altering the intravesicular milieu might also profoundly influence antigen processing, antimicrobial drug delivery, and drug activity. Here we report the first example of an organism proliferating within phagolysosomes that maintain a relatively neutral pH for a sustained period of time. We inoculated P388D1 macrophages with fluorescein isothiocyanate (FITC)-labeled Histoplasma capsulatum or zymosan. Using the ratio of fluorescence excitations at 495 and 450 nm, we determined that vesicles containing either virulent or avirulent FITC-labeled H. capsulatum yeasts had a pH one to two units higher than vesicles containing either zymosan or methanol-killed H. capsulatum. The difference in pH remained stable for at least 5.5 h postinoculation. Longer-term studies using cells preincubated with acridine orange indicated that phagolysosomes containing live Histoplasma continued to maintain a relatively neutral pH for at least 30 h. Many agents raise the pH of multiple vesicles within the same cell. In contrast, H. capsulatum affects only the phagolysosome in which it is located; during coinoculation of cells with unlabeled Histoplasma and labeled zymosan, organelles containing zymosan still acidified normally. Similarly, unlabeled zymosan had no influence on the elevated pH of vesicles housing labeled Histoplasma. Thus, zymosan and Histoplasma were segregated into separate phagolysosomes that responded independently to their phagocytized contents. This localized effect might reflect an intrinsic difference between phagosomes housing the two particle types, active buffering by the microbe, or altered ion transport across the phagolysosomal membrane such that acidification is inhibited.

Intracellular parasitism by Histoplasma capsulatum: fungal virulence and calcium dependence.

Histoplasma capsulatum is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States. A "dimorphic" fungus, H. capsulatum exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation. Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions. To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt CBP1 in H. capsulatum using a telomeric linear plasmid and a two-step genetic selection. The resultingcbp1 yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.

Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.

Location of the initial cleavage sites in mouse pre-rRNA.

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

Molecular mycology: a genetic toolbox for Histoplasma capsulatum.

Research in medical mycology has traditionally been a mix of exciting biology and frustrating genetics, although the excitement has been steadily increasing as genetic obstacles have been successfully overcome. Now, a variety of fungal pathogens can be studied using molecular techniques derived from classical bacterial and yeast genetics, but with selective and strategic adaptations. Histoplasma capsulatum is the best-studied of the primary pathogens known as 'dimorphic' fungi, and tailored molecular genetic strategies are beginning to reveal a repertoire of genes and gene products intimately associated with pathogenesis.

Phagosome-lysosome fusion in P388D1 macrophages infected with Histoplasma capsulatum.

The issue of whether or not phagocytized Histoplasma capsulatum yeasts evade phagosome-lysosome fusion (P-LF) has been debated by several investigators. To resolve this problem, yet avoid drawbacks associated with the conventional assays of P-LF (electron microscopy and the acridine orange assay), we used fluorescein isothiocyanate-labeled dextran (FITC-dextran) to monitor P-LF in the macrophage-like cell line P388D1.D2. Controls indicated that FITC-dextran could be used to distinguish between evasion of P-LF by Toxoplasma gondii and phagolysosome formation following ingestion of Saccharomyces cerevisiae. Phagosomes containing H. capsulatum clearly fused with FITC-dextran-labeled lysosomes at a rate comparable to that observed for S. cerevisiae. This was true for several strains of H. capsulatum including two avirulent strains derived in this laboratory. Varying the dose of H. capsulatum did not alter the percentage of phagolysosomes formed. Our results indicate that H. capsulatum is one of a small number of organisms which is able to survive in phagolysosomes.

Measurement of intracellular Ca2+ in cultured arterial smooth muscle cells using Fura-2 and digital imaging microscopy.

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility.

Rickettsialpox: report of an outbreak and a contemporary review.

Rickettsialpox is a mild illness characterized by the appearance of a primary eschar at the site of a mite bite followed by fever, headache, and a papulovesicular rash. It can be confused with a variety of illnesses including several other rickettsial diseases and chickenpox. R. akari, the etiologic agent, is a rickettsia belonging to the spotted fever group (SFG) of rickettsial illnesses. In spite of significant serologic cross-reactivity with other SFG agents, there is no convincing evidence of cross-immunity to these agents after recovery from rickettsialpox. Tetracyclinie is the drug of choice in the treatment of this disease.

