Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus.

The clinical course and laboratory evaluation of 21 patients coinfected with human immunodeficiency virus (HIV) and Ehrlichia chaffeensis or Ehrlichia ewingii are reviewed and summarized, including 13 cases of ehrlichiosis caused by E. chaffeensis, 4 caused by E. ewingii, and 4 caused by either E. chaffeensis or E. ewingii. Twenty patients were male, and the median CD4(+) T lymphocyte count was 137 cells/microL. Exposures to infecting ticks were linked to recreational pursuits, occupations, and peridomestic activities. For 8 patients, a diagnosis of ehrlichiosis was not considered until > or =4 days after presentation. Severe manifestations occurred more frequently among patients infected with E. chaffeensis than they did among patients infected with E. ewingii, and all 6 deaths were caused by E. chaffeensis. Ehrlichiosis may be a life-threatening illness in HIV-infected persons, and the influence of multiple factors, including recent changes in the epidemiology and medical management of HIV infection, may increase the frequency with which ehrlichioses occur in this patient cohort.

Identification of Ehrlichia chaffeensis morulae in cerebrospinal fluid mononuclear cells.

We report a case of ehrlichiosis in a 72-year-old man who developed extreme lethargy, acute renal failure requiring hemodialysis, and respiratory insufficiency requiring intubation. Lumbar puncture performed on the second day of hospitalization revealed significant cellular pleocytosis. Ehrlichia morulae were tentatively identified in mononuclear cells in routinely processed Wright-stained cytospin preparations of cerebrospinal fluid (CSF). Identification was confirmed by a specific immunocytochemical staining procedure. Subsequent identification specifically as Ehrlichia chaffeensis morulae was established by polymerase chain reaction analysis, which revealed E. chaffeensis-specific DNA in CSF, bone marrow, and blood samples; by indirect fluorescent-antibody analysis, the patient developed an antibody titer of 32,768 against E. chaffeensis antigen. The patient responded to intravenous therapy with doxycycline and dexamethasone. Subsequently, neurologic, hematologic, renal, and pulmonary status had returned to baseline at follow-up 12 weeks after admission. To our knowledge, this is the first identification of E. chaffeensis morulae in CSF cells in an infected patient.

Tumor-targeted delivery of 8-hydroxyquinoline.

RIF-1 mouse tumors express high levels of beta-glucuronidase activity relative to most normal tissues. The high activity can be exploited for targeting specific drugs preferentially to tumor tissues. In this study we examined the kinetics of 8-hydroxyquinoline (8-OHQ) accumulation in tumor and in several normal tissues resulting from the in vivo deconjugation of 8-hydroxyquinolyl-glucuronide (8-OHQ-GlcA). Tumors were acidified with D-glucose and NaHCO3 prior to the administration of 8-OHQ-GlcA; subsequently the deconjugated aglycone, 8-OHQ, accumulated preferentially in tumors and reached peak levels between 30 and 60 min after the 8-OHQ-GlcA injection. Mild hyperthermia of 30 min at 43 degrees C to the tumors further increased their peak 8-OHQ levels by a factor of 2-3. Some normal tissues, mostly kidney, liver, and colon, also accumulated 8-OHQ, but the aglycone appeared early in the normal tissues (near 30 min post-injection) and was significantly reduced by 60 min when 8-OHQ remained high in the tumor. Administration of 8-OHQ-GlcA alone, without prior tumor acidification, failed to produce measurable accumulations of 8-OHQ in tumors and in normal tissues. Tissue clearance of 8-OHQ is mediated primarily by the enzymatic reconjugation of 8-OHQ via UDP-glucuronosyltransferase (UDPGT). UDPGT activity was high in liver, kidney, and bowel, but low in the RIF tumor, spleen, muscle, and brain. Hyperthermia had only a modest effects on UDPGT activity: a heat dose of 30 min at 45 degrees C reduced activity less than 60%. Thus, preferential accumulation and prolonged retention of 8-OHQ in RIF tumors may be caused by a combination of factors: a) high tumor beta-glucuronidase activity, b) selective tumor acidification during hyperglycemia, c) low tumor UDPGT activity, and d) other factors, such as tumor blood flow.

