Intracellular survival of Staphylococcus aureus within cultured enterocytes.

BACKGROUND: Little is known about the mechanisms involved in bacterial translocation from the intestinal lumen to extraintestinal sites. Because Staphylococcus aureus can colonize the intestinal tract, and because the intestinal tract is a reservoir for antibiotic resistant S. aureus, experiments were designed to clarify the interactions of S. aureus with cultured intestinal epithelial cells, and assays included measurements of bacterial internalization, enterocyte apoptosis, and epithelial barrier function. METHODS AND RESULTS: Mature, confluent enterocytes were incubated 1 h with S. aureus, and the gentamicin protection assay was used to quantify intracellular bacterial survival at various time intervals up to 120 h later. Enterocyte apoptosis was assessed using Annexin V, and the permeability of confluent enterocyte cultures was measured by transepithelial electrical resistance and by transmigration of Escherichia coli across confluent enterocytes.S. aureus was internalized by cultured enterocytes and remained viable for up to 120 h within both HT-29 and Caco-2 enterocytes. S. aureus intracellular survival was associated with enterocyte apoptosis and with decreased transepithelial electrical resistance across confluent Caco-2 enterocytes. S. aureus intracellular survival over time was also associated with increased E. coli transmigration across confluent Caco-2, but not HT-29, enterocytes. CONCLUSIONS: S. aureus appeared to survive within cultured enterocytes for prolonged time periods, up to several days. Survival of S. aureus within host eukaryotic cells, such as enterocytes, might facilitate persistence of S. aureus in infected tissue despite appropriate antibiotic therapy.

Effect of tumor necrosis factor alpha, interferon gamma, and interleukin-4 on bacteria-enterocyte interactions.

BACKGROUND: Little is known about the mechanisms involved in bacterial translocation from the intestinal lumen to extraintestinal sites. Because the cytokine cascade associated with sepsis, inflammation, and trauma has been shown to affect intestinal epithelial permeability, experiments were designed to clarify the effects of selected cytokines on bacterial adherence to and internalization by cultured HT-29 and Caco-2 enterocytes. METHODS: Mature, confluent enterocytes were pretreated 48 to 72 h with tumor necrosis factor alpha (TNF-alpha), interferon gamma, (IFN-gamma), or interleukin-4 (IL-4). Adherence of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, and Escherichia coli was measured by enzyme-linked immunosorbent assay and bacterial internalization was quantified by the gentamicin protection assay. Enterocyte permeability was measured by transepithelial electrical resistance and by flux of 40-kDa fluorescent dextran. Bacterial transmigration across confluent enterocytes was measured using enterocytes cultivated on permeable supports. RESULTS: TNF-alpha, IFN-gamma, and IL-4 had variable effects on bacterial adherence to HT-29 and Caco-2 enterocytes, although the most consistent finding was increased bacterial adherence associated with INF-gamma. However, none of these cytokines had a noticeable effect on bacterial internalization by either Caco-2 or HT-29 enterocytes. In addition, none of these cytokines had a noticeable effect on the permeability of confluent enterocytes as measured by transepithelial electrical resistance or dextran flux. Bacterial transmigration across confluent HT-29 enterocytes was not altered by TNF-alpha, IFN-gamma, or IL-4; however, IL-4 consistently decreased bacterial transmigration across confluent Caco-2 enterocytes. CONCLUSIONS: IFN-gamma may augment the epithelial adherence of selected species of enteric bacteria, and IL-4 may act as a barrier-sustaining agent to decrease bacterial migration across the intestinal epithelium.

Integrin expression, enterocyte maturation, and bacterial internalization.

BACKGROUND: Little is known about the molecular mechanisms involved in the translocation of enteric bacteria. Adhesion molecules mediate interactions between some enteric pathogens and mammalian cells, but no such interactions have been identified for enterocytes and normal enteric bacteria. Using enteric pathogens, adhesion molecule expression has been linked to bacterial internalization and to enterocyte differentiation. Therefore, experiments were designed to study enterocyte integrin expression and differentiation, as well as enterocyte internalization of Salmonella typhimurium, Proteus mirabilis, and Escherichia coli. MATERIALS AND METHODS: Relative expression of the alpha2, alpha3, and beta1 integrin subunits on Caco-2 and HT-29 enterocytes (mature and immature) was measured by ELISA. Bacteria-enterocyte surface interactions were observed by light and scanning electron microscopy. Bacterial internalization by enterocytes was quantified using the gentamicin protection assay. RESULTS: Expression of the alpha2, alpha3, and beta1 integrin subunits was consistently increased in immature compared to mature Caco-2 enterocytes; however, compared to mature enterocytes, immature HT-29 enterocytes had similar expression of alpha3 and beta1 but decreased alpha2. Compared to untreated mature enterocytes, bacterial internalization was increased in immature enterocytes as well as mature enterocytes with lateral membranes artifactually exposed. However, there was no difference in bacterial internalization between immature enterocytes and mature enterocytes treated to expose the lateral membrane. CONCLUSIONS: Bacterial internalization by enterocytes appeared to be due to factors other than integrin expression or enterocyte differentiation. Exposure of the lateral enterocyte membrane may play an important role in facilitating bacterial internalization by enterocytes.

