Establishing a direct role for the Bartonella bacilliformis invasion-associated locus B (IalB) protein in human erythrocyte parasitism.

The invasion-associated locus A and B genes (ialAB) of Bartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of the lacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (rho), and cytochrome b content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.

Hemin-binding surface protein from Bartonella quintana.

Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from B. quintana bind hemin and that a approximately 25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100 degrees C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library. The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis (31.7% identity). High-stringency Southern blots indicate that all five pathogenic Bartonella spp. possess hbpA homologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E. coli. Intact B. quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized from B. quintana.

Supplemental protein plus ruminally protected methionine and lysine for primiparous beef cattle consuming annual rye hay.

Primiparous beef cows (n = 35 Bos taurus, average initial BW of 498 kg) were allotted to treatments in a split-plot designed experiment to determine the effects of supplemental ruminally protected amino acids on cow and calf productivity and metabolic changes in the cows. Cows were fed chopped annual rye hay at 1.5% of BW. The following treatments were used: 1) .8 kg soybean hulls, 1.4 kg ground corn, .6 kg soybean meal (CON); 2) 1.4 kg ground corn, 1.4 kg soybean meal (PRO); 3) PRO plus ruminally protected methionine and lysine (supplied 5 and 10 g, respectively; PRO1); and 4) PRO with twice the level of ruminally protected amino acids in PRO1 (PRO2). Cow weight gain was not different (P > .26) among treatments and averaged 1.2 kg/d for the 45 + 6 d before parturition. After parturition, cow weight gain did not differ (P > .47) between CON and PRO treatments, but it decreased quadratically (P < .01) with increasing level of ruminally protected amino acids. Total milk yield, protein, and fat (4 h) were greater (P < .05) for cows consuming PRO supplements than for CON, whereas CON cattle tended (P = .11) to lose less body condition. Total milk protein showed a quadratic increase (P < .05) in response to level of ruminally protected amino acids that was the inverse of the quadratic response noted for cow weight gain. Serum urea N concentration was greater (P = .07) for cattle consuming additional protein. Metabolic hormones were not affected (P > .18) by dietary treatment, but they responded (P < .05) to changes in physiological state. Supplements with additional protein supported greater (P = .0001) milk urea N concentration and output. Milk urea N concentration increased (P < .05) and milk IGF-I decreased (P < .05) as the lactation period progressed. The measurement of CON and PRO diets revealed that supplements with additional soybean meal had greater (P < .05) DM and N degradation; the extent of forage DM and NDF degradation was similar (P > .05) among treatments. Production shifted away from body weight gain to increased milk protein production when daily supplementation of ruminally protected methionine and lysine increased from 5 and 10 to 10 and 20 g, respectively. This shift in production was not reflective of changes in the metabolic regulators measured.

Audit of postoperative pain control. Influence of a dedicated acute pain nurse.

The inadequacies of conventional intramuscular opioid analgesia have fueled an expansion in the use of patient-controlled analgesia and epidural analgesia after surgery. This is not always accompanied by increased education and specialist supervision of ward staff and patients. A survey in our hospital prior to the appointment of an Acute Pain Nurse showed an unacceptable incidence of side effects when epidural analgesia was employed on ordinary surgical wards. More surprisingly, efficacy of patient-controlled analgesia was found to be low. Frequent review of patients and regular education of ward staff by a specialist Pain Nurse have achieved a substantial reduction in side effects of epidural analgesia and improvement in efficacy of patient-controlled analgesia. We have shown that the advantages of patient-controlled analgesia can be largely negated by failure to address deficiencies in knowledge of pain management among ward staff and patients.

Cervical cytology: a randomized comparison of four sampling methods.