Enhanced glycosyltransferase activity during thermotolerance development in mammalian cells.

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N-acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau-phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of thermotolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O-glycans, alpha 1,2-fucosyltransferase and alpha 2,6-sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat-induced activation of O-glycan-specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.

Effect of age and renal function on cefonicid pharmacokinetics.

Cefonicid (15 mg/kg) was administered intravenously at a constant rate of infusion over 15 min to 10 geriatric patients (mean age, 77 years) and to 4 young subjects (mean age, 35 years). Model-dependent and noncompartmental pharmacokinetic parameters were calculated and found to be congruous; noncompartmental data are reported. Significant differences in the values for area under the curve, mean residence time, total body clearance, and renal clearance were observed between the geriatric and young groups. Mean elimination half-life values were 9.59 and 4.88 h for the geriatric and young groups, respectively. Total body and renal clearances were inversely correlated to age and directly correlated to creatinine clearance. Free fraction was not correlated to albumin concentration but was correlated exponentially to total cefonicid concentration. Despite the prolonged half-life values observed in our geriatric patients, the difference in mean trough concentrations was slight. Daily administration of a 15-mg/kg dose should provide adequate concentrations in serum and should not produce appreciable accumulation in geriatric patients.

Tumor-targeted cell killing with 8-hydroxyquinolyl-glucuronide.

Many tumors show elevated levels of hydrolytic enzymes that may be associated with invasive processes. The RIF-1 murine tumor has levels of beta-glucuronidase that are more than four times higher than those in liver. Elevated tumor glucuronidase levels can be used as a basis for tumor-targeted therapy when systemically administered glucuronides of cytotoxic drugs are deconjugated preferentially at the tumor site. In this study we have used 8-hydroxyquinoline (8-OHQ) as a model compound for such a tumor-targeting concept. We showed that RIF tumors and spleen had the highest beta-glucuronidase activity in C3H mice; for example, RIF tumors released approximately seven times more phenolphthalein per gram of tissue from its glucuronide than liver, when compared under identical conditions. In vitro, low concentrations of 8-OHQ that might be achievable in vivo, ranging from 1 to 10 microM reduced cell survival by four orders of magnitude, while 1 mM 8-hydroxyquinolyl-glucuronide (1 h, 37 degrees C) resulted in only modest (S = 54%) cytotoxicity. Combination treatments of 8-OHQ (2.5 or 5 microM) with either hyperthermia or X radiation did not significantly change the slope of survival curves for RIF tumors in vitro, but suggest that targeted 8-OHQ toxicity combined with local hyperthermia and/or irradiation may be useful for significantly increasing therapeutic gains in vivo.

Pediatric viral gastroenteritis.

Rotavirus and Norwalk agent cause most viral gastroenteritis in the pediatric population. Infection results in osmotic diarrhea, which can cause dehydration and acidosis. Oral rehydration therapy has been shown worldwide to be a safe and effective means of treating this disease. Formula feeding should be resumed as soon as possible; breast feeding should be maintained throughout the illness. Admission criteria are based on weight loss, serum sodium level and ability to rehydrate orally on an outpatient basis.

Heat protection by sugars and sugar analogues.

We have examined the relative ability of 16 sugars and sugar analogues to reduce cell killing by hyperthermia of 40 min, 45 degrees C. In general, sugars were added to the culture medium 6 h prior to heating at a concentration of 100 mM (400 mosmol). The results show that D-hexoses, L-hexoses, methylated or thiolated sugars and disaccharides significantly protected cells against thermal damage, increasing survival by factors of 10 to 100. The degree of protection varied for specific sugars and could not be predicted on the basis of sugar conformation or the number of hydroxyl groups. Relative heat protection was partially dependent on the survival assay technique (pre- and post-plating); consistently lower cell survival was measured when cells were subcultured after hyperthermia, both in medium-control and sugar-protected cells. However, the time dependence of heat protection appeared independent of pre- and post-plating. Cell survival after heating was not increased by two sugars: (a) D-idose, and (b) 2-deoxy-D-galactose. The latter sugar, curiously, was also a heat protector but only when cells were trypsinized after hyperthermia. Both of these sugars were relatively more toxic at 37 degrees C under identical treatment conditions. The lack of protection by these two sugars is not understood. Another reported non-sugar heat protector, sodium butyrate, was included as an additional control. Heat protection by butyrate was not observed in CHO cells. The accumulation of intracellular free sugar was measured by gas chromatography after incubating cells for 6 h, 37 degrees C with talose, idose, L-galactose or 1-O-methyl-D-glucose. All of these sugars were found in high concentrations inside of cells. The data are consistent with the hypothesis that polyhydroxy compounds must accumulate intracellularly for cellular heat protection.