Cognitive and motor functioning in Parkinson disease: subjects with and without questionable dementia.

BACKGROUND: The nature of cognitive performance in subjects with Parkinson disease (PD) without dementia is controversial, perhaps because of failure to exclude subjects with unrecognized very mild dementia. OBJECTIVE: To compare cognitive and motor functioning in well-characterized subjects with PD without overt dementia with healthy elderly control subjects. DESIGN: Subjects' conditions were evaluated clinically and psychometrically at entry into a longitudinal study of cognitive and motor performance in elderly subjects. Measures included a global dementia staging scale, the Washington University Clinical Dementia Rating; psychometric tests, including Logical Memory, Digit Span, Associate Learning, Information, Block Design, Digit Symbol, Trail-making A, Crossing-off, Boston Naming Test, and Word Fluency; and motor measures, including finger tapping, gait velocity, reaction time, and movement time. SETTING: A university-based research facility. SUBJECTS: There were 3 groups of subjects: healthy elderly control subjects (n=43), subjects with PD without dementia (n=58), and subjects with PD with questionable dementia (n=22), each evaluated at time of entry. RESULTS: As expected, both PD groups were impaired on motor measures (gait velocity, finger tapping, and movement time) compared with the healthy elderly control group. Neither PD group showed slowing in reaction time. The subjects with PD with questionable dementia were more impaired on Logical Memory, Block Design, Digit Symbol, and Trailmaking A compared with the subjects with PD without dementia. Although free of clinically evident cognitive dysfunction (Clinical Dementia Rating score, 0), the PD group without dementia was impaired with respect to the healthy elderly control group on all measures from the psychometric assessment except Digit Span, Associate Learning, and Word Fluency. CONCLUSIONS: The PD group without dementia showed global cognitive impairments in comparison with the healthy elderly control group, possibly because the healthy elderly control subjects represented idealized aging. Although the deficits were of small magnitude, this finding suggests that PD may predispose to subclinical cognitive impairment. Longitudinal follow-up is required to determine whether subjects with PD destined to develop overt dementia can be distinguished from those who do not.

Motor dysfunction in mildly demented AD individuals without extrapyramidal signs.

BACKGROUND: Although not as prominent as cognitive decline, motor dysfunction occurs in AD, particularly in the later stages of the disease. OBJECTIVE: To determine whether early-stage AD is also characterized by motor impairment. METHODS: We examined very mildly (Clinical Dementia Rating [CDR] 0.5) and mildly (CDR 1) demented AD individuals in comparison with healthy elderly control individuals (CDR 0) on a variety of nonmotor cognitive and psychomotor measures and on four motor measures (gait velocity, finger tapping, reaction time, movement time). To minimize the possibility of extrapyramidal dysfunction contaminating the groups, only individuals who were clinically free of extrapyramidal signs were included in the study. RESULTS: Mildly demented AD individuals were slowed on all motor measures except for finger tapping. No evidence of motor dysfunction was found in the very mildly demented AD group. As expected, both AD groups were impaired on the nonmotor cognitive and psychomotor tests. CONCLUSIONS: These results indicate that AD alone, in the absence of clinically confirmed extrapyramidal dysfunction, is associated with motor slowing in a stage-dependent manner. It remains to be determined whether this motor slowing represents a general characteristic of mild AD or indicates other neuropathology such as PD or the Lewy body variant of AD.

Patterns of brain atrophy in frontotemporal dementia and semantic dementia.

OBJECTIVE: To identify and compare the patterns of cerebral atrophy associated with two clinical variants of frontotemporal lobar degeneration (FTLD): frontotemporal dementia (FTD) and semantic dementia (SemD). METHODS: Twenty patients with FTLD were classified as having FTD (N = 8) or SemD (N = 12) based on current clinical criteria. Both groups showed a similar spectrum of behavioral abnormalities, as indicated by the neuropsychiatric inventory. T1-weighted MRI was obtained for each patient and 20 control subjects. The regions of focal gray matter tissue loss associated with both FTD and SemD, as well as those differing between the two groups were examined using voxel-based morphometry. RESULTS: Regions of significant atrophy seen in both groups were located in the ventromedial frontal cortex, the posterior orbital frontal regions bilaterally, the insula bilaterally, and the left anterior cingulate cortex. The FTD, but not the SemD, group showed atrophy in the right dorsolateral frontal cortex and the left premotor cortex. The SemD, but not the FTD, group showed tissue loss in the anterior temporal cortex and the amygdala/anterior hippocampal region bilaterally. CONCLUSIONS: Although FTD and SemD are associated with different overall patterns of brain atrophy, regions of gray matter tissue loss in the orbital frontal, insular, and anterior cingulate regions are present in both groups. The authors suggest that pathology in the areas of atrophy associated with both FTD and SemD may underlie some the behavioral symptoms seen in the two disorders.