OBJECTIVE: The purpose of this study was to compare smear quality and endocervical cell recovery of four cervical smear sampling devices. STUDY DESIGN: Two thousand fifteen patients undergoing routine cervical smears at the University of Tennessee Obstetrics and Gynecology clinics were randomly assigned to a cotton swab-spatula, Cytobrush-spatula, Cervex-Brush, or Bayne Pap Brush. The cytopathology laboratory, blind to method, used specific criteria to grade smears as being optimal, adequate, marginal, or inadequate. Statistical analysis was by the chi 2 and analysis of variance tests. RESULTS: No statistical differences occurred among the groups for nonpregnant patients. For pregnant patients smear quality was improved with both Cytobrush-spatula and Bayne Pap Brush versus cotton swab-spatula (p = 0.0301 and 0.0004, respectively); cotton swab-spatula had fewer endocervical cells than the Cytobrush-spatula (p = 0.0001), Cervex-Brush (p = 0.0288), and Bayne Pap Brush (p = 0.0081). CONCLUSIONS: The cotton swab-spatula and Cytobrush-spatula appear to be the most effective screening methods for nonpregnant and pregnant patients, respectively.

Inhalation induction of anaesthesia with isoflurane: effect of added carbon dioxide.

Fifty children underwent inhalation induction of anaesthesia with isoflurane and nitrous oxide in oxygen with or without the addition of 5% carbon dioxide. Addition of carbon dioxide resulted in more rapid induction and significantly reduced the incidence and severity of airway related problems.

Gross and histologic examination of tubal ligation failures in a residency training program.

A previous study from this institution revealed laparoscopic tubal sterilization failure rates of 26.5 per 1000 and 45.5 per 1000, respectively, for the tubal ring and spring-loaded clip in procedures performed by residents in training. In an effort to identify potential anatomical reasons for this unacceptably high failure rate, 20 patients becoming pregnant after laparoscopic tubal occlusion underwent bilateral salpingectomy. Gross and histologic evaluation of the surgical specimens demonstrated improper application of the occlusive device in all cases. Seventeen patients were found to have nonoccluded or partially occluded tubes on one or both sides, with all occlusive devices located in the infundibular segment. Two patients were missing tubal rings on one side, and the remaining patient had a tubal ring misapplied to the round ligament. Sixteen residents who had completed a 1-month rotation on the ambulatory surgery service were given a standardized interview to assess their knowledge of proper sterilization techniques as well as their training experience. The frequency of incorrect responses given to four specific questions concerning proper placement of the tubal ring and spring-loaded clip ranged from 43.8-81.2%. The sterilization failure rate at this institution appears to be directly related to the resident surgeon's lack of understanding of the operative technique. Realizing that our institution is not unlike most other resident training programs, we developed a standardized education program including formal instruction of residents in proper sterilization technique and have altered supervisory guidelines for attending surgeons.

Fatal paradoxical thrombo-embolism during anaesthesia.

Deep venous thrombosis is a recognised complication of trauma to the leg and may lead to pulmonary thrombo-embolism, but paradoxical thrombo-embolism is rare. A case of fatal paradoxical thrombo-embolism which followed a leg injury in a previously fit 36-year-old male is presented and contributory factors are reviewed.

Outcome after general anaesthesia for repair of fractured neck of femur. A randomized trial of spontaneous v. controlled ventilation.

One hundred and fifty-two patients undergoing surgery for fractured neck of femur were randomly allocated to receive either general anesthesia with spontaneous ventilation with nitrous oxide and halothane in oxygen or general anaesthesia with controlled ventilation with fentanyl, nitrous oxide and halothane in oxygen. Atracurium was used to provide muscle paralysis in 65% of the latter group, the remainder receiving no neuromuscular blocking agent other than suxamethonium for intubation. Patients were followed up for 6 months. Mortality and outcome were not significantly different between the groups. Overall mortality at 4 weeks was 5.2%, and at 6 months was 15.1%--figures which are considerably lower than in some other comparable studies. This study does not support the suggestion that general anaesthesia with controlled ventilation is associated with increased postoperative mortality.