Protection against thermal cell death in Chinese hamster ovary cells by glucose, galactose, or mannose.

The addition of D-glucose, D-galactose, or D-mannose to culture medium increased survival of heated Chinese hamster ovary cells in a concentration- and time-dependent manner. Heat protection by sugars was not immediate but required prior incubation in the sugar medium before hyperthermia. The degree of heat protection conferred by each sugar and its time dependence differed characteristically: galactose protection appeared rapidly (within 1 hr) and was proportional to the galactose concentration in the medium up to 0.3 M. Glucose and mannose were less effective heat protectors at 0.3 M concentrations when compared with galactose, but cell survival after 40 min, 45 degrees, at concentrations below 0.1 M was similar in the three hyperosmotic sugar media. Heat protection by 0.3 M glucose under hyperosmotic conditions (600 mOSM) became apparent only after a preincubation period of at least 5 hr, 37 degrees. Under isoosmotic conditions (300 mOSM, 10% medium-10 mM morpholinopropanesulfonic acid-250 mM glucose), heat protection by glucose appeared more rapidly (3 hr), but the time dependence of heat protection was not eliminated. Under the same isoosmotic conditions in galactose medium, the survival of heated cells was not measurable. The 45 degrees survival curve in hyperosmotic galactose medium (6 hr, 37 degrees prior to heating, 0.3 M) was characterized by a DO of 10 min (controls, 3 min) and a quasithreshold dose of 17 min (controls, 17.5 min). When galactose-loaded cells were returned to fresh medium, heat protection decayed rapidly; cell survival measured 1 or 6 hr later showed a small degree of residual heat protection. A 5-hr incubation period at 37 degrees in galactose-supplemented medium resulted in a major intracellular accumulation of free galactose with smaller accumulations of glucose, sorbitol, and dulcitol. A similar incubation in glucose medium showed only minor intracellular elevations of polyols or sugars. A flow-cytometric analysis of the age distribution showed that incubation in the sugar-supplemented media slightly reduced the fractional cell population in G1 with concomitant gains in both the S- and G2-phase populations. Thus, heat protection by galactose or glucose is probably neither the result of a redistribution of cells in the cycle, nor does it always require the intracellular accumulation of free sugars and polyols. The greater degree of heat protection by galactose and its rapid manifestation under hyperosmotic conditions may be related to the intracellular accumulation of free galactose and/or its metabolites.

Microbiology for orthopaedic surgeons: selected aspects.

The treatment and prevention of orthopedic sepsis is based on the principles of any surgical sepsis and the factors that influence the chronicity of infection and microbiology. In the past, the field of orthopedic sepsis has been neglected and has retained the methodologies of previous years without a sharp focus on the principles and pathophysiology of infectious disease. Over the last ten years there has been an increasing interest in orthopedic sepsis along with significant changes in our concepts of antibiotics, principles of treatment, and microbiology. In this article the authors hope to identify the practical uses of clinical microbiology in diagnosing and managing orthopedic infections. The surgeon can use microbiologic techniques in many ways: (a) to determine sterility of wounds, (b) to detect the presence of infection, (c) to estimate the timing of primary closure of acute and chronic wounds and the application of skin grafts, and (d) as guides for the surgeon and infectious disease physician in the choice of appropriate antibiotics.

Effect of step-down heating on the cytotoxicity of adriamycin, bleomycin, and cis-diamminedichloroplatinum.

