Chloramphenicol clearance in typhoid fever: implications for therapy. We prospectively studied the pharmacokinetics of intravenous Chloramphenicol succinate (CS) in children (age 6 months-14 years) with culture proven typhoid fever (n = 30) and non typhoidal illnesses (n = 10). CS was administered in three different dosage regimens (50, 75 and 100 mg/kg/d-q 6 hourly). Liver function tests were monitored. Plasma trough and peak chloramphenicol concentrations were measured by HPLC analysis after 42 hrs. The 50 mg/kg/day dosage schedule was terminated midway through the study, as blood levels were consistently low and two patients with typhoid relapsed, children with typhoid had significantly lower clearance of CS in comparison with those with non-typhoidal illness (0.29 +/- 0.1 versus 0.5 +/- 0.37 1/kg/hr, P 0.05). There was no significant difference between mean peak and trough concentrations of chloramphenicol on 100 mg/kg/day and 75 mg/kg/day in children with typhoid. However, two children on 100 mg/kg/day dosage developed trough concentrations greater than 20 mcg/ml. No correlation was found between CS clearance and serum bilirubin, SGPT (alanine transaminase) and alkaline phosphatase. Our data show altered clearance of CS in children with typhoid and suggests that 75 mg/kg/day may be a safer dose in children with hepatic dysfunction in typhoid.


Multidrug resistant Salmonella typhi in Delhi. In 1990, we isolated 158 strains of Salmonella typhi from blood cultures of patients suffering from typhoid fever. Seventy nine (50%) of these isolates were found to be simultaneously resistant to chloramphenicol, ampicillin and cotrimoxazole. These strains were also resistant to streptomycin and tetracycline, but sensitive to gentamicin, amikacin and cephalexin. The minimum inhibitory concentrations of chloramphenicol and trimethoprim for a representative number of these strains were found to be greater than 1024 micrograms/ml and greater than 128 micrograms/ml respectively. Majority of the multidrug resistant (MDR) strains tested against cefotaxime (23/23), ciprofloxacin (38/38) and amoxycillin plus clavulanic acid (23/24) were sensitive to these drugs.


Circulating immune complexes in leukaemias and lymphomas. Circulating immune complexes (ClC) were estimated in 78 patients of leukaemias and lymphomas by Clq deviation ELISA and PEG assay. In all leukaemias a significant elevation in ClC was seen at the time of first presentation. While in ALL a decrease occurred on therapy as partial or complete remission was achieved, no such fall was seen in AML or CML-BC when treated. ClC levels were much higher in non-Hodgkins lymphoma than in Hodgkins disease and showed a direct correlation with B symptoms and activity of the disease. The ClC levels were highest in null-ALL followed by those in common ALL and T-ALL. The mean duration of remission in patients of ALL without elevation in ClC was much longer than in those with ClC.


Iron nutritional status of preschool children. Low hemoglobin and low MCHC levels were indicative of high incidence of iron deficiency in preschool children. The extent of iron deficiency as assessed by serum ferritin and free erythrocyte protoporphyrin showed a different trend. While FEP levels were highly suggestive of extensive iron deficiency (in 40-45% of children below the age of 5 years), low serum ferritin was seen in only 16-20% of children. The discrepant finding of high serum ferritin, and high erythrocyte protoporphyrin despite low MCHC in the present study, possibly reflects iron deficiency status along with chronic infection resulting in hyperferritinemia and hyperprotoporphyrinemia. It may be also due to associated folate deficiency resulting in non utilization of iron leading to the elevated levels of protoporphyrin.


Attitude of children towards medication and health in urban and rural schools of Tamil Nadu. In most cases of Pediatric practice, medicine is prescribed to the child without taking into consideration his likes and dislikes. The medicines are usually thrust upon them. Though our foremost aim is to cure the patient, we should give due importance to make the process of cure as comfortable as possible. Keeping this in mind, opinion poll of around nine hundred school students from rural and urban population of age ranging from 11 years to 16 years was taken. The result is analysed and suggestion is given to rationalise the treatment for better compliance.


Hemostatic profile in neonatal septicemia. Hemostatic profile was studied in 25 full term non-asphyxiated neonates with blood culture-proven septicemia. Observations were compared with that of 25 healthy, non-asphyxiated, full term, birth weight and age-matched controls. Detailed coagulation tests & platelet studies were done in each of the 50 neonates by standard techniques. Hemostatic defects occurred in 96% of the septicemic neonates and none in the control group irrespective of the occurrence of clinical bleeding. The coagulation tests were deranged in 805 and platelet function tests in 92% of patients. These tests were significantly deranged in septicemic neonates as compared to control group.


Participatory health communication and action through women groups in rural areas. Twelve women groups in 10 villages of block Beri were identified and activized through participatory health communication actions for mother and child development. Women could undertaken responsibilities on key health problems concerning mothers and children. In these villages over 58% of antenatal mothers now used home made clean packets for delivery and chose right place for delivery. Most of them (60%) now initiate breast feeding immediately after delivery as against 23% in the year 1988. Over 54% of women now drink chlorinated water and almost same proportion used sugar salt solution in diarrheal diseases. Thus women have become self reliant in chlorination of wells and pots as also in diarrheal diseases management. Practice of hand washing has been almost universalized.


Prevalence and abuse of psychoactive substances in children and adolescents. The present study has been carried out in the slum areas of Gorakhpur city, covering a population of 10,187 in the four colleges of Gorakhpur. Five hundred and eighty children and adolescents in urban slums, and 750 college students between 10-18 years were studied by means of a questionnaire card for detection of prevalence rate and various others co-relates of abuse of psychoactive substances. Overall, prevalence of abuse of psychoactive substances was 25% in slum areas, and 18% in college students. Abuse of tobacco was most frequent (50.3% & 72.5%) followed by that of alcohol (11.7% & 16.2%) in both the groups respectively. Cannabis was also used by some children (0.6%). More abusers were from Hindu families with low educational status and low family income. Surprisingly no one was found to be abusing tablets and street drugs.


Epidemiological co-relates of low birth weight in rural Tamil Nadu. A total of 328 consecutive births born between July and September 1990 were analysed. The rate of LBW was 24.6%. The mean birth weight was 2.72 kg (+/- 0.44 kg). Association between LBW and parity, mother's age, mother's height, gestational weight, risk status at pregnancy and antenatal care was observed. These results indicate that there is a need to strengthen maternal services to address the problem of LBW in India.


Epidemiological evaluation of oral polio vaccine efficacy in Delhi. Forty seven cases of poliomyelitis and 94 controls were studied for immunization status. Unmatched analysis with one control per case and two controls per case was done to find out the ratio of the odds of immunization in diseased individuals as compared with the nondiseased (odds ratio). This ratio (OR) was used further to calculate oral polio vaccine efficacy. OPV efficacy was found to be 93% with 95% confidence limits of 75-98%.


Citrobacter sepsis in infants.

Citrobacter sepsis in infants. A study of blood cultures from 320 cases of neonatal sepsis showed 136 (42.5%) to be positive for bacterial growth; of these 82 (60.29%) isolates being gram negative bacilli. Citrobacter was the commonest gram negative bacilli isolated. Other commonly isolated gram negative organisms were Pseudomonas, Klebsiella, Salmonella typhimurium, Acinetobacter and Escherichia coli. Antibiotics susceptibility pattern revealed the isolates to be resistant to commonly used antibiotics.


Oral rehydration therapy in severely malnourished children with diarrheal dehydration. Fifty patients of grade III & IV malnutrition with diarrhoeal dehydration were rehydrated using the WHO recommended ORS. Serum sodium and potassium levels were estimated at admission and 24 hours later. Forty seven patients were successfully rehydrated orally. In 7 patients the level of dehydration at initial assessment was overestimated. Periorbital edema developed in 25.5% of the patients rehydrated. No patient had cardiac failure or convulsions during therapy. Though persistent hyponatremia and hypokalemia were found in 10.6% and 19.15% cases respectively after rehydration, the incidence decreased as compared to the pre-hydration levels and was comparable to that found in malnourished children without diarrhea who served as controls in the present study. Oral rehydration was discontinued in three patients due to development of excessive vomiting in one case and paralytic ileus in two. Thus WHO ORS can be used safely in children with severe malnutrition but constant monitoring is required.


Influence of maternal nutritional status on mode of delivery and asphyxia neonatorum. To assess the influence of maternal malnutrition on the mode of delivery and asphyxia neonatorum, a cross sectional survey of 615 women in the age group of 20-28 yrs at the time of delivery was done. Women with chronic ailments and complicated pregnancies were excluded. The mothers were then classified into three groups based on Weight Height Product Index (WHPI) namely well nourished (WN), moderately malnourished (MMN) and severely malnourished (SMN). The proportion of asphyxiated babies among the three groups did not differ (P greater than 0.05). Abnormal deliveries like caesarean section were more common among SMN group compared to WN group ((P less than 0.01). No such difference was made out between MMN and WN groups (P greater than 0.05).


Failure to isolate verotoxin producing Escherichia coli from patients of haemolytic uremic syndrome. Haemolytic Uremic Syndrome (HUS) been defined as the simultaneous occurrence of acute renal failure in children with haemolytic anemia and thrombocytopenia. This clinical condition that has been recognized is an important cause of acute renal failure in children.


Perinatal risk factors in neonatal infections. Perinatal risk factors were studied among 50 cases of neonatal septicemia and 200 matched normal neonates during one year period. The consanguinity among parents, birth order and sex of the baby did not increase the risk for developing septicemia. There was significant increase in the risk for septicemia when the duration of labour was more than 24 hours (P less than 0.01), time interval between rupture of membrane and delivery of baby was more than 12 hours (P less than 0.001), liquor was meconium stained or foul smelling (P less than 0.001) and delivery was operative (P less than 0.01). The neonatal factors identified with risk for septicemia were preterm delivery (P less than 0.01), low birth weight (P less than 0.01), birth asphyxia (P less than 0.001) assisted ventilation (P less than 0.001) and intravenous alimentation (P less than 0.02). Identification of high risk pregnancies and appropriate management can minimize many of the above risk factors which in turn will reduce the occurrence of neonatal sepsis.