Absence of cognitive impairment or decline in preclinical Alzheimer's disease.

OBJECTIVE: To determine whether clinically nondemented elderly individuals with pathologically confirmed preclinical AD are characterized by cognitive decline as measured by psychometric tests before death. METHODS: Psychometric performance was examined retrospectively in 14 individuals who were nondemented at time of death and grouped in accordance with their neuropathologic findings: 1) Healthy brain (n = 9) was characterized by the absence of senile plaques or by only patchy neocortical deposits of plaques; 2) preclinical AD (n = 5) was characterized by neuritic and diffuse plaques distributed throughout the neocortex. All individuals showed neurofibrillary pathologic change in medial temporal lobe structures. For comparison, we also evaluated 10 individuals who died in the earliest symptomatic stage of dementia of the Alzheimer type (DAT). All individuals had been assessed by clinical and psychometric measures during life. The psychometric measures yielded a standardized factor score that represented global cognitive performance. RESULTS: At the last assessment before death, individuals with very mild DAT were impaired on the factor score and on individual psychometric measures with respect to the nondemented individuals. Those nondemented individuals with preclinical AD did not differ in performance from those with healthy brains. For individuals with at least three psychometric assessments during life, there was no decline in performance for either those with healthy brains (n = 5) or preclinical AD (n = 3), although decline was evident for very mild DAT individuals (n = 5). CONCLUSIONS: Pathologically confirmed preclinical AD is not associated with cognitive impairment or decline, even on measures shown to be sensitive to very mild DAT.

Mucus and surfactant synthesis and secretion by cultured hamster respiratory cells.

Procedures for the selective isolation and cultivation in monolayer of respiratory cells have been developed. This technique requires repeated protease treatment and gradient centrifugation of hamster tracheal or lung tissues and permits the establishment of proliferating cultures of epithelial cells with biologic specialization. Mucus synthesis was monitored in cultured tracheal cells by incorporation of 3H-labeled N-acetyl-D-galactosamine and 14C-serine into glycoprotein as determined by trichloroacetic acid precipitation of growth medium followed by acrylamide gel electrophoresis. For comparative purposes tracheal explants and several established cell lines were also examined. Synthesis and secretion of the glycoprotein macromolecule by tracheal cell monolayers appeared to be regulated by vitamin A since its addition to the culture medium significantly increased both the number of cell-associated granules and glycoprotein secretion. Lung-originated cell cultures were grown to confluence and radio-labeled with 3H-choline in serum-free medium for 24 hr to examine surfactant synthesis. Cell monolayers and growth medium were then extracted by the Folch method, and total radioactive phosphatidylcholine as well as disaturated phosphatidylcholine were determined by thin-layer chromatography and alumina gel fractionation of osmium tetroxide-reactive phospholipid, respectively. Data indicate that these cultures have a marked ability to synthesize and secrete surfactant when compared to other established cell lines. In addition, naturally transformed cells that arose during passage and senescence of the primary cultures were analyzed for their biosynthetic capabilities.

Demonstration of substance P in aortic nerve afferent fibers by combined use of fluorescent retrograde neuronal labeling and immunocytochemistry.

Combined use of the intraaxonal retrograde transport of the fluorescent marker 'true blue' with substance P (SP) immunocytochemistry has been used to trace the nodose ganglion projections of SP-containing neurons of the aortic depressor nerve. It has been found that (1) SP immunoreactive (SP-I) cell bodies are clearly demonstrable in clusters in the rostral part of the nodose ganglion without the aid of colchicine pretreatment; (2) 'true blue' is retrogradely transported to the nodose ganglion following its application to the central cut end of the aortic nerve; (3) 'true blue' fluorescence and SP fluorescent immunoreactivity can be visualized in the same tissue section and certain cell bodies in the nodose ganglia contain both SP-I and retrogradely transported 'true blue'. These results indicate that the aortic nerve which projects from the aortic arch baro- and/or chemoreceptors to brainstem vasomotor centers contains SP-I afferent fibers which emanate form the nodose ganglion.