The cytotoxicity of a number of anticancer drugs can be potentiated by mild hyperthermia. Since the cytotoxicity of low-hyperthermic temperatures in the range of 39-43 degrees can be potentiated by using a step-down heating (SDH) sequence (short times at greater than or equal to 45 degrees followed by longer times at lower temperatures), we investigated the combined effects of SDH (specifically 45 degrees, 10 min, followed by time at 41.5 degrees) and three chemotherapeutic drugs known to be more toxic at elevated temperatures. In agreement with previous observations with Chinese hamster cells, we found that cultured RIF tumor cells showed an increased rate of cell killing at 41.5 degrees when the low-temperature heat treatment was preceded by an acute heat shock of 10 min, 45 degrees. Similarly, RIF cells showed enhanced cytotoxicity of Adriamycin, bleomycin, and cisplatin at 41.5 degrees over that at 37 degrees, as reported previously for hamster cells. When SDH and drug exposures were combined, the rate of cell killing was greater than that observed with the drugs at a constant 41.5 degrees. SDH alone reduced the Do on the 41.5 degrees survival curve from approximately 500 min to 100 min after 10 min at 45 degrees. Adriamycin cytotoxicity (0.3 microgram/ml) at 41.5 degrees was characterized by a Do of approximately 160 min for treatment times of 1 to 4 hr; with SDH, this was reduced to 52 min. Similarly, bleomycin (5 micrograms/ml) and cisplatin (0.5 microgram/ml) showed a decrease in Do by factors of 1.4 (68 to 48 min) and 2 (25.2 to 13.5 min), respectively, with the SDH sequence when compared with drug toxicity at 41.5 degrees alone. However, the sensitization to cisplatin was the same when 10 min at 45 degrees was followed by drug exposure at 37 degrees, or at 41.5 degrees. The results indicate that Adriamycin and bleomycin, but not cisplatin, cytotoxicity was increased by SDH over that achievable with 41.5 degrees alone. Possible clinical implications are discussed briefly.

Exposure to pretreatment hypothermia as a determinant of heat killing.

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).

Brain abscess due to Nocardia caviae. Report of a fatal outcome associated with abnormal phagocyte function.

Nocardia is an uncommon cause of human disease. We report a patient with a fatal brain abscess who had abnormal phagocyte function as measured by neutrophile chemotaxis, adherence, phagocytic function, and chemiluminescence in vitro and by Rebuck skin window in vivo. Antimicrobial agar dilution susceptibility suggested minocycline was more active than sulfamethoxazole.

Cefotaxime treatment of pelvic inflammatory disease.

We studied cefotaxime in the treatment of gonococcal and nongonococcal pelvic inflammatory disease. Cefotaxime was uniformly effective against gonococcal pelvic inflammatory disease. However, 4 of 11 patients with nongonococcal pelvic inflammatory disease had a suboptimal response.

Mannose in body fluids as an indicator of invasive candidiasis.

Using gas-liquid chromatography, we measured five mannose in the serum of six nondiabetic patients with autopsy-proven invasive candidiasis. In all patients serum mannose concentrations were higher than mannose levels found in serum from normal adults and children or from patients with catheter-associated candidemia, mucosal candidiasis, and other mycoses. Spinal fluid from two patients with Candida meningitis showed increased free mannose as compared to seven non-inflammatory spinal fluid samples. However, free mannose in the serum of poorly controlled diabetics (blood glucose of greater than or equal to 300 mg/dl) did overlap concentrations in patients with invasive candidiasis. In vitro culture of Candida albicans demonstrated increasing concentrations of mannose associated with growth of the organism. We conclude that physical and chemical assay for mannose in body fluids may be a useful technique to assist in the diagnosis in invasive candidiasis.

Purified Clostridium difficile cytotoxin stimulates guanylate cyclase activity and inhibits adenylate cyclase activity.