Retrospective evaluation of gynecologic cytodiagnosis. II. Interlaboratory reproducibility as shown in rescreening large consecutive samples of reported cases.

Two laboratories exchanged and rescreened a large sample of cases with cervicovaginal smears they had consecutively accessioned to examine the reproducibility of gynecologic cytodiagnosis under optimum conditions. At least a "working agreement" (diagnoses within +/- 1 category on a ten-category scale) was achieved in diagnoses of normal, benign reaction and squamous abnormality (from minimal dysplasia though invasive cancer) in 18,859 cases (96.8%), of endometrial abnormality in 21 cases (42%) and of "unsatisfactory" in 99 cases (20.7%). Larger differences occurred in greater than or equal to 30% of cases except in the categories of "normal" and "benign reaction," reaching a maximum of 82% for moderate dysplasia. Reexamining 382 cases decreased disagreement by category to the 20% to 65% range only in the five categories of dysplasia plus carcinoma in situ. Agreement was not predicated on the presence of endocervical cells or squamous metaplasia; the basis for "unsatisfactory" calls was not uniform. Comparison of the laboratories' diagnoses with referee diagnoses or, on 178 cases, with tissue diagnoses also demonstrated differences in diagnostic criteria.

Adenoid cystic carcinoma of the cervix: a report of 17 cases.

Seventeen cases of adenoid cystic carcinoma of the cervix are presented. The long-term survival was in early stage IB-IIA. Patients with more than stage IIB had no survival rate over 5 years. One thing of interest was the fact that lung metastasis was 29.4%.

Paget's disease of the anal mucosa in association with carcinoma demonstrating mucoepidermoid features.

A case of Paget's disease of the anal mucosa with an underlying carcinoma demonstrating mucoepidermoid features is reported. The histopathologic characteristics of both extramammary Paget's disease and mucoepidermoid carcinoma are noted. Special histochemical staining procedures were consistent with the diagnosis. The actual histogenesis of extramammary Paget's disease and mucoepidermoid carcinomas are controversial topics that are briefly discussed in this paper.

Cervical cancer occurrence in Memphis and Shelby County, Tennessee, during 25 years of its cervical cytology screening program.

This review of the cytologic history of 430 women with histologically proved invasive cancer of the uterine cervix indicates that several significant subgroups can be defined. One major subgroup consists of 142 patients or 33% of the total group in whom invasive cancer developed within 1 to 5 years, with an average of 2.6 years, after one or more negative cytologic findings. This appears to be a significant proportion of the occurrence of invasive cervical cancer and warrants consideration in the recommendation of screening intervals. The second major subgroup, consisting of 88 patients (20.5%), had a delay in diagnosis averaging 9.9 years after the first abnormal cytologic finding. In some of these patients either the cytologic examination results reverted to negative for a period or the biopsy material did not confirm the cytologic abnormalities. The disease progressed to invasion. These findings appear to bear out the importance of close follow-up of patients with any abnormal cytologic finding in the intraepithelial neoplasia category even though intermittent cytologic studies may appear negative.

Routine gynaecological clinic in northern Ghana.

A routine gynaecological screening clinic was conducted in a town in northern Ghana, West Africa. The presence of cervical intraepithelial neoplasia and other gynaecological disorders were documented. Results show that it is possible to detect pre-malignant lesions, and to provide timely therapy, even in the remote regions of Ghana. More health care professionals in remote areas of the Third World are needed in order to provide such care in the future.

Retrospective evaluation of gynecologic cytodiagnosis. I. Reproducibility using an experimental diagnostic scale.