Are benign cellular changes on a Papanicolaou smear really benign? A prospective cohort study.

OBJECTIVES: To determine the underlying prevalence of cervical intraepithelial neoplasia (CIN) in women with benign cellular changes on a Papanicolaou smear, and to evaluate follow-up strategies to identify women at high risk for serious underlying pathology. METHODS: Nonpregnant women aged 18 to 75 years with benign cellular changes on a Papanicolaou smear were recruited from primary care clinics of an urban teaching hospital. The subjects (N = 132) were tested at baseline for the presence of human papillomavirus using the polymerase chain reaction technique, and underwent repeated cervicovaginal smears at 3, 6, and 9 months. At 12 months colposcopy was performed. The main study outcome was the proportion of subjects with CIN as determined by colposcopic biopsy specimens. We determined the sensitivity, specificity, and predictive values of historical risk factor information, human papillomavirus testing, and repeated cervicovaginal smears for the detection of CIN. RESULTS: Cervical intraepithelial neoplasia was found in 30 of 132 women, of whom 27 (20%) had low-grade CIN (CIN I) and 3 (2%) had high-grade CIN (CIN II). Underlying CIN was significantly more common in women younger than 35 years or who had a history of Trichomonas infection or venereal warts, a positive human papillomavirus test result, or abnormal follow-up smears. However, no follow-up strategy combined high sensitivity with a low referral rate for colposcopy. CONCLUSIONS: The prevalence of underlying high-grade CIN in women with benign cellular changes is low. However, further prospective studies in other settings are needed before routine follow-up can unequivocally be recommended.

Fine-needle aspiration of normal tissue from enlarged salivary glands: sialosis or missed target?

Salivary gland enlargement by hypertrophy of normal-appearing parotid or submaxillary tissue is known as sialosis. It can be idiopathic, or may be associated with malnutrition, diabetes, bulimia, or alcoholism. When normal tissue is aspirated from an enlarged gland, one is tempted to diagnosed sialosis. We performed 26 such aspirates over a 5-yr period. In all cases, the cytology featured abundant acinar and ductal tissue in a clean (noninflammatory) background. Six cases were excluded when the records showed no return visits to the clinic. The remaining 20 patients included 9 men and 11 women, aged 24-92 yr (median 56), who harbored 12 parotid (2 bilateral) and 8 unilateral submaxillary enlargements. Clinical findings included ethanol abuse (2), diabetes (1), and previously diagnosed head and neck carcinoma (3). In six patients, the duration of the mass was described as months or years. Excision (6), reaspiration (3), radiographic evaluation (2), and clinical follow-up of patients not evaluated by other means (9 cases with median follow-up of 6 months) revealed no malignancies. One excised gland contained a pleomorphic adenoma measuring 0.5 cm in diameter. This had been diagnosed by repeat fine-needle aspiration (FNA) prior to surgery. We suggest that sialosis is a meaningful FNA diagnosis in patients who are carefully examined, skillfully aspirated, and reasonably followed.

Cervical cytology findings in women infected with the human immunodeficiency virus.

It has been suggested that immunocompromised, HIV-infected patients are at risk of developing HPV infection and SIL. The well documented role of HPV and SIL in cervical carcinogenesis should lead to frequent, careful evaluation of HIV infected women. Forty-four cervical smears from 23 patients (20 HIV and 3 AIDS) were reviewed. While 11 of the 23 patients produced negative smears, 11 had abnormal cytological findings on at least one occasion. Sixteen smears (36 percent) from 10 patients (43%) showed evidence of HPV and/or SIL. Two smears (two patients) were assigned to the benign epithelial atypia category. (One of these showed keratosis which may indicate HPV infection.) Six smears (three patients) represented either a severe Trichomonas, fungal (Candida sp.), or Herpes infection. Three smears were deemed unsatisfactory for diagnosis due to severe acute inflammation or obscuring blood. Five biopsies were available. In four, histologic findings supported the original cytologic diagnosis. One patient with a negative smear had a biopsy showing condyloma. This study further supports an association of HPV and/or cervical dysplasia with HIV. Careful evaluation and follow-up of HIV-infected women is essential.

Atypia in breast fine-needle aspiration smears correlates poorly with the presence of a prognostically significant proliferative lesion of ductal epithelium.