Antibiotic-associated pseudomembranous colitis has been linked with Clostridium difficile toxin. We examined the effect of toxins from four strains of C. difficile isolated from patients with pseudomembranous colitis on colonic adenylate (EC 4.6.1.1) and guanylate cyclase (EC 4.6.1.2) activities. Partially purified toxins had a cytotoxic effect on hamster fibroblasts in culture at a concentration of 10 ng/ml. Likewise, these toxins enhanced colonic guanylate cyclase activity two- to threefold, with the maximal stimulation being at 10 ng/ml. These toxins also enhanced guanylate cyclase activity in ileum, cecum, and duodenum. Both the cytotoxic activity on hamster fibroblasts and the enhancement of hamster guanylate cyclase activity were inhibited by antiserum to C. difficile toxin. These same toxins inhibited adenylate cyclase activity at a 100-ng/ml concentration, but had no effect at 10 ng/ml. They also had no effect at any concentration on colonic Na+-K+ adenosine triphosphatase. To be sure that the findings were not due to a contaminant, a purified C. difficile cytotoxin was used, and the same findings were found with the pure cytotoxin (at a 100-fold-lower concentration). The data suggest that activation of guanylate cyclase may be a factor in the pathogenesis of antimicrobial-associated pseudomembranous colitis.

Rosaramicin versus penicillin G in experimental Pneumococcal meningitis.

Rosaramicin, a new macrolide antibiotic, was compared with penicillin G in the treatment of pneumococcal meningitis in rabbits. Animals were infected intracisternally with 10(4) colony-forming units of Streptococcus pneumoniae type III (rosaramicin minimal inhibitory/bactericidal concentrations, 0.25/0.5 mug/ml; penicillin G minimal inhibitory/bactericidal concentrations, 0.03/0.06 mug/ml). Treatment was instituted 96 h later. Infusion of rosaramicin at 25 mg/kg per h intravenously for 8 h produced a peak cerebrospinal fluid (CSF) drug concentration of 1.54 mug/ml (range, 0.87-3.6 mug/ml). During this infusion the numbers of pneumococci in CSF decreased from 6.2 +/- 0.5 to 3.36 +/- 1.12 log(10) colony-forming units per ml. Penicillin G, infused at 30 mg/kg per h for 8 h, reached a similar concentration in CSF but caused a greater reduction (P < 0.01) in CSF bacteria, from 6.4 +/- 0.36 to 1.3 +/- 0.67 log(10) colony-forming units per ml. Penicillin G, at 100 mg/kg per day intramuscularly for 5 days, cured 7 of 10 rabbits with pneumococcal meningitis. A higher dose, 300 mg/kg per day for 5 days, was no more efficacious: 11 of 14 rabbits were cured. Rosaramicin at 100 mg/kg per day intramuscularly for 5 days cured only 5 of 15 rabbits with meningitis, but a higher dosage regimen of that drug (250 mg/kg per day intramuscularly) produced acute, fulminant enterocecitis and death within 48 h in seven of eight rabbits. No cytotoxin was detected in the feces of one rabbit with acute enterocecitis. Thus the efficacy of rosaramicin in experimental pneumococcal meningitis, measured by bacterial clearance from CSF and by treatment outcome, was less than that of penicillin G. In addition, high-dose parenteral rosaramicin caused acute, fulminant enterocecitis in a high proportion of treated rabbits.

D-Mannose in human serum, measured as its aldononitrile acetate derivative.

We report a procedure for determining D-mannose in serum. After removal of proteins and lipids, the carbohydrate-containing fraction is treated to form the aldononitrile acetate derivative and analyzed by gas--liquid chromatography with a flame-ionization detector. D-Mannose and D-glucose had retention times of 31 and 34 min, respectively. Day-to-day reproducibilities (CV) of between 2 and 12% for mannose were attainable for quantities of 100--900 ng. Related hexoses and derivatives do not interfere. The sensitivity was such that 10 mg/L could be detected in a 0.1-mL sample of serum. This method may be of use in diagnosing invasive Candida infection.

Evaluation of a vented drainage system in prevention of bacteriuria.

A vented urinary drainage system was compared to an otherwise identical non-vented system in a prospective, randomized, double-blind study. Among the 316 female patients evaluated there was a significant reduction in the rate of bacteriuria after 10 days using the vented system (66 per cent in the non-vented group versus 26 per cent in the vented group, p less than 0.05), while no significant difference could be demonstrated among the 190 male patients. We used urine hemoglobin as an indicator of mucosal trauma that might predispose to bacteriuria and no significant difference could be shown between the 2 drainage systems.