Effect of G-6 PD deficiency on sickle cell disease in Saudi Arabia. High incidence of G6PD deficiency has been reported in areas of the eastern province of Saudi Arabia where sickle cell gene is also prevalent. This study was conducted to assess the co-incidence of this enzymopathy with Hb S and its influence upon the clinical and hematological expression of sickle cell disease. Eighty three children with SS disease, 145 patients with sickle cell trait and 100 random cord blood as samples with normal Hb AF, and an FS electrophoretic pattern respectively were examined. The frequency of interaction of G6PD deficiency with Hb S was found significantly increased but no effect of this enzyme defect was discerned on the clinical and hematological status of homozygous sickle cell disease.


Contrasting excitatory and inhibitory effects of adenosine in blood pressure regulation. Administration of adenosine results in profound hypotension without the expected activation of reflex sympathetic and renin mechanisms in most animal models. This action can be explained by the vasodilatory and neuroinhibitory effects of adenosine. It is generally considered an inhibitory neuromodulator because it inhibits the release of virtually all neurotransmitters studied and produces hyperpolarization of neurons. In contrast, adenosine produces vasoconstriction of some vascular beds, including the renal and pulmonary circulations. Renal vasoconstriction is caused by activation of A1 receptors and involves an interaction with angiotensin II. In other vascular beds adenosine releases eicosanoids, including thromboxane, also resulting in vasoconstriction. Adenosine-induced vasoconstriction is transient and species dependent. Neither the receptor type, the molecular mechanisms of these actions, nor their significance to pathophysiological processes have been defined. Adenosine also has an apparent excitatory effect in the nucleus tractus solitarii. Microinjections of adenosine into this brain stem nucleus lead to decreased sympathetic tone and hypotension similar to those produced by the excitatory amino acid glutamate. The mechanism that explains this action has recently been explored and involves the release of glutamate by adenosine. Adenosine also stimulates afferent fibers mediating sympathetic activity, including renal and myocardial afferent nerves, and carotid and aortic chemoreceptors. Afferent nerve activation seems to be more pronounced in humans and may explain most of the cardiovascular and respiratory actions of adenosine in this species. Finally, animal studies suggest that endogenous adenosine plays a role in the regulation of the baroreceptor reflex and restrains the full expression of renin-dependent hypertension.


Trandolapril inhibits atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. The effects of trandolapril (0.25 mg/kg body wt per 48 hours) on aortic atherosclerosis were examined in the Watanabe heritable hyperlipidemic rabbit treated from 3 to 12 months of age. Trandolapril caused a significant decrease in atherosclerotic involvement of the intimal surface of total aorta from 56.3 +/- 5.0% in control Watanabe rabbits to 35.0 +/- 4.1% with treatment (p less than 0.01). The largest reductions were observed in descending thoracic aorta where 21.8 +/- 5.7% of intimal surface was involved in the trandolapril-treated animals versus 54.4 +/- 7.7% in the control group (p less than 0.01). Significant decreases also occurred in ascending aorta/arch and abdominal aortic segments. Cholesterol content of descending thoracic aorta was also significantly reduced in the trandolapril-treated rabbits. The atherosclerotic plaques in aorta from trandolapril-treated rabbits appeared to contain less foam cells and relatively greater amounts of connective tissue than those from control animals. These studies indicate that trandolapril inhibits aortic atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. The similarity in results between the current study and that using captopril suggests that the antiatherosclerotic action of trandolapril and captopril represents a class effect related to angiotensin converting enzyme inhibition.


Renin expression in renal ablation.

Renin expression in renal ablation. To determine whether expression of the renin-angiotensin system (RAS) is influenced by the degree of renal ablation, male Sprague-Dawley rats underwent uninephrectomy, 1 1/3 nephrectomy, or sham operation. Renin and angiotensinogen messenger RNA (mRNA) were not different among the three groups 2 weeks after surgery. The time course of expression of renin mRNA after 1 1/3 nephrectomy showed no difference versus controls at 2 and 4 weeks and a decrease at 6 weeks after surgical ablation. Because nephrons adjacent to the infarcted area in the 1 1/3 nephrectomy may be hypoperfused and a source of increased renin synthesis, intrarenal distribution of tissue renin content, renin mRNA, and immunostainable renin were examined in separate groups of rats subjected to 1 1/3 nephrectomy. The kidney was divided into two pieces, one containing the scar and scar-adjacent tissue and the other portion the tissue distant from the scar. Tissue renin content, renin mRNA, and immunostainable renin were significantly greater in the scar-adjacent tissue compared with the nonscar tissue. Immunoreactive renin was seen in the juxtaglomerular apparatuses as well as in vascular elements proximal to the juxtaglomerular apparatus and within mesangial cells of some glomeruli of the scar-adjacent tissue. In conclusion, immunostainable renin, tissue renin content, and renin mRNA were increased in scar-adjacent tissue after 1 1/3 nephrectomy. We speculate that this unique scar-associated redistribution of renin may play a pathophysiological role in the progression of renal disease.


Collagen deposition and the reversal of coronary reserve in cardiac hypertrophy. The aim of this study was to clarify how collagen deposition or medial hypertrophy of the vascular wall affects the coronary dilator reserve in pressure-overloaded hearts and whether inhibition of collagen deposition reverses the abnormalities after relief of pressure overload. We used ascending aortic banding and debanding methods and superimposed beta-aminopropionitrile in some of the banded rats (50 mg/kg i.p., twice a day). Ten weeks of banding increased in vivo peak systolic left ventricular pressure and produced medial hypertrophy, an increase in collagen deposition in the myocardial and perivascular tissues, and myocardial hypertrophy in the banded group without beta-aminopropionitrile treatment. Superimposition of beta-aminopropionitrile treatment on banding inhibited the increase in collagen deposition. In the groups debanded after the 10-week banding period, both with and without beta-amino-propionitrile treatment, medial and myocardial hypertrophy regressed 4 weeks after debanding. We estimated coronary dilator reserve in Langendorff preparations perfused with modified Tyrode's solution containing oxygenated bovine red blood cells and serum albumin. The ratio of reactive peak flow after brief ischemia-to-resting flow decreased in both of the banded groups. After debanding, the ratio remained lower in the banded group without beta-aminopropionitrile treatment than in the control group. However, debanding in the group with beta-aminopropionitrile treatment increased the ratio to a level similar to that of the control group. Thus, in pressure-overloaded cardiac hypertrophy with coronary hypertension, coronary reserve seems to be determined by medial hypertrophy independently of collagen deposition, but collagen deposition plays an important role in the reversal of vasodilator reserve after relief of the overload.


Cerebral glucose utilization and blood flow in adult spontaneously hypertensive rats. Not only blood pressure but also behavioral activity, brain morphology, and cerebral ventricular size differ between young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. This suggests that cerebral blood flow and cerebral metabolism may vary between these two rat strains. To test this hypothesis, we measured local cerebral glucose utilization in 31 brain areas of 26-30-week-old rats. Local cerebral blood flow was also assessed in these same areas. Cerebral glucose utilization was measured by the 2-deoxyglucose method; cerebral blood flow was determined by the iodoantipyrene method. In virtually all gray matter structures, the apparent rate of glucose utilization was lower in SHR than in normotensive WKY rats; the interstrain differences varied significantly among structures and were statistically significant (uncorrected t tests) in 14 of 28 gray matter areas. Local cerebral blood flow was fairly similar in the two rat strains. The coupling of blood flow to glucose utilization varied significantly among brain areas in normotensive WKY rats as well as in SHR. In a number of gray matter structures, the coupling of flow to metabolism differed between hypertensive and normotensive animals. These data suggest that for many brain areas, either glucose utilization or glucose partitioning differs between WKY rats and SHR.


Effects of L-arginine on forearm vessels and responses to acetylcholine. This study was designed to investigate the effects of L-arginine (the substrate of endothelium-derived nitric oxide) in human forearm vessels. We examined whether intra-arterial infusion of L-arginine dilated forearm vessels and augmented vasodilatory responses to acetylcholine in young, healthy humans. The left brachial artery was cannulated for drug infusions and direct measurement of arterial pressure. Forearm blood flow was measured by a strain gauge plethysmograph. Intra-arterial infusions of L-arginine at 10, 20, 40, and 60 mg/min increased forearm blood flow from 4.7 +/- 0.6 to 4.9 +/- 0.5, 5.7 +/- 0.5, 7.2 +/- 0.8, and 8.2 +/- 0.9 ml.min-1.100 ml-1, respectively (n = 8, p less than 0.01), whereas D-arginine at the same doses did not alter forearm blood flow (n = 7). Intra-arterial infusions of acetylcholine (n = 7) (4, 8, 16, and 24 micrograms/min) and sodium nitroprusside (n = 5) (0.2, 0.4, 0.8, and 1.2 micrograms/min) increased forearm blood flow dose dependently (p less than 0.01 for both). Arterial pressure was not altered with infusions of these drugs. Responses to acetylcholine were augmented with simultaneous intra-arterial infusion of L-arginine at 10 mg/ml (p less than 0.01) but not with D-arginine. Responses to sodium nitroprusside were not altered by L-arginine. These results in human forearm resistance vessels support the notion that vasodilation induced by acetylcholine is a result of the conversion from L-arginine to endothelium-derived nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)


Phosphoramidon-sensitive effects of big endothelins in the perfused rabbit kidney. The enzymatic conversion of human big endothelins (1, 2, and 3) to their respective active metabolites (endothelin-1, -2, and -3) was investigated in the perfused rabbit kidney through the pressor- and eicosanoid-releasing properties of these peptides. Intra-arterial bolus injections of endothelin-1 and -2 (5-50 pmol), endothelin-3 (100-250 pmol), and big endothelin-1 and -2 (100-250 pmol) into the kidney produced dose-dependent increases of perfusion pressure, whereas big endothelin-3 was inactive at doses up to 1,000 pmol. Endothelin-1 and -2 (10 nM), endothelin-3 (100 nM), and big endothelin-1 and -2 (100 nM) are potent enhancers of prostacyclin release without inducing any release of thromboxane B2 in the perfused kidney. In contrast, big endothelin-3 did not trigger the release of eicosanoids. A metalloprotease inhibitor, phosphoramidon (100 microM, 60 minutes), reduced the prostanoid release and pressor responses induced by big endothelin-1 and -2 without affecting the response induced by endothelin-1, -2, and -3. These results suggest the presence of a phosphoramidon-sensitive endothelin converting enzyme that converts the precursors of endothelin-1 and -2, but not of endothelin-3, in the renal vasculature of the rabbit.