Proliferative lesions of breast duct epithelium are associated with an increased risk of subsequent carcinoma. Fine-needle aspiration criteria for these lesions are poorly defined; most studies are retrospective and do not clearly use the classification of Page and Rogers. Suggested criteria for "atypia" include crowded, enlarged, overlapping nuclei in three-dimensional groups or sheets, loss of cohesion, occasional single cells, a homogeneous cell population, chromatin changes, and increased cellularity in older patients. Our prospective series of 1,925 aspirations included 717 breast cases, of which 25 (3.5%) were considered sufficiently atypical to possibly represent proliferative lesions, but were not suspicious for carcinoma. Fifteen patients with histologic follow-up formed the basis for this study. All had physical examinations and mammogram results consistent with fibrocystic change. Their ages ranged from 30 to 70 years (median age, 44 years). Cytologic changes of atypia were cataloged. Histologically, six cases (40%) showed prognostically significant lesions (moderate, florid, or atypical hyperplasia and one lobular carcinoma in situ). Many (60%) cytologically provocative lesions may originate in prognostically trivial lesions. At this time our limited understanding of the cytologic presentation of these lesions indicates that surgical excision is essential in all instances. Efforts to recognize and properly classify these proliferations in cytologic material should continue.

Hyperplasia of type II pneumocytes in acute lung injury. Cytologic findings of sequential bronchoalveolar lavage.

The organizing stage of acute lung injury syndrome is characterized, in part, by a reactive hyperplasia of type II pneumocytes. These cells may be recovered in large numbers and in an excellent state of preservation by bronchoalveolar lavage. It was noted recently that such cells in lavage samples may mimic adenocarcinoma. To examine the dynamics of type II pneumocyte shedding in acute lung injury, we studied 62 bronchoalveolar lavage samples from 38 patients with acute onset of the adult respiratory distress syndrome (median age, 54 years). A single lavage was performed in 25 patients, whereas from two to five studies were performed in 13 individuals. The timing of lavage ranged from 1 to 435 days after the onset of respiratory distress. Type II pneumocytes were present in 12 specimens as follows: 6 of 24 (25%) samples from days 1 to 3, 4 of 9 (44%) samples from days 4 to 10, and 2 of 11 (18%) samples from days 21 to 32. Specimens obtained after day 32 never contained such cells. Cytologically, type II pneumocytes may resemble the cells of acute radiation injury. Alternatively, they may be smaller, with an increased nuclear-cytoplasmic ratio, nuclear membrane irregularities, and prominent nucleoli, thus resembling the cells of adenocarcinoma. They may shed singly or in groups. A spectrum of changes from small cells to large, fully hyperplastic type II cells may be seen. Recognition of the morphologic features of type II pneumocytes and careful clinical correlation usually will suffice to prevent a false diagnosis of malignancy. Sequential lavage provides a means to study pulmonary epithelial changes after lung injury.

Processing of needle rinse material from fine-needle aspirations rarely detects malignancy not identified in smears.

When preparing FNA smears, we recover material left in the needle hub by forcefully striking the open hub against a slide. Material in the syringe tip is expressed by repeated forceful blasts of air (needle unattached). We investigated the utility of recovering additional material by rinsing the needle and syringe. Saline was used to flush the needle and syringe tip repeatedly. All material was processed by cytocentrifugation. We studied 159 needle rinse (NR) specimens from 152 patients (breast = 70, lymph node = 30, lung = 15, soft tissue = 14, salivary gland = 12, thyroid = 12, liver = 5, branchial cleft cyst = 1). Malignancy was identified in 21 FNAs (13%) from 21 patients (14%). All were diagnosed in smears (9 lung, 5 liver, 4 lymph node, 2 breast, 1 soft tissue). NR material identified 16 of these (76%). No case with benign smears (n = 138) showed malignancy in NR material. We conclude that if good technique is applied to preparation of smears and recovery of material from the needle hub and syringe tip, NR material will rarely identify additional malignancies. It thus represents an inefficient allocation of technical and human resources within the laboratory. However, NR may provide additional slides for special stains and may be useful for clinicians who do not always prepare high quality smears. Furthermore, the ease with which FNA of palpable masses can be repeated suggests that in the small number of cases requiring special stains, additional material can be readily obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactive type II pneumocytes in bronchoalveolar lavage fluid from adult respiratory distress syndrome can be mistaken for cells of adenocarcinoma.

A growing body of literature illustrates that bronchoalveolar lavage is a reliable and efficient means of diagnosing primary and secondary malignancies in the lung. Its safety in severely compromised patients often makes it preferable to other biopsy procedures. However, a variety of reparative and degenerative pulmonary disorders may result in cytologic alterations so severe that pneumocytes resemble cells of malignancy. We describe four patients with the adult respiratory distress syndrome from whom lavage fluid showed gland-like groups of malignant-appearing cells morphologically consistent with adenocarcinoma. Transbronchial biopsy sections in one case and lavage fluid electron microscopy in another showed that these pseudomalignant cells were reactive Type II pneumocytes with surface microvilli, cell junctions, and numerous cytoplasmic myelin figures. Careful clinicopathologic correlation is the best way to ensure accurate diagnosis in these cases.