Costs and use of mental health services before and after managed care.

This paper tracks access, utilization, and costs of mental health care for a private employer over nine years during which mental health benefits were carved out of the medical plan and managed care was introduced. Prior to the carve-out, mental health costs increased by around 30 percent annually; in the first year after the change, costs dropped by more than 40 percent; in the six follow-up years, costs continued to decline slowly. This cost reduction was not attributable to decreased initial access, as the number of persons using any mental health care increased following the change. Instead, the cost reduction was the result of (1) fewer outpatient sessions per user, (2) reduced probability of an inpatient admission, (3) reduced length-of-stay for an inpatient episode, and (4) substantially lower costs per unit of service.

More evidence for the insurability of managed behavioral health care.

Debate continues about the cost and use of mental health services under managed care, as legislators consider various "parity" bills. This descriptive research replicates, broadens, and expands previously published case studies of single employers' data on cost and treatment prevalence in a large, diverse, national sample whose varied point-of-service benefits were provided by thirty employers representing multiple industries. Of those covered, 59,005 received treatment over the seven years studied. Of particular note is the pattern of increased use, increased care within the managed behavioral health organization network, and long-term cost reductions.

The effect of dopamine on glomus cell membranes in the rabbit.

Rabbit carotid bodies were excised and the membrane potentials (MPs) of the glomus cells were recorded in vitro. Upstream injections of dopamine (DA; 25-250 micrograms) elicited a slow, long lasting depolarization in 76% of the cells studied. The remaining 24% were hyperpolarized by DA. Regardless of polarity, the DA-induced potential was associated with a significant increase in input resistance (Ro). On the other hand, changes in MP which were produced artificially by passing direct current through the recording electrode had little effect on Ro. It is concluded that DA alters the MP and Ro of glomus cells and that this may serve to modulate synaptic excitability within the carotid body.

Redox changes in the mouse carotid body during hypoxia.

The carotid body of the mouse is ideally suited for in vitro photometric and fluorometric studies using transillumination. It is small, thin and translucent. Photometric recordings were made by using a dual wavelength method. Wavelengths of 550 nm (maximal absorbance, MA) and 540 nm (isosbestic point, IP) were employed to study cytochrome c, 600 nm (MA) and 620 nm (IP) for a pigment presumed to be cytochrome aa3, 580 nm (MA) and 570 or 542 nm (IP) to control for possible effects of hemoglobin (Hb). Exposure of the organ to NaCN (10-50 nM) reduced cytochromes c and putative aa3. Hypoxia (produced by superfusing with a medium equilibrated with 100% N2) reduced cytochrome c but tended to oxidize the presumed cytochrome aa3. These effects were apparent at a medium pO2 of 100 torr or less. Fluorometry revealed reduction of NADH under both cyanide exposure and hypoxia. There was little or no interference by Hb when the preparations were carefully washed of remaining red cells. The precise site of the recorded mitochondria is unknown: they could have been located in the parenchymal cells (type I and/or II), nerve terminals, smooth muscle fibers, etc. Resolution of this point will need using dissociated carotid body cells. Further, the possible presence of a special respiratory pigment responding at a wavelength similar to that exciting cytochrome aa3 has not been discarded. Its study would require isolating carotid body mitochondria.

Postpartum cerebellar herniation in von Hippel-Lindau syndrome.

PURPOSE: To describe exacerbation during pregnancy of cerebellar hemangioblastoma in von Hippel-Lindau syndrome. METHOD: Case-report. A 21-year-old woman with von Hippel-Lindau syndrome was found on routine ocular examination to have severe papilledema 1 week after giving birth. RESULTS: Immediate magnetic resonance imaging disclosed a large cerebellar cyst from hemangioblastoma causing cerebellar tonsillar herniation. Immediate neurosurgical intervention was life saving. CONCLUSION: Worsening of intracranial hemangioblastoma during pregnancy in cases of von Hippel-Lindau syndrome should be realized and periodic neurologic and ophthalmologic observation is warranted.