Impaired renal vascular reactivity in prehypertensive Dahl salt-sensitive rats. We have previously shown that renal vascular resistance is less in Dahl salt-sensitive rats than salt-resistant rats fed 1% NaCl diets; however, renal vascular resistance increases before nonrenal vascular resistance as salt-sensitive rats develop hypertension when fed 8% NaCl diets. When salt-resistant rats are given 8% NaCl diets, renal vascular resistance decreases. The current study reports effects of atrial natriuretic peptide, nitroprusside, norepinephrine, angiotensin II, and endothelin-1 on renal and nonrenal vascular resistance in prehypertensive salt-sensitive and salt-resistant rats given 1% NaCl diets; doses used did not affect blood pressure. Resistance of nonrenal vessels in salt-sensitive and salt-resistant rats responded similarly to dilators or constrictors. However, atrial natriuretic peptide and nitroprusside decreased renal vascular resistance of salt-resistant rats (by 65%, p less than 0.01) but not that of salt-sensitive rats. Norepinephrine, angiotensin II, and endothelin-1 increased renal vascular resistance in salt-sensitive rats by 126%, 135%, and 135%, respectively (p less than 0.01); norepinephrine and angiotensin II did not change renal vascular resistance of salt-resistant rats, but endothelin-1 decreased renal vascular resistance in salt-resistant rats by 30% (p less than 0.01). Reactivity of nonrenal blood vessels in prehypertensive salt-sensitive and salt-resistant rats was similar when infused with dilators or constrictors in doses used. By contrast, renal vessels of salt-sensitive rats did not dilate in response to atrial natriuretic peptide and nitroprusside but were hypersensitive to norepinephrine and angiotensin II. Endothelin-1 caused renal vasoconstriction in salt-sensitive rats and renal vasodilation in salt-resistant rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiovascular and plasma catecholamine responses to exercise in blacks and whites. The purpose of the present study was to assess possible racial differences in cardiovascular and plasma catecholamine responses to dynamic exercise. A biracial group of normotensive college-age men (15 blacks, 15 whites) were tested for maximal oxygen uptake, resting blood pressure, and heart rate. Subjects then rode a cycle ergometer at 25%, 50%, and 75% of peak oxygen uptake (6 minutes at each stage). Blood pressure and heart rate were measured during supine rest, seated rest, and at each stage of exercise with an automated blood pressure monitor. At each stage, venous blood was sampled to allow determination of plasma norepinephrine and epinephrine, and cardiac output was measured with the carbon dioxide rebreathing technique. The results indicated that resting blood pressure was similar for blacks and whites (114/68 versus 115/68 mm Hg, respectively). Blacks exhibited greater systolic and diastolic blood pressures during submaximal dynamic exercise. However, blacks also showed a trend toward a positive parental history of hypertension, which has been associated with an increased pressor response. Racial differences did not exist for heart rate or cardiac output, but blacks had higher values for total peripheral resistance both at rest and during exercise. Although no overall racial differences were seen for plasma catecholamine concentrations at rest, blacks had significantly lower levels of norepinephrine (1,275 versus 1,556 pg/ml) and higher levels of epinephrine (306 versus 216 pg/ml) than whites at the highest work rate. The current study confirms the increased pressor response to exercise in normotensive blacks. Blacks had an elevation in total peripheral resistance that was not accompanied by an increase in plasma norepinephrine levels.


Variability between current definitions of 'normal' ambulatory blood pressure. Implications in the assessment of white coat hypertension. The assessment of white coat hypertension is complicated by the lack of generally agreed-on normal limits of ambulatory blood pressure. To assess the influence of four of these limits on the prevalence of white coat hypertension and the corresponding distribution of left ventricular hypertrophy, we performed 24-hour ambulatory blood pressure monitoring and echocardiographic studies in 346 untreated patients with essential hypertension and 47 age-matched normotensive control subjects. The upper limits of normal daytime ambulatory blood pressure were lower using standards drawn from clinically normotensive populations than using standards drawn, partly or entirely, from general populations. The prevalence of white coat hypertension differed markedly using the different standards, being 12.1%, 16.5%, 28.9%, and 53.2% (chi 2 = 346.0, p less than 0.0001). Left ventricular mass index averaged 77 g/m2 in the control group, 85 g/m2 in the two groups with white coat hypertension defined by using standards drawn from normotensive populations (both comparisons not significant versus control group), and 90 and 98 g/m2 in the two groups with white coat hypertension defined by using the other two standards (both p less than 0.01 versus control group). The prevalence of echocardiographic left ventricular hypertrophy was 0% in the control group, 2.4% and 3.5% in the two groups with white coat hypertension defined by using standards drawn from normotensive populations, and 9.0% and 14.7% in the other two groups with white coat hypertension (p less than 0.05 and p less than 0.01, respectively, versus control group).(ABSTRACT TRUNCATED AT 250 WORDS)


Increased waist/hip ratio, metabolic disturbances, and family history of hypertension. To test whether nonhypertensive subjects with a two-generation positive family history of hypertension (PFH) are characterized by disturbed glucose metabolism, 16 men (38 +/- 6 years old) with PFH and 25 subjects matched for age and with negative family histories of hypertension (NFH) were recruited. Blood pressure; serum lipids; erythrocyte transmembrane sodium transport; and the glucose, plasma insulin, and C-peptide responses to an oral glucose tolerance test were investigated. Subjects with PFH had higher blood pressure, body weight, body mass index (BMI), waist/hip ratio (WHR), and abdominal sagittal diameter than subjects with NFH. Baseline blood glucose, plasma insulin, serum lipids, and transmembrane sodium transport did not differ between the two groups. Blood glucose levels at 90 and 120 minutes after oral glucose were significantly higher in subjects with PFH than in controls. Blood glucose adjusted for BMI and WHR at 90 minutes was significantly related to a PFH. Plasma insulin level at 90 minutes during the glucose load was significantly higher in subjects with PFH. In multivariate analysis, WHR was significantly related to baseline blood pressure, insulin, and cholesterol, whereas BMI was significantly associated with the insulin response to the oral glucose tolerance test. Transmembrane sodium transport was significantly related to blood pressure only. In conclusion, subjects with PFH are characterized by increased body weight and BMI, increased visceral fat accumulation, and an altered blood glucose response to an oral glucose load. It was also shown that WHR was related to blood pressure and that BMI was more related to cholesterol and response to glucose loading than a PFH was.


Regulation of vasopressin receptors in deoxycorticosterone acetate-salt hypertension. Since arginine vasopressin may play a role in mineralocorticoid hypertension, we examined the effects of deoxycorticosterone acetate (DOCA)-salt on vasopressin V1 and V2 receptor binding and their second messengers, inositol phosphate and adenylate cyclase, respectively, in liver and kidney to determine whether altered vasopressin receptor binding is pathogenetic in mineralocorticoid hypertension. The mean arterial blood pressure of mineralocorticoid (DOCA-salt)-treated rats (163 +/- 1 mm Hg) was increased compared with control salt-treated rats (salt) (122 +/- 1 mm Hg) and water-treated rats (120 +/- 1 mm Hg; p less than 0.001). Mineralocorticoid treatment also increased plasma sodium, osmolality, and vasopressin concentration (p less than 0.001). In the hypertensive animals, there was a reduction in hepatic V1 (DOCA-salt, 91 +/- 12; salt, 132 +/- 13; and water, 145 +/- 13 fmol/mg protein; p less than 0.05) and renal V2 receptor binding density (DOCA-salt, 53 +/- 5; salt, 93 +/- 9; and water, 95 +/- 9 fmol/mg protein; p less than 0.01), although receptor affinities remained unaltered. In contrast, the density of renal V1 receptors was increased by mineralocorticoid treatment (DOCA-salt, 24 +/- 2; salt, 16 +/- 2; water, 18 +/- 1 fmol/mg protein; p less than 0.05), although the affinity was unchanged. Downregulation of V2 receptors was associated with a decrease in maximum cyclic adenosine monophosphate levels (DOCA-salt, 19 +/- 4; salt, 49 +/- 6; water, 53 +/- 9 pmol.mg protein-1.10 min-1; p less than 0.05), whereas changes in V1 receptor levels were not associated with changes in maximum inositol phosphate levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Association of red blood cell sodium leak with blood pressure in recombinant inbred strains. Red blood cell Na+ content as well as ouabain-resistant Na+ and Rb+ (K+) transport (susceptible or resistant to inhibition by loop diuretics) were determined in spontaneously hypertensive rats (SHR) and normotensive Brown Norway (BN) rats the erythrocytes of which were incubated in either saline or Mg(2+)-sucrose medium. Elevated ouabain-resistant Na+ net uptake contrasted with slightly decreased red blood cell Na+ content in SHR compared with BN rats. Acceleration of furosemide- and bumetanide-sensitive Na+ fluxes contributed to enhanced ouabain-resistant Na+ influx into SHR erythrocytes in saline medium, whereas higher furosemide- or bumetanide-resistant Na+ efflux caused greater ouabain-resistant Na+ efflux in Mg(2+)-sucrose medium. Furosemide- and bumetanide-resistant Rb+ leaks were augmented in SHR erythrocytes. The association of the disclosed ion transport alterations with blood pressure was examined in 20 recombinant inbred strains derived from F2 SHR x BN hybrids. Ouabain-resistant Na+ uptake as well as furosemide- and bumetanide-resistant Na+ inward leaks (but not red blood cell Na+ content or furosemide- and bumetanide-sensitive Na+ net uptake) cosegregated with systolic and pulse pressures but not diastolic pressure of the recombinant inbred strains. In contrast, neither ouabain-resistant Na+ efflux nor any component of ouabain-resistant Rb+ uptake correlated positively with blood pressure of the recombinant inbred strains. Increased ouabain-resistant Na+ influx was compensated for by accelerated ouabain-sensitive Na+ extrusion because red blood cell Na+ content was not elevated in the hypertensive strains. Thus, high cell Na+ turnover rates might be related to genetic hypertension if an altered Na+ inward leak would be less effectively compensated for in tissues involved in cardiovascular regulation.