A system for the exchange of specimens between two independent laboratories ("A" and "B") was designed to enable them to review a consecutive series of one another's gynecologic smears. Another cytopathologist "refereed" diagnoses differing beyond agreed limits and examined all smears called carcinoma in situ or a more severe lesion by either laboratory. A ten-numeral gradient nomenclature scale was developed to code all cytodiagnoses for data processing. This analysis of the reproducibility of the experimental diagnostic system is based on all participants' examination of 100 selected specimens, half contributed by reviewing laboratory A and half by reviewing laboratory B, with the target value of each specimen established by the contributor. Intralaboratory reproducibility, expressed by a correlation coefficient (r), ranged from 0.78 to 0.93. After adjusting for straightforward squamous-cell lesions, r ranged from 0.88 to 0.91. Intralaboratory agreement +/- 1 scalar category, defined as a "working consensus," ranged from 77% to 84%. Interlaboratory correlation ranged from r = 0.74 to 0.94. Adjustment for squamous-cell lesions exerted less effect than it had on intralaboratory reproducibility. Interlaboratory agreement within one category ranged from 58% (laboratory B reviewing specimens contributed by laboratory A) to 92% (referee examining specimens contributed by laboratory B). Initially, laboratory A tended to classify a given specimen higher on the scale than did laboratory B and the referee, but as the study progressed, both laboratories tended to converge toward a more congruent diagnostic position, with referee greater than A greater than B. The level of discrimination attained by all participants validates use of this system to compare a larger series of diagnoses.

Human ovarian 17 beta-hydroxysteroid oxidoreductase activity: a comparison of normal and polycystic ovarian tissues.

Ovarian tissue and ovarian venous blood were obtained from women undergoing wedge resection, and ovarian tissue was obtained from normally menstruating women who had an oophorectomy for medical reasons. A morphological evaluation was made of the wedged tissue. 17 beta-Hydroxysteroid oxidoreductase levels were determined as the 17-ketoreductase (17-KR) and 17 beta-dehydrogenase activities in both normal and wedged tissue. Plasma androstenedione (A) and testosterone (T) levels were measured in the ovarian venous blood. Low, but measurable, 17 beta-hydroxysteroid oxidoreductase activity was found in the mitochondria, microsomes, and cytosol. With whole, cell-free homogenates, mean 17-KR activity was not significantly different in normal tissues and polycystic tissues; mean 17-KR activity of corpora lutea was significantly greater than that of the other tissues. Mean 17 beta-dehydrogenase activity was not significantly different from 17-KR activity in the tissues studied. Ovarian venous A levels were higher than the reported mean ovarian venous A levels of normal women with only one exception; approximately half of the ovarian venous T levels were higher than reported mean ovarian venous T levels of normal women. The morphology of the wedge sections did not correlate well with the biochemical data.

The long-term course of carcinoma in situ of the uterine cervix.

The malignant potential of carcinoma in situ (CIS) of the uterine cervix has been the subject of great controversy. Despite refinements and additions to knowledge in this area, few reports on the long-term course of the disease have appeared in the past decade. Recent developments in diagnostic and therapeutic techniques coupled with changes in the patient population with this disease have prompted renewed interest in conservative management. Results of long-term observation of a group of patients followed initially without ablative therapy are reported. The data indicate that CIS of the uterine cervix is not the inevitably progressive disease that it has been considered to be. Unequivocal invasive cancer develops in only a small percentage of cases and can be controlled, if not cured, by current therapeutic modalities. The intraepithelial lesion, however, tends to persist despite conization, and eventually requires ablative therapy in most cases. Conservative procedures should be regarded as temporizing, at least until their long-term benefits can be recorded.

Polycystic ovarian disease, maturation arrest of spermiogenesis, and Klinefelter's syndrome in siblings of a family with familial hirsutism.

A family with hirsutism in five generations in which polycystic or bilaterally enlarged ovaries were documented in three different sibships of two generations is described. Two brothers of one of the women with polycystic ovaries had a low or low-normal plasma follicle-stimulating hormone level and one of these had oligospermia due to maturation arrest of spermiogenesis. A third brother had Klinefelter's syndrome. Other abnormal features in the family included precocious adrenarche, beardless males, eunuchoidism, and prepubertal grand mal seizures.