Oligodendrocytes and myelin formation along the optic tract of the developing hamster: an immunohistochemical study using the Rip antibody. The monoclonal antibody Rip recognizes an antigen specific to oligodendrocytes and their processes (Friedman et al: Glia 2:380, 1989). We have used this antibody to document the appearance of oligodendrocytes and the sequence of ensheathment of axons along the optic tract (OT) and within its major target areas in neonatal (P3-P21) and adult hamsters. Myelination of axons in the visual pathway follows an overall proximo-distal gradient. On P3, immunopositive, pre-ensheathing oligodendrocytes are detected in the OT ventral to the lateral geniculate body (LGB) whereas myelin segments are present around OT axons by P5. The first pre-ensheathing oligodendrocytes are detected medially in the LGB on P7 and myelinated axons in the overlying OT by P11. In the superior colliculus, pre-ensheathing oligodendrocytes are present in the optic fiber layer (SO) on P7, but not in the superficial gray layer (SGS) until P11. Myelination of axons within SO proceeds along a marked rostro-caudal gradient. On P14, axons in rostral SO are heavily myelinated; thereafter, ensheathment continues caudally within the SO and the SGS. The progressive invasion of oligodendrocytes along the proximo-distal axis of the optic pathway, and the corresponding myelination of OT axons, are discussed in the context of a possible inhibitory role of oligodendrocytes in regulating the regenerative propensity of retinotectal axons.


Immuno-electron microscopical localization of vimentin and glial fibrillary acidic protein in mouse astrocytes and their precursor cells in culture. Immuno-electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP-vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP-vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell.


Serotonin uptake by astrocytes in situ.

Serotonin uptake by astrocytes in situ. Co-localization of glial fibrillary acidic protein (GFAP) and radioactivity was examined after intraventricular injection of [3H]5-HT in adult rat brains. Radioactivity localized over GFAP-positive astrocytes was seen, especially when image-enhancing techniques were applied to the data. Also slices prepared from astrogliotic hippocampi of rats pretreated with kainic acid showed a twofold increased uptake of [3H]5-HT compared to control slices. This indicates that the uptake of [3H]5-HT seen in primary astrocyte cultures also occurs for astrocytes in situ. Also, as with astrocyte cultures, only some of the GFAP(+) astrocytes in situ showed localization of radioactivity, supporting the concept of intraregional heterogeneity of astrocyte functions.


Glial response to dorsal root lesion in the irradiated spinal cord. Exposure of the rat lumbar spinal cord to X-rays during the early postnatal period results in a marked reduction in the glial populations within the irradiated region. The present study was undertaken to determine what effects this reduction of glia, particularly astrocytes, has on the pattern and characteristics of the scar formation that follows root injury in the normal spinal cord. Morphological assessments 60 days following injury of the right L4 dorsal root revealed a distinct difference in the extent of the astrocyte response between the irradiated and the nonirradiated rats. In the nonirradiated animals, a thick astrocytic scar composed of multiple layers of astrocyte processes formed over the dorsal horn and adjacent portions of the dorsal surface of the cord. This astrocytic response was not confined to the surface of the spinal cord but extended also into the root, i.e., into regions normally considered as PNS. In irradiated rats, the astrocytes did not form a thick scar nor did they extend into the injured root. Instead, they formed a glia limitans and were at most only one or two layers thick over the region of cord comparable to that occupied by the thick astrocytic scar in the nonirradiated rats. Mechanisms involved in the glial response of the irradiated spinal cord to dorsal root injury are discussed, particularly with regard to the possible positive effect that this reduction in scar formation may have on regrowth of injured dorsal root axons into the spinal cord environment.


Current issues in profiling quality of care. Profiling provider performance for the assessment of quality involves a number of issues related to selection of appropriate quality measures, subsequent data collection and analysis, and selection of standards of comparison. This article emphasizes the limitations of current data systems for this purpose and discusses hierarchical modeling as the optimal analytic approach for analyzing resulting data. Mention is made of the difficulties of achieving large enough sample sizes for statistical significance at the individual provider level. Finally, the article discusses feasible options for profiling quality.


Physician-based measures of Medicare access. Physician payment reforms implemented in January 1992 have dramatically changed the way payments for services are determined under the Medicare Part B program. This paper presents new measures of access using physician-level data that provide a baseline for monitoring changes in access that might occur as these payment reforms unfold and that allow us to examine recent access trends. Our results suggest that Medicare beneficiaries as a group currently have a high degree of access to care, and that access generally improved between 1986 and 1990.


Rural and urban hospital closures, 1985-1988: operating and environmental characteristics that affect risk. Due to congressional concern that rural hospitals were particularly disadvantaged by Medicare's Prospective Payment System, the U.S. General Accounting Office investigated the role of Medicare and other factors in hospitals' risk of closure. This paper reports on the findings of that study, which compared the risk of closure among urban and rural hospitals during 1985 to 1988, the period after implementation of PPS. When hospital operating and environmental characteristics were held constant, the odds of closure in rural and urban areas differed significantly only for private nonprofit hospitals. Although a number of factors were associated with hospitals' higher risk of closure, we did not find evidence that Medicare was a major factor associated with financial distress or closure during the 1985 to 1988 period.


Zidovudine's impact on resource use by patients with symptomatic HIV illness: a large sample analysis. This study used a longitudinal data set of 4,957 patients and 39,455 patient-months of observation, drawn from 17 months of data on patients with symptomatic HIV disease identified from the New York State Medicaid Program. Multivariate regression analysis was used to evaluate the effect of AZT on the use of several medical services. The results show that AZT produced substantial reductions in resource use. Patients who took AZT were hospitalized fewer days per month and, including the cost of the drug, had Medicaid expenditures that were several hundred dollars lower per month. The reduction in total expenditures is largely accounted for by a reduction in inpatient expenditures and an increase in pharmacy expenditures. These effects were temporary, however, as the impact of the drug decreased with each month of use. There were no declines in expenditures and utilization after approximately 7 to 9 months of AZT use.


The process and outcome of hospital care for Medicaid versus privately insured hospital patients. Some have argued that low Medicaid payment rates compromise the accessibility and quality of medical care for Medicaid beneficiaries. In this study we compare the process and outcome of hospital care for Medicaid versus privately insured hospital patients. We studied 4,033 emergency patients admitted with a principal diagnosis of acute myocardial infarction, to Massachusetts hospitals in 1987. After we statistically adjusted for differences among patients relating to clinical and demographic characteristics and the type of hospital where treatment occurred, we found that the Medicaid patients had longer hospital stays but were less likely to receive three selected coronary procedures. Moreover, after controlling for confounding variables, we found the risk of death for Medicaid patients to be almost twice as high as for privately insured patients.


Improving organ donation: compensation versus markets. Proposals suggesting monetary compensation of organ donors to increase donation rates for transplantation are founded on the ethical premise that the principal criterion for organ procurement policy should be patients' health and not the personal preferences and philosophies of policymakers. In this paper, we argue that a market-based organ procurement system is superior to both the current altruistic-based system and a system based on compensation without a market. A market system would address both the problem of potential donors refusing to donate and that of their never being asked, whereas the altruistic system addresses neither problem and a system of compensation addresses only the former. Empirical evidence suggests that the latter (not being asked) is the predominant cause of the current shortage of organs. Our discussion shows that collection rates are likely to be substantially higher under a market system.


Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria. Oligosaccharide side chains of human colonic mucins contain O-acetylated sialic acids and glycosulfate esters. Although these substituents are considered to protect the chains against degradation by bacterial glycosidases, sialate O-acetylesterase, N-acetylneuraminate lyase, and glycosulfatase activities have been found in fecal extracts. To better define the source of these activities, we measured extracellular and cell-bound sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities produced by 23 isolates of human fecal bacteria grown anaerobically in a hog gastric mucin culture medium; these represented dominant populations of fecal anaerobes, facultative anaerobes, and the subset of mucin oligosaccharide-degrading bacteria. Every strain produced sialidase and high levels of arylesterase, and all but five facultative anaerobes produced sialate O-acetylesterase. Sialic acids containing 2 mol or more of O-acetyl ester per mol of sialic acid were cleaved from mucin glycoproteins more slowly by sialidases of mucin oligosaccharide-degrading stains than were sialic acids containing 1 or 0 mol, and only N-acetyl- and mono-O-acetylated sialic acids were recovered from enzyme digests of a mucin containing di-O-acetylated sialic acids. No detectable N-acetylneuraminate lyase activity was produced by any strain, but low activity was induced by increasing the glycoprotein-bound sialic acid concentration in the culture medium of six Escherichia coli strains. Using lactitol-6-sulfate as a substrate, we found weak glycosulfatase activity in the partially purified, concentrated enzyme mixture in the culture supernatants of four mucin oligosaccharide-degrading strains but in none of the unconcentrated culture fractions. We conclude that the presence of two or more O-acetyl groups on sialic acids inhibits enteric bacterial sialidases but that production of sialate O-acetylesterases by several populations of enteric bacteria lessens the likelihood that mucin oligosaccharide chains terminating in O-acetylated sialic acids are protected from degradation. Sialate O-acetylesterases have a role in bacterial degradation of mucin glycoproteins in the human colon.


Effects of alginase on the natural history and antibiotic therapy of experimental endocarditis caused by mucoid Pseudomonas aeruginosa. The exopolysaccharide (alginate) of mucoid strains of Pseudomonas aeruginosa is believed to be an important virulence factor. The ability of an alginate-deploymerizing enzyme (alginase) to modify the polymorphonuclear leukocyte (PMN)-directed and antibiotic-mediated phagocytosis and killing of mucoid P. aeruginosa was studied both in vitro and in vivo. In vitro, pretreatment of a mucoid P. aeruginosa strain (144MR) resulted in a significant enhancement of PMN phagocytosis and killing of the organism (P less than 0.05), to levels similar to that observed with its nonmucoid mate, strain 144NM. Moreover, alginase treatment of the mucoid strain 144MR caused a substantial removal of bacterial cell surface alginate as assessed by immunofluorescence staining with a murine monoclonal antialginate antibody. The experimental endocarditis model was used to evaluate the in vivo effect of alginase in modifying the course of a deep-seated pseudomonal infection caused by mucoid strain 144MR. In right-sided endocarditis, in which PMNs normally mediate spontaneous clearance of the organism from cardiac vegetations (A. S. Bayer, J. Yih, C. Y. Chiu, and C. C. Nast, Chemotherapy 35:278-288, 1989), the presence of the alginate exopolysaccharide on strain 144MR was associated with an inability to reduce intravegetation pseudomonal counts over a 13-day postinfection period; in contrast, right-sided vegetations infected with the nonmucoid strain 144NM underwent significant reductions in bacterial densities over this same time (P less than 0.05). Administration of alginase intravenously (i.v.) (750 enzyme units per day for 7 days) to animals with right-sided endocarditis caused by the mucoid strain 144MR was associated with a significant reduction in intravegetation pseudomonal counts (P less than 0.05), to levels similar to that seen with endocarditis caused by the nonmucoid strain. In left-sided endocarditis caused by mucoid strain 144MR, animals received either no therapy, amikacin (20 or 40 mg/kg twice a day for 7 or 14 days), or amikacin plus alginase (750 U/day [i.v.]). The coadministration of alginase for 14 days with the higher-dose amikacin regimen rendered more left-sided vegetations culture negative than those in animals receiving the antibiotic alone for 7 or 14 days (P = 0.001 and 0.056, respectively). These salutary effects of alginase in vivo were paralleled by the ability of the enzyme to remove the exopolysaccharide from the surface of mucoid pseudomonal cells within cardiac vegetations, as assessed by transmission electron microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)


Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci. Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.


Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis. The P.69 Bordetella pertussis protective antigen was expressed by use of the trc promoter from the chromosome of a Salmonella typhimurium aro vaccine strain, BRD509, by integrating the prn gene, encoding the 93-kDa precursor of this protein, into the aroC locus. P.69 was detected on the cell surface of the S. typhimurium strain (BRD640) by agglutination and immunoelectron microscopy. BALB/c mice immunized orally or intravenously with BRD640 showed a significant level of protection against an aerosol challenge with virulent B. pertussis, compared with control animals. No anti-P.69 antibodies in the serum or anti-P.69 antibody-secreting cells in the lungs were detected in BRD640-vaccinated animals, although cells isolated from spleens showed a P.69-dependent cell proliferative response. In contrast, low levels of anti-P.69 antibodies in the serum and anti-P.69 antibody-secreting cells in the lungs were detected in immunized mice following a B. pertussis challenge.


Tumor necrosis factor and interleukin-6 in Candida albicans infection in normal and granulocytopenic mice. We administered a neutralizing monoclonal antibody to tumor necrosis factor (TNF) during infection with Candida albicans in normal and granulocytopenic mice. Mice were rendered granulocytopenic (less than 0.1 x 10(9) granulocytes per liter) with cyclophosphamide. Growth of C. albicans from the kidneys was significantly increased in normal mice treated with the antibody to TNF, compared with that in control mice, after 36 h (3.6 x 10(4) +/- 1.2 x 10(4) CFU per kidney versus 9.1 x 10(3) +/- 6.2 x 10(3) CFU per kidney; P less than 0.05) and after 72 h (3.7 x 10(6) +/- 2.7 x 10(6) CFU per kidney versus 2.3 x 10(4) +/- 1.3 x 10(4) CFU per kidney; P less than 0.01). In granulocytopenic mice, the antibody to TNF had no effect on the growth of C. albicans from the kidneys. Furthermore, our study showed that the cytokines TNF and interleukin-6 (IL-6) were produced in a dose-dependent manner during C. albicans infection. TNF was detectable between 6 and 60 h, with peak levels at 24 h. Both TNF and IL-6 levels were significantly higher in cyclophosphamide-treated mice than in normal mice. Heat-inactivated C. albicans induced a TNF response different from that induced by viable C. albicans, with an early peak occurring at 3 to 4 h and declining to non-detectable levels after 15 to 24 h. Peak levels of TNF obtained with heat-inactivated C. albicans were lower than those obtained with viable C. albicans. Our study demonstrates that TNF and IL-6 are produced systemically during C. albicans infection and suggests that TNF is essential for granulocyte antifungal activity in vivo.


Group B Streptococcus type II polysaccharide-tetanus toxoid conjugate vaccine. Group B streptococci (GBS) are the most common cause of bacterial sepsis and meningitis in neonates in the United States. Although the capsular polysaccharide of GBS is an important virulence factor, it is variably immunogenic in humans. In this report, we have increased the immunogenicity of GBS type II polysaccharide by coupling it to tetanus toxoid (TT). Like other GBS capsular polysaccharides, the type II polysaccharide has side chains terminating in sialic acid. Controlled periodate oxidation of native II polysaccharide resulted in the conversion of 7% of sialic acid residues to an analog of sialic acid, 5-acetamido-3,5-dideoxy-D-galactosyloctulosonic acid. TT was conjugated to free aldehyde groups created on the oxidized sialic acid residues by reductive amination. Serum from rabbits vaccinated with type II-TT conjugate (II-TT) vaccine contained antibodies specific to type II polysaccharide as well as to TT, whereas rabbits vaccinated with uncoupled native type II polysaccharide failed to produce a type-specific antibody response. Antibodies elicited by II-TT vaccine were serotype specific and mediated phagocytosis and killing in vitro of type II GBS by human peripheral blood leukocytes. Serum from rabbits vaccinated with II-TT vaccine provided 100% protection in a mouse model of GBS type II infection. Antibodies induced by II-TT vaccine were specific for the native but not desialylated type II polysaccharide, suggesting that an important antigenic epitope of II-TT vaccine was dependent on the presence of sialic acid. Therefore, the coupling strategy which selectively modified a portion of the sialic acid residues of types II polysaccharide before coupling the polysaccharide to TT preserved the epitope essential to protective immunity and enhanced the immunogenicity of the polysaccharide.


Immunoelectron microscopic studies reveal differences in distribution of sialo-oligosaccharide receptors for Mycoplasma pneumoniae on the epithelium of human and hamster bronchi. Long-chain sialo-oligosaccharides with poly-N-acetyllactosamine backbones (Ii antigen type) are major host cell receptors for the human pathogen Mycoplasma pneumoniae. Previous immunofluorescence studies of the human bronchial epithelium, using sequence-specific monoclonal antibodies to the branched I-type and linear i-type backbones, have indicated that sialylated and nonsialylated long-chain sequences of both types are richly expressed on the ciliated cells, where they are polarized at the apical aspects. These sequences are lacking in the goblet cells. In the present study, the display of these oligosaccharides has been investigated by electron microscopy (immunogold labelling) in the human bronchial epithelium and in that of the hamster, an animal model commonly used for M. pneumoniae infection. In the human bronchial epithelium, the long-chain branched sequences have been detected along the entire length of the cilia and on microvilli, whereas the linear sequences are confined to the microvilli and the basal aspects of the cilia. On the ciliated epithelial cells of the hamster, by contrast, the branched and linear sequences (sialo- and asialo-) have been detected exclusively on microvilli. A further striking difference is that in the hamster these structures are expressed in abundance on the goblet cells and in the intracellular globules. We suggest that the latter finding may partly explain the relatively large doses of M. pneumoniae required to establish experimental infection in the hamster, as the receptor-bearing secreted mucus may have a protective role in binding to the microorganisms, leading to their clearance by bronchociliary action.


Identification and characterization of a surface protein-releasing activity in Streptococcus mutans and other pathogenic streptococci. Surface proteins of Streptococcus mutans have been reported to be released into the culture filtrate at concentrations that vary with the growth conditions. The reason for this is not clear. The present study attempts to investigate the mechanism of the protein release. The results showed that whole cells and raffinose-stabilized protoplasts of S. mutans NG8, when incubated in buffers, were capable of releasing their surface proteins in a pH-dependent manner with optimal release at pH 5 to 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the released proteins were very complex. Two proteins, adhesin P1, which has been previously shown to interact with a human salivary agglutinin, and glucosyltransferase have been identified among the released proteins. The release of adhesin P1 and other proteins was found to be inhibited by heat, Cu2+,Zn2+, and thiol-blocking reagents. The inhibition by heat and Cu2+ was irreversible, whereas that by the thiol-blocking reagents was reversible. EDTA, phenylmethylsulfonyl fluoride, and N-p-tosyl-L-lysyl-chloromethyl ketone had no effect on the release of P1, indicating that the release was probably not due to proteolytic activity. Adhesin P1 from Cu(2+)-inactivated S. mutans NG8 protoplasts could be released by mixing with fresh whole cells and protoplasts, but not the culture filtrate, of a P1-negative mutant of NG8, suggesting that the enzyme is located on the cell surface. This P1-releasing activity was also detected in two other strains of S. mutans and one strain each of S. gordonii, S. agalactiae, S. pneumoniae, and S. pyogenes. The biological role(s) of this enzyme activity remains to be determined. However, owing to its ability to release virulent surface proteins from the cell, it may play an important role in cell surface modulation among the pathogenic streptococci.


Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice. The decreased synthesis of hepatic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of gluconeogenesis, that occurs during endotoxemia was shown previously in rats to occur at the transcriptional level. In the current study, the exogenous administration of human recombinant tumor necrosis factor (TNF), a proximal mediator of endotoxic shock, reduced the PEPCK transcription rate, mRNAPEPCK levels, and PEPCK enzyme activity in a time- and dose-dependent manner in CD-1 mice. Comparable amounts of circulating TNF were measured in mice 2 h after injection of human recombinant TNF (10(5) U) or a 50% lethal dose of Escherichia coli endotoxin (20 mg/kg). Direct action of TNF to decrease the PEPCK transcription rate was confirmed in vitro with H-4-II-E Reuber hepatoma cells, in which a dose-dependent inhibition of PEPCK transcription was observed with 1 to 100 U of TNF per ml. A role for TNF-elicited changes in PEPCK gene expression during endotoxemia was confirmed by the protective effect of rabbit polyclonal antibodies to recombinant murine TNF. C57BL/6 mice passively immunized with anti-TNF 4 h prior to endotoxin challenge exhibited normal PEPCK enzyme activity. Neutralization of circulating TNF with anti-TNF failed, however, to prevent the hypoglycemia commonly observed during endotoxemia, suggesting the participation of other mediators. Anti-TNF treatment reduced circulating interleukins 1 and 6 at 3 and 6 h after endotoxin treatment, respectively. These results suggest that during endotoxemia, the development of hypoglycemia is multifaceted and that several cytokines are most likely involved. The findings from the Reuber hepatoma cell model afford an opportunity in future work to map putative cytokine response elements in the PEPCK promoter responsible for perturbed hormonal regulation of the gene during endotoxemia.


Adhesion to and invasion of cultured human cells by Bartonella bacilliformis. Bartonella bacilliformis was tested for its ability to adhere to and invade tissue culture cell monolayers. The parasite was able to efficiently bind and penetrate human dermal fibroblasts, human laryngeal epithelium, and human umbilical vein endothelial cells. Exposure of the organism to immune serum prepared against a crude Bartonella extract containing cell wall and membranous material resulted in decreased ability of the parasite to invade host cells. There was also an overall reduction in the invasiveness of bartonellae and total host cell association when human laryngeal epithelial cells and human umbilical vein endothelial cells were preexposed to cytochalasin D, indicating an active involvement of host cells in the uptake of bartonellae. Transmission electron microscopy revealed the presence of bartonellae inside and outside intracellular vacuoles. These data suggest that a surface-associated factor is involved in the invasion process and that internalization of the parasite by host cells involves a microfilament-dependent process similar to phagocytosis.


Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.


Early expression of cytokine mRNA in mice infected with Listeria monocytogenes. Protective immunity first becomes evident at 3 to 4 days after inoculation of mice with a sublethal dose of Listeria monocytogenes. Recent evidence suggests that production of gamma interferon (IFN-gamma) occurs earlier (within the first 24 h of infection). The purpose of this study was to define better the sequence of cytokine mRNA expression during the early stages of L. monocytogenes infection. Cytokine mRNA expression was detected by polymerase chain reaction-assisted amplification of RNA extracted from the spleen cells of individual mice euthanized at 0.5 to 120 h after L. monocytogenes challenge. By using this method, mRNAs for tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-2, IL-4, IL-5, and IFN-gamma were detected in RNA from the spleen cells of uninfected mice. The intensity of the bands for IFN-gamma, however, was increased greatly at 16 h after intravenous injection of 5 x 10(4) CFU (nearly 1 50% lethal dose) of L. monocytogenes. IL-6 and granulocyte-macrophage colony-stimulating factor mRNAs were not detected in spleen cell RNA from uninfected mice but were induced within 30 and 60 min, respectively, after inoculation with L. monocytogenes. Increased amounts of mRNAs for IFN-gamma, IL-6, and granulocyte-macrophage colony-stimulating factor were detected after injection of viable, but not killed, L. monocytogenes. IL-3 mRNA was not detected at any time in RNA extracted from the spleen cells of uninfected or L. monocytogenes infected mice. These results suggest that infection with L. monocytogenes elicits a detectable cytokine mRNA response within the first few hours of infection.


Experimental infection of severe combined immunodeficient beige mice with Mycobacterium paratuberculosis of bovine origin. Severe combined immunodeficient beige mice were inoculated orally and intraperitoneally with a bovine strain of Mycobacterium paratuberculosis to explore their potential as laboratory animal models in the study of paratuberculosis (Johne's disease). Control animals were similarly inoculated with heat-killed M. paratuberculosis. In the mice inoculated intraperitoneally, focal lesions and acid-fast bacilli were first detected in the livers (4 weeks postinfection) and later in the spleens and intestines of the test but not the control animals. No bacteria were seen in the hearts, kidneys, or lungs. At 12 weeks postinfection, all test mice had significant losses in body weight compared with those in controls (P less than 0.05), a characteristic sign of bovine paratuberculosis. Tumor necrosis factor alpha was not detected in the serum. Histologic lesions were seen in the intestines, livers, and spleens of the animals in the orally inoculated test group after 26 weeks of infection. Our results suggest that the severe combined immunodeficient beige mouse may be a useful model for the investigation of paratuberculosis and cachexia and the evaluation of antimycobacterial drugs.


The glucuronoxylomannan of Cryptococcus neoformans serotype A is a type 2 T-cell-independent antigen. The humoral immune response of inbred mice to immunization with the glucuronoxylomannan (GXM) of Cryptococcus neoformans was investigated both serologically and in plaque-forming cells (PFCs). The T-helper-cell-independent quality of the GXM was demonstrated by using BALB/c nu/nu mice. Primary and secondary dose responses to three antigenic forms of GXM, (i) the native antigen, (ii) a GXM-bovine serum albumin protein conjugate, and (iii) a cryptococcal whole-cell vaccine, revealed a lack of isotype class switching and anamnestic responses. Both the levels of complement-fixing anti-GXM antibody in serum and the PFC responses in the athymic mice showed no significant differences from those in the wild-type controls. However, T cells are involved in the suppression of the primary response to GXM. When BALB/cBy mice were given rabbit anti-mouse thymocyte serum along with 0.5 microgram of GXM, both antibody levels in serum and PFC responses were significantly increased over those of control mice that received GXM and normal rabbit serum. In addition, T cells were also shown to enhance the primary immune response to GXM. BALB/cBy mice were given GXM and anti-mouse thymocyte serum on day 1. On day 2, the experimental group was given anti-mouse thymocyte serum and the control group was given saline. On day 5, comparison of the PFC responses and anti-GXM antibody titers of the two groups revealed a significant increase in the immune response of the control over the experimental group. The type 2 T-cell-independent quality of GXM was also demonstrated in CBA/cHN xid mice. These mice lack the Lyb+ subset of B cells and are unable to respond to type 2 T-independent antigens but respond normally to type 1 T-independent antigens. Type III pneumococcal polysaccharide, a type 2 T-independent antigen, was used as a negative control, and trinitrophenyl-lipopolysaccharide, a type 1 T-independent antigen, was used as a positive control. The CBA/cHN xid mice failed to respond to either type III pneumococcal polysaccharide or GXM but did not respond to immunization with trinitrophenyl-lipopolysaccharide. BALB/cBy mice responded normally to all three antigens.


Unipolar reorganization of F-actin layer at bacterial division and bundling of actin filaments by plastin correlate with movement of Shigella flexneri within HeLa cells. Shigella flexneri causes bacillary dysentery, an invasive disease of colonic epithelial cells in humans. The capacity of bacteria, once they have entered into a cell and escaped the phagocytic vacuole, to spread intracellularly and directly to adjacent cells without further extracellular passage is a key factor in invasion of the epithelial layer. Movement of intracellular bacteria is dependent upon the polymerization of actin; concentration of the formed filaments to one end of the bacterium is associated with initiation of movement. This movement may lead to the formation of a protrusion at the cell surface through which the bacterium passes to an adjacent cell. Development of these protrusions in infected HeLa cells is described, with emphasis on two critical observations. First, initiation of movement is coupled with bacterial division since elongation of the bacterial body is associated with relocalization of the previously uniformly distributed layer of actin to one pole of the bacterium. Second, the actin-bundling protein plastin appears to bundle the actin filaments just posterior to the bacterium, producing an ongoing contraction of the cylindrical actin tail that may be associated with forward movement of the bacterium within the protrusion.


Genetic relationships of penicillin-susceptible and -resistant Streptococcus pneumoniae strains isolated on different continents. Sixty-six strains of Streptococcus pneumoniae isolated in different parts of the world, 46 resistant and 22 susceptible to penicillin, were subdivided by multilocus enzyme electrophoresis into 28 distinct electrophoretic types (ETs). The ETs to which penicillin-susceptible strains were assigned differed from those containing resistant isolates of the same serotype. Five common clones could be recognized among the penicillin-resistant bacteria by combining the ETs, the antigenic properties of penicillin-binding proteins PBP 1a and 2b, and the tetracycline and chloramphenicol resistance profiles. Two clones were found in Finland and were associated with capsular serotypes 6B and 23F, respectively. Two clones were from Spain (type 6B and 9V, respectively). The fifth clone was isolated in South Africa and in Spain and contained both serotype 23F isolates and one type 19F strain. The other resistant strains were represented by rare isolates distributed among 12 other ETs, confirming that resistance to penicillin has evolved by multiple branches. Because capsular type was mixed in several ETs, the results also demonstrate that it may vary among very closely related pneumococci.


Staphylococcus saprophyticus hemagglutinin is a 160-kilodalton surface polypeptide. Many strains of Staphylococcus saprophyticus cause direct hemagglutination of sheep erythrocytes. For a high proportion of clinical isolates, a surface protein (Ssp) that is apparently not involved in this property has been described. In this study, S. saprophyticus CCM883, a hemagglutinating but Ssp-negative strain, was used for the identification, purification, and characterization of a 160-kDa surface polypeptide that appears to be the major component of the hemagglutinin. Expression of the protein required the addition to the growth medium of EDTA in micromolar quantities, suggesting an inhibitory role for some unidentified metal ion. The protein was purified by means of Sephacryl S-300 chromatography, and antisera were raised in rabbits. Antibody against this protein inhibited the hemagglutination of two other, unrelated strains and was used to demonstrate, by electron microscopy, the presence of the protein on the surface of the cells. In a confirmatory experiment, the purified antigen was incubated with erythrocytes and binding was detected by the Western immunoblot technique with the antibody to the 160-kDa polypeptide. These experiments indicate that this surface protein is the hemagglutinin of S. saprophyticus.


SCID-Hu mice immunized with a pneumococcal vaccine produce specific human antibodies and show increased resistance to infection. Seventy-eight severe combined immunodeficiency (SCID) mice were administered intraperitoneally 1 x 10(7) to 9 x 10(7) human peripheral blood mononuclear cells (PBL) in five experiments. Human immunoglobulin G (IgG) was detected in 70 to 88% of these SCID-PBL-Hu mice after cell transplantation, and all four subclasses were present. The total concentration of human IgG varied from less than 1 to 10.2 g/liter. The SCID-PBL-Hu mice with high concentrations of human IgG regularly had mono- or oligoclonal human IgG bands in serum, as demonstrated by agarose gel electrophoresis. Of the SCID-PBL-Hu mice that were immunized with a 23-valent pneumococcal polysaccharide vaccine, 63 to 78% developed a significant human IgG antipneumococcal antibody response, whereas only very low levels of human IgM and no human IgA antipneumococcal antibodies could be detected. Twelve to twenty-two percent of the SCID-PBL-Hu mice showed signs of leakiness; these mice developed a significant mouse IgM antipneumococcal antibody response and no human antibodies. SCID-PBL-Hu mice were challenged intraperitoneally with 10 50% lethal doses of Streptococcus pneumoniae serotype 4 to study the protective effect of immunization with pneumococcal vaccine. The immunized SCID-PBL-Hu mice showed less bacteremia than did all control groups, and survival was 45 to 60%. None of the unimmunized SCID-PBL-Hu mice survived.


Experimental verocytotoxemia in rabbits. The clinicopathologic effects of intravenously administered purified verocytotoxin 1 (VT1; Shiga-like toxin 1) in 2-kg male rabbits was studied. The 50% lethal dose was 0.2 micrograms of protein per kg of body weight (2 x 10(4) 50% cytotoxic doses per kg). The clinical features included nonbloody diarrhea and a progressive flaccid paresis, usually culminating in death. The histopathology was characterized by edema and hemorrhage in the mucosa and submucosa of the cecum and edema, hemorrhage, and neuronal necrosis in the brain and gray matter of the spinal cord. Thrombotic microangiopathy, the characteristic histopathologic renal lesion in the hemolytic-uremic syndrome, was also found to be the underlying lesion in verocytotoxemic rabbits. To determine the specific distribution of VT1 in rabbit tissues, purified 125I-labelled VT1 was administered intravenously to 20 rabbits (both immunologically naive and VT1-immune rabbits). The highest specific uptake of 125I-VT1 was in the spinal cord, brain, cecum, colon, and small bowel in unimmunized animals but in the liver, spleen, and lungs in immune animals. Immunofluorescent staining of cecal and spinal cord tissues after intravenous administration of VT1 showed evidence of specific vascular endothelial cell binding of the toxin. The striking correlation of the central nervous system and gastrointestinal localization of 125I-VT1 with the sites of known histopathology is consistent with direct toxin-mediated injury to these tissues, initiated by the specific binding of VT1 to the vascular endothelium. We conclude that the vascular damage induced by VT1 in affected rabbit tissues is similar to that seen in the kidneys and other tissues in patients with verocytotoxin-producing Escherichia coli-associated hemolytic-uremic syndrome. This suggests that although the rabbit model fails to replicate human hemolytic-uremic syndrome, it is useful for studying the pathogenesis of the vascular lesions in verocytotoxin-producing E. coli-associated diseases.


Retrovirus-induced immunodeficiency in mice exacerbates gastrointestinal candidiasis. Dysfunction of neutrophils in patients infected with human immunodeficiency virus is at least partly responsible for secondary microbial diseases in these individuals, including invasive gastrointestinal (GI) candidiasis. Immunoregulatory disturbances associated with the development of AIDS in human immunodeficiency virus-infected patients exacerbates Candida albicans infection of the upper GI tract and frequently leads to oropharyngeal and esophageal candidiasis. In this article, we present the first report of a murine model of invasive GI candidiasis associated with an AIDS-related murine immunodeficiency syndrome that results from infection of C57BL/6 mice with a previously described retrovirus complex (LP-BM5). Mice of the inbred strain were infected with C. albicans by oral-intragastric inoculation as infants and with the retrovirus by the intraperitoneal route 30 days later. Control mice of the same strain were infected with C. albicans as above and subsequently infected with the avirulent, ecotropic helper virus (MBI-5). Animals were killed 90 days after retroviral challenge. Total and differential blood cell counts, CD4+ T-cell counts in the spleen, and the histopathology of the gastric mucosa of experimental and control animals were determined. The virulent LP-BM5-infected animals developed murine AIDS and showed eruptive and suppurative lesions, with associated C. albicans mainly in regions of the cardial-atrium fold of the stomach. Well-defined abscesses with entrapped C. albicans hyphae were observed in the region of the cardial-atrium fold of control mice. A significant increase in the number of C. albicans CFU in homogenized and plated segments of the GI tract was recognized in mice with murine AIDS versus the control animals. The murine model of GI candidiasis reported here permits examination of the nature of C. albicans interaction with the gastric mucosa both in the immunocompetent host under conditions in which the yeast exists predominantly as a commensal organism and in the immunosuppressed host during progressive stages of AIDS induced by a retroviral infection.


Induction of procoagulant activity on human endothelial cells by Streptococcus pneumoniae. The inflammatory response in infection caused by gram-negative organisms involves induction of procoagulant activity (PCA) on human endothelial cells. Although infections caused by gram-positive organisms are also associated with fibrin formation and thrombosis, the bacterial determinants inducing PCA are unknown. This study shows that intact pneumococci and the pneumococcal cell wall efficiently induce PCA on human endothelial cells. Upon exposure of endothelial cells to pneumococci, PCA was first detectable at 30 min, peaked at 2 h, and disappeared by 6 h. The specific activities of encapsulated and unencapsulated strains for induction of PCA were equivalent. Purified pneumococcal cell walls were as potent as endotoxin in induction of PCA. The ability to induce a procoagulant state on endothelial cells is a new biological activity of gram-positive cell walls which promotes the participation of the coagulation cascade in the inflammatory response to disease caused by gram-positive organisms.


Immunocytological responses in porcine proliferative enteropathies. The ileum, colon, and mesenteric lymph nodes of pigs naturally affected by either of the two major forms of proliferative enteropathy, namely, intestinal adenomatosis or hemorrhagic enteropathy, were examined for immunocytological responses to infection by immunocytochemistry, using antibodies directed against elements of the porcine immune system. In both forms, there was mucosal proliferation of immature enterocytes which lacked substantial major histocompatibility complex class II expression and a marked accumulation of immunoglobulin A (IgA) at the apical cytoplasm of affected enterocytes in association with intracellular Campylobacter-like organisms. In intestinal adenomatosis, there was only a mild infiltration of CD8+ and CD25+ T cells in the intestinal lamina propria. In hemorrhagic enteropathy, there was a moderate infiltration of CD8+ and CD25+ T cells and IgM+ B cells in the lamina propria. In rats and humans, villous enterocytes are thought to act as antigen-presenting cells, with major histocompatibility complex class II molecules present on their surface, capable of initiating a T-cell response (particularly of CD8+ T cells) in response to bacterial antigens. Therefore, the selection of immature crypt cells by the intracellular Campylobacter-like organisms for entry and multiplication may represent a remarkable microbial adaptation associated with local immunomodulation and enhanced bacterial survival. The accumulation of IgA within affected enterocytes may represent a reduced capability of the cells to process nonspecific IgA or an accumulation of specific IgA.


Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. A cytotoxigenic Clostridium difficile strain that fails to produce toxin A but causes hemorrhage and bloody fluid accumulation in ligated ileal loops of rabbits and hemorrhage and diarrhea in hamsters is described. The lack of reaction of DNA from this strain in hybridization studies with a toxin A gene-specific 4.5-kb probe and polymerase chain reaction studies with six toxin A-specific primers indicate the absence of the toxin A gene. The cytotoxin produced by this strain was not responsible for the enterotoxic or hemorrhagic activity and shared characteristics with toxin B, i.e., its cytotoxicity was neutralized by antibodies to toxigenic strains of C. difficile and Clostridium sordellii. Polymerase chain reaction studies with toxin B-specific primers showed that the DNA from this strain produced a 690-bp product in addition to the expected 591-bp product.


Killing of Coccidioides immitis by human peripheral blood mononuclear cells. The ability of human peripheral blood mononuclear cells (MNL) obtained from healthy donors to kill the fungus Coccidioides immitis was examined in vitro with an assay that uses a single fungal particle per well. MNL killed 25.0% +/- 3.5% of a coccidioidal arthroconidial target, compared with the 4.7% +/- 2.9% killed by polymorphonuclear leukocytes obtained from the same donors (P = 0.012). Arthroconidial killing by MNL was not dependent on donor delayed dermal hypersensitivity to spherulin. Killing of another fungal target, Candida glabrata, was not significantly different between MNL and polymorphonuclear leukocytes (P = 0.783). Depletion of monocytes from MNL with Sephadex G-10 resulted in a significant reduction in arthroconidial killing (21.4% +/- 13.6% versus 2.4% +/- 3.4%; P = 0.025), while enrichment of monocytes by Percoll density gradient centrifugation or plastic adherence resulted in significantly increased arthroconidial killing compared with that by MNL (P = 0.005 and 0.001, respectively). Killing of 96-h spherules by MNL was 7.3% +/- 3.1%, significantly less than the 21.4% +/- 2.8% killing of arthroconidia in the same experiments (P = 0.016). Incubation of MNL with human recombinant gamma interferon or tumor necrosis factor alpha did not result in increased MNL killing of coccidioidal arthroconidia under various conditions. These results suggest that MNL have an inherent ability to kill coccidioidal arthroconidia in vitro which is not dependent on prior host exposure to C. immitis. This activity appears to reside in peripheral blood monocytes.


Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi. The uptake mechanism for the spirochete Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by electron microscopy for human and murine phagocytes. Spirochetes of both a low- and a high-passage strain were preferentially internalized by coiling rather than by conventional phagocytosis. The spirochetes engulfed by coiling phagocytosis were found to disintegrate in an organelle exclusion zone without evident participation of lysosomes. Preincubation of B. burgdorferi with monoclonal antibody to the spirochetal OspA enhanced phagocytosis in general but did not consistently influence the uptake mechanism. Quantitative and kinetic differences concerning the phagocytic rate and mechanism were evident between cells from different lineages, different human individuals, and mice and humans. In general, when few phagocytes participated in spirochete uptake, the active cells displayed a high ratio of coiling versus conventional phagocytosis. These results suggest that coiling phagocytosis of B. burgdorferi plays a critical role in the control of spirochetal infection. More detailed studies on the molecular basis of this phagocytic mechanism may lead to new insights into the pathogenesis of Lyme borreliosis, a disease which is frequently characterized by the host's inability to eliminate the pathogenic spirochete.


Identification of a 58-kilodalton cell surface fibrinogen-binding mannoprotein from Candida albicans. Treatment of both yeast (blastoconidia) and hyphal (blastoconidia with germ tubes) cells of Candida albicans with beta-mercaptoethanol (beta ME) releases a complex array of cell wall-bound proteins and glycoproteins. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting with fibrinogen-anti-fibrinogen antibody allowed the identification of a 58-kDa mannoprotein (mp58) in both extracts which specifically interacts with human fibrinogen. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) for short periods or with beta ME abolished or significantly reduced binding of fibrinogen. A rabbit polyclonal antiserum was raised against the purified mp58 species released by beta ME from germinated blastoconidia (PAb anti-mp58). By Western blotting, the antiserum cross-reacted with the homologous 58-kDa fibrinogen-binding mannoprotein present in beta ME extracts from blastoconidia, and by indirect immunofluorescence, the antiserum labelled both yeast cells and hyphae, yet reactivity was found primarily on the cell surface of filamentous forms. Immunostaining of human infected tissue sections with PAb anti-mp58 showed that the mp58 species is also expressed in vivo; in this case, the species is in the forms of both yeast and hyphal elements similarly labelled by the antiserum. Purified immunoglobulin G fraction from the antiserum did not alter the binding of fibrinogen as determined by a modified enzyme-linked immunosorbent assay and Western blotting. The N- and O-glycosidically linked carbohydrates represent 18 to 20% and 3 to 4%, respectively, of the molecular mass of the mp58. O-linked sugar residues may be involved in the interaction of the molecule with fibrinogen.


Early activation of splenic macrophages by tumor necrosis factor alpha is important in determining the outcome of experimental histoplasmosis in mice. Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.


Similarity between the group B and A streptococcal C5a peptidase genes. Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.


Analysis of adhesion and cytotoxicity of Tritrichomonas foetus to mammalian cells by use of monoclonal antibodies. The relationship of Tritrichomonas foetus adhesion to mammalian cells and cytotoxicity to these targets was investigated. High-adherence and low-adherence lines of T. foetus, derived by repeated adhesion to HeLa cells, showed high and low cytotoxicity, respectively, to HeLa cells. When parasites were separated from targets by membranes (0.4-microns pore size), no cytotoxicity was detectable. Monoclonal antibodies elicited against T. foetus that lowered adhesion also lowered parasite-mediated cytotoxicity. Flow cytometry experiments revealed that the levels of an adhesion- and cytotoxicity-blocking antibody bound to the surface of high-adherence clones of T. foetus were higher than those in low-adherence clones. Western blots of parasite extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with an anti-T. foetus antibody. A molecule with a molecular weight of approximately 190,000 composed of subunits with molecular weights of approximately 140,000 to 150,000 and approximately 65,000 was identified. Immunoprecipitation experiments with metabolically labeled T. foetus and the same antibody confirmed that similar subunits were synthesized by the parasite. These results indicate that adhesion of T. foetus to mammalian cells is an important step in cytotoxic damage of these targets and that a surface adhesin on the parasite is involved in the adhesion mechanism.


Specific lung mucosal and systemic immune responses after oral immunization of mice with Salmonella typhimurium aroA, Salmonella typhi Ty21a, and invasive Escherichia coli expressing recombinant pertussis toxin S1 subunit. Pertussis toxin (PT) is considered an essential protective component for incorporation into new generation vaccines against Bordetella pertussis, the causative agent of whooping cough. Traditionally, antipertussis vaccination has employed an intramuscular route. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunization with live vaccine carrier strains of Salmonella spp. or Escherichia coli. Recombinant S1 subunit of pertussis toxin was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, in the human typhoid vaccine strain Salmonella typhi Ty21a, and in E. coli CAG629 containing the Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness of epithelial cells. Expression of recombinant PT S1 subunit (rPT-S1) did not affect in vitro invasiveness of the tested strains, which retained the ability to adhere to and invade the embryonic human intestinal cell line HI-407. Following oral immunization of mice with the live vaccine strains expressing rPT-S1, immunoglobulin G (IgG), IgA, and IgM responses were monitored. IgG specific to PT was detected in serum samples of mice, while IgG and IgA specific to PT were detected in lung washes after oral immunization with living Salmonella spp. or E. coli (pWR110) expressing rPT-S1. Utilization of live oral vaccines expressing B. pertussis antigens, which stimulate both a systemic and lung mucosal response, may provide an attractive alternative to purified component vaccines against whooping cough.


Role of Yersinia enterocolitica Yst toxin in experimental infection of young rabbits. We constructed a Yst-negative mutant of Yersinia enterocolitica W1024 by reverse genetics, and we compared the virulence of the yst+ and yst isogenic strains in an experimental oral infection of the young rabbit. The rabbits infected with the yst+ strain suffered from the diarrhea and lost weight, and most of them died. By contrast, the occurrence of diarrhea, weight loss, and death in the group of rabbits infected with the yst mutant was as low as that in the group of uninfected rabbits. Bacteria from both strains were excreted in the feces and induced a serum antibody response against Yop proteins. The yst mutant disappeared more rapidly from the feces. We conclude that the enterotoxin Yst is a major factor involved in the Y. enterocolitica-associated diarrhea in the young rabbit. Given the similarity with the symptoms observed for children, this result suggests that Yst could also be an important factor in diarrhea in young children infected with Y. enterocolitica.


Rhodopseudomonas sphaeroides lipid A derivatives block in vitro induction of tumor necrosis factor and endotoxin tolerance by smooth lipopolysaccharide and monophosphoryl lipid A. Rhodopseudomonas (Rhodobacter) sphaeroides diphosphoryl lipid A is a relatively inert species of lipid A but has been shown to antagonize the effects of toxic lipopolysaccharide (LPS) both in vivo and in vitro. The antagonist and its monophosphoryl derivative were examined for the ability to block tumor necrosis factor synthesis and reverse tolerance induction in vitro in macrophage cultures stimulated with bioactive preparations of smooth LPS, rough LPS, diphosphoryl lipid A, and monophosphoryl lipid A. Inhibition of agonist activity and reversal of tolerance by these novel penta-acylated lipid A antagonists provides new insight into macrophage-LPS interactions.


Adhesion of glucosyltransferase phase variants to Streptococcus gordonii bacterium-glucan substrata may involve lipoteichoic acid. Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3H]thymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp+ and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria.