Maintenance of imaginal discs of Drosophila melanogaster in chemically defined media.

A phosphate-buffered saline and a chemically defined synthetic medium for in vitro maintenance of imaginal discs of Drosophila melanogaster were developed. The composition of the chemically defined medium was varied in order to optimize the incorporation of tritiated uridine into RNA and tritiated amino acids into acid-insoluble protein. The optimal ranges obtained were: pH, 6.75-7.35; osmolarity, 285-345 milliosmoles/liter; sodium concentration, 40-60 mM/liter; potassium concentration, 40-60 mM/liter; magnesium concentration, 0.5-3.5 mM/liter; calcium concentration, 0.3-1.5 mM/liter; and inorganic phosphate concentration, 1.5-4.0 mM/liter. The phosphate-buffered saline is superior to a commonly used insect Ringer solution in maintaining total RNA and acid-insoluble protein synthesis in culture. The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.

Microcloning and replica plating of mammalian cells.

A technique for cloning single mammalian cells has been developed by means of a microculture method (0.010 milliliters per culture). A technique of replica plating has also been developed for use with clones of single cells in microcultures; this technique permits the screening of desired properties of many clones simultaneously before the growth of stock cultures. With these techniques an investigator can with limited budget, space, and personnel perform research using tissue culture systems.

Lymphocyte phenotype and function in pseudolymphoma associated with Sjögren's syndrome.

Lymph node (LNL) and salivary gland lymphocytes (SGL) from three patients with pseudolymphoma and primary Sjögren's syndrome (1(0)SS) were characterized with monoclonal antibodies to demonstrate (a) a predominance of T cells (greater than 80%) reactive with anti-T cell antibodies OKT4 (greater than 70%) and OKT8 (less than 20%); (b) a high prevalence of activation antigens (greater than 50% of cells reactive with antibody OKT10 and anti-Ia antibody); (c) polyclonal B cells (8-15% of all cells expressing kappa or lambda); and (d) a specific B cell subset defined by reactivity with antibody B532 that was not present in their peripheral blood. In vitro functional studies showed that both SGL and LNL provided T helper activity for immunoglobulin synthesis and that this activity could be abolished by treatment with antibody OKT4 plus complement. The SGL and LNL exhibited little natural killer, antibody-dependent cellular cytotoxicity, or cytotoxic T cell activity. Normal karyotype was observed in SGL, LNL, and peripheral blood lymphocytes (PBL) from these patients. These findings indicate that pseudolymphoma in 1(0)SS results from the infiltration of salivary glands and extraglandular tissues by nonneoplastic T helper cells. Monoclonal antibodies provide an important tool to distinguish pseudolymphoma from non-Hodgkins (B cell) lymphomas that have a markedly elevated incidence in 1(0)SS patients. Our finding of T helper cells in pseudolymphoma tissues supports the hypothesis that chronic stimulation of B cells by helper T cells leads to eventual escape of a malignant B cell clone.

Phase I clinical comparative study of monoclonal antibody KS1/4 and KS1/4-methotrexate immunconjugate in patients with non-small cell lung carcinoma.

A Phase Ia clinical trial was undertaken to evaluate and compare murine monoclonal antibody KS1/4 and KS1/4-methotrexate immunoconjugate in patients with Stage IIIB or IV non-small cell carcinoma of the lung. Six patients received KS1/4 alone and five patients received KS1/4-methotrexate conjugate. The maximal total dose received per patient in both groups was 1661 mg. Mild to moderate side effects in both groups included fever, chills, anorexia, nausea, vomiting, diarrhea, anemia, and brief transaminasemia. One patient who received antibody alone had an apparent acute immune complex-mediated reaction. Ten of 11 patients had a human anti-mouse response. Posttreatment carcinoma biopsies revealed binding of monoclonal antibody KS1/4 and deposition of C3d and C4c complement fragments. Monoclonal antibody binding and complement deposition correlated with increasing doses of infused antibody. There was one possible clinical response.

The distinction of small cell and non-small cell lung cancer by growth in native-state histoculture.

Histological analysis remains the primary method of distinguishing between small cell (SCLC) and non-small cell lung cancer (NSCLC). This distinction has significant impact therapeutically because of their relative difference in chemoresponsiveness (J.D. Minna et al., Principles and Practice of Oncology, pp. 396-474, 1981). Yet for at least 10% of lung tumors, pathologists will disagree upon the classification (A.R. Feinstein et al., Am. Rev. Respir. Dis., 101: 671-684, 1970). Furthermore, current neuroendocrine markers lack specificity for SCLC although the presence of these markers may help predict chemosensitivity (S.L. Graziano et al., J. Clin. Oncol., 7: 1375-1376, 1989; S.B. Baylin, J. Clin. Oncol., 7: 1375-1376, 1989; C.L. Berger et al., J. Clin. Endocrinol. Metab., 53: 422-429, 1981; A.F. Gazdar et al., Cancer Res., 45: 2924-2930, 1985). In vitro growth characteristics may more accurately reflect biological properties of aggressiveness and susceptibility to chemotherapy. In this study, 3-dimensional gel-histoculture was used to retrospectively distinguish between NSCLC and SCLC. Tumor explants from 78 patients with NSCLC and 13 patients with SCLC were grown in gel-supported histocultures with an overall success rate of 92%. These 2 tumor types were distinguishable by their 3-dimensional in vitro tissue architecture. In addition, proliferation rates were measured by histological autoradiography after 4-day incorporation of [3H]dThd. The percentage of cells labeled in the most proliferatively active regions of the autoradiograms was termed the growth fraction index (A.F. Gazdar et al., Cancer Res., 45: 2924-2930, 1985; R.A. Vescio et al., Proc. Natl. Acad. Sci. U.S.A., 84: 5029-5033, 1987; R.M. Hoffman et al., Proc. Natl. Acad. Sci. U.S.A., 86: 2013-2017, 1989). The mean growth fraction index for pure small cell lung cancer was 79 +/- 10%, differing markedly from that of 35 +/- 19% for mixed small cell/large cell tumors, adenocarcinoma (38 +/- 16%), large cell undifferentiated carcinoma (40 +/- 18%), and squamous cell carcinoma (33 +/- 15%) (P less than 0.001 in each case). We therefore conclude that 3-dimensional gel-histoculture is a useful means of distinguishing pure SCLC from NSCLC, which may improve treatment decision making.

Apoptosis in breast carcinomas detected with monoclonal antibody to single-stranded DNA: relation to bcl-2 expression, hormone receptors, and lymph node metastases.

Precise quantitation of apoptotic cells in solid tumors is necessary to determine the role of apoptosis in cancer growth, prognosis, and treatment. In this study, the intensity of apoptotic death was determined in 91 breast carcinomas with a novel cellular marker of apoptosis based on the staining of histological sections with a monoclonal antibody (MAb) to single-stranded DNA. Staining of apoptotic cells with the MAb reflected the decreased thermal stability of DNA induced by the digestion of nuclear proteins, as demonstrated by the elimination of staining in sections reconstituted with histones before heating. The high sensitivity and specificity of apoptosis analysis with the MAb is based on the central role of protease activation in the mechanism and control of apoptosis. Apoptotic indexes (AIs) in breast carcinomas ranged between 0 and 46%. Most of the carcinomas had relatively low AIs, whereas 29 cases were classified as carcinomas with intensive apoptosis (AI >/= 10%). The high level of apoptotic cell death was associated with negative immunostaining for bcl-2 protein, the loss of estrogen and progesterone receptors, high proportion of cells in S-phase, and increased risk of lymph node metastases. There was no correlation between AI and tumor size or p53 immunostaining. Lymph node metastases were detected in 59% of patients with high levels of apoptosis in primary carcinomas and in only 21% of patients with AIs below 10% in primary carcinomas. Thus, the high sensitivity of the MAb assay made it possible to identify a subset of breast carcinomas with intensive apoptosis and markers of poor prognosis. These results demonstrate that the measurement of apoptosis in breast carcinomas provides valuable prognostic information.

Scintigraphic evaluation of diabetic osteomyelitis: concise communication.

We have reviewed the three-phase bone scans, radiographs, and histologic findings of 39 diabetic patients with serious foot problems. The sensitivity and specificity of bone scans were 83% and 75%, respectively, for osteomyelitis of the small bones of the foot. The positive and negative predictive values were 87% and 69%, respectively. The radiographs were less sensitive (62%) and less specific (69%), with predictive values of 80% (positive) and 47% (negative).

Synergistic induction of apoptosis in breast cancer cells by tamoxifen and calmodulin inhibitors.

Breast cancer cells are relatively resistant to the induction of apoptosis (AP) and drug regimens which readily activate apoptotic death, may enhance the antitumor effect. Rapid and intensive induction of apoptosis was observed in estrogen receptor positive and negative breast cancer cell cultures treated with tamoxifen (TMX) combined with the calmodulin antagonists trifluoperazine (TFP) or W7. TMX (1-5 microM) alone or calmodulin antagonists alone did not induce apoptosis. Importantly, intensive apoptosis was also induced by TMX and TFP in the cells obtained from primary human breast carcinomas. Inhibition of the Ca2+ calmodulin signaling pathway is an effective way to activate apoptotic death in epithelial cells. Combination of TMX with non-toxic calmodulin inhibitors may increase the preventive and therapeutic effects of TMX.

Spindle cell lipoma in a perianal location.

The authors report a case of spindle cell lipoma that occurred in the perianal region with involvement of the external anal sphincter. This is a very rare location for this benign lipoma. This case extends the range of sites in which this neoplasm is found to arise.

Intrauterine latent herpes simplex virus infection: I. Spontaneous abortion.

Herpes simplex virus (HSV, probably type 2) antigen was detected in nonpregnant and pregnant endometria, placentae, umbilical cords, and neonatal tissues (companion paper) by avidin-biotin complex immunohistochemical studies. HSV cytologic abnormalities were not detected in any of the 380 cases examined: included were specimens from therapeutic and spontaneous abortions (200 cases) and endometrial curettage (180 cases). The presence of inflammation was not correlated with HSV positivity. Endometrial HSV positivity was significantly correlated with normal late secretory phase (40 per cent of specimens positive), abnormal secretory phase (67 per cent positive), and therapeutic (33 per cent positive) versus spontaneous (26 per cent positive) abortions. Placental HSV positivity was significantly correlated with spontaneous (39 per cent positive) versus therapeutic (14 per cent positive) abortions and with blighted ova (67 per cent positive). No significant correlation was found between HSV positivity and a clinical history of oral or genital HSV infection in either the patient or the male partner. The data support the concept of a subclinical latent intrauterine endometrial HSV infection that is hormonally regulated and can produce transplacental infection of the embryo or fetus, with variable consequences.

Intrauterine latent herpes simplex virus infection: II. Latent neonatal infection.

Herpes simplex virus (HSV, probably type 2) antigen has been detected in endometria and abortion tissue (companion paper) and in placentae, umbilical cords, and fetal and neonatal organs by avidin-biotin complex immunohistochemical studies. HSV cytologic abnormalities were not detected in any of the 12 normal and 64 abnormal cases analyzed, nor was HSV detected by culture or electron microscopy in selected cases. Antigen was present in single epithelial and, rarely, mesenchymal cells of various organs. Clinically unexplained fetal or neonatal problems associated with HSV antigen positivity included intrauterine death, fetal growth retardation, cystic brain degeneration, hydrops, interstitial pneumonitis, necrotizing enterocolitis, hepatitis, encephalitis, myocarditis, and renal failure. Maternal floor infarct of placenta and calcifying funisitis are the manifestations of intrauterine HSV infection in most cases. Maternal history of HSV infection was uncommon. It is concluded that intrauterine HSV infection may persist in the fetus and neonate in a latent fashion without cytologic abnormalities or detectable virus. This latent infection may be associated with intrauterine and neonatal death, organ damage, and neonatal disease.

Polypoid peritoneal metastases from carcinoid neoplasms.

Two cases of lethal carcinoid neoplasia that involved unusual polypoid intraperitoneal metastases rather than the usual flat, sclerotic foci are reported. This type of intraperitoneal polypoid carcinomatosis should alert both the surgeon and pathologist to the possibility of carcinoid neoplasia.

The future anatomic pathology laboratory for molecular diagnosis and prognosis: circa 1990-95.

Molecular biology provides analytical tools for the study of human disease. These clinical assays can help make a diagnosis, predict a prognosis, or elucidate the pathogenesis of poorly understood diseases. The detection and/or quantitation of specific genes, messenger RNA's, and their protein products provide diagnostic and prognostic information for physicians. Illustrative applications of some of these techniques in human virus infection and breast cancer will suggest the future direction that an anatomic pathology laboratory should consider if it is to provide optimal and competitive service to physicians.

Apoptosis in human breast and gastrointestinal carcinomas. Detection in histological sections with monoclonal antibody to single-stranded DNA.

We report application of a novel immunohistochemical procedure for the staining of apoptotic (AP) cells in paraffin sections using monoclonal antibody (MAb) to single-stranded DNA. MAb differentiated between apoptosis and necrosis and in contrast to in situ end labelling specifically stained only AP cells. AP carcinoma cells stained with the antibody were detected in 32 of 58 infiltrating human breast carcinomas and in 9 of 15 colon adenocarcinomas. Stromal cells stained with the MAb were observed in all carcinomas, including those in which no AP carcinoma cells were detected. There was a strong positive correlation between the presence of AP cells, loss of hormone receptors and a high proliferation rate in breast carcinomas. AP cells were present in 80-87% of receptor-negative carcinomas, while most of receptor-positive breast carcinomas did not contain AP cells. Apoptosis in tumor cells was detected significantly more frequently among breast carcinomas with high, than among carcinomas with low S-phase fraction. AP cells were present in 93-95% of breast carcinomas which were receptor-negative and had a high S-phase fraction. Immunostaining demonstrated a strong positive correlation between the loss of bcl-2 protein and intensive apoptosis in breast carcinomas. Association between apoptosis and markers of poor prognosis in breast cancer (loss of hormone receptors, intensive proliferation, loss of bcl-2 protein) indicates that apoptotic cell death is typical of more aggressive carcinomas.

Thyroid nodules indeterminate by needle biopsy.

In our experience with 121 patients 18 (15 percent) thyroid nodules studied by needle biopsy were considered indeterminate relative to the presence of a low-grade, well-differentiated carcinoma. For 11 of the 18 patients, operation was performed with carcinoma identified in two (18 percent). Although experience reduced this problem, the frequency of carcinoma justifies operation for patients with indeterminate thyroid nodules by needle biopsy, unless other factors dictate otherwise. Inadequate results of fine-needle aspiration biopsy requires a determination of therapy on the basis of other clinical factors. However, permanent disappearance or great reduction in size following aspiration of cystic nodules, repeat biopsy, and biopsy with large needles are important in supporting nonoperative therapy. The indeterminate and inadequate cases must be considered in assessing reports of the use of needle biopsy of thyroid nodules. The large size of a thyroid nodule and previous external radiation therapy are factors supporting operative treatment. Improved selection of patients with benign thyroid nodules for thyroid hormone suppression therapy is needed--thyroid-releasing hormone testing may be of help.

Femoral trabecular-pattern index and bone mineral content measurement by photon absorption in senile osteoporosis.

A comparative study of the usefulness of the femoral trabecular-pattern index (Singh index) and bone mineral content measurement by photon absorption in senile osteoporosis was performed in 106 white women, seventy to ninety-five years old. In 17% of the subjects adequate hip roentgenograms could not be obtained. Interpretation of the Singh index was not found to be simple and easily reproducible. A positive correlation was noted between the bone mineral content and the Singh index. The incidence of previous vertebral and non-vertebral fracture was noted to be inversely related to both bone mineral content and the Singh index, but neither method clearly separated subjects with fracture from those without fracture.

Atypical squamous cells of undetermined significance. Current laboratory practices of participants in the College of American Pathologists Interlaboratory. Comparison Program in Cervicovaginal Cytology.

OBJECTIVE: To evaluate current laboratory practices and rates for atypical squamous cells of undetermined significance (ASCUS), a category of epithelial cell abnormality in the Bethesda System. DESIGN: Questionnaire surveys were mailed in December 1993 and March 1994. SETTING: Cytopathology laboratory participants in the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology (PAP). RESULTS: Most responding laboratories (82.5%) limited the use of "atypia" terminology to abnormalities of undetermined significance. Nearly half of the laboratories employed only the term ASCUS for squamous epithelial changes in this category. The median rate of ASCUS in 1993 was 2.8%, with 10% of laboratories reporting rates greater than 9.0%. The median squamous intraepithelial lesion rate was 2.0%, with a median ASCUS-squamous intraepithelial lesion ratio of 1.3. The majority of laboratories qualified a portion of ASCUS cases and issued recommendations for follow-up when appropriate. Fifty-six percent of laboratories surveyed included patients diagnosed with ASCUS in follow-up programs. Laboratories estimated that about 20% (median response) of patients with ASCUS smears had a squamous intraepithelial lesion or equivalent diagnosis made within a year's follow-up. CONCLUSIONS: The ASCUS category is used by the majority of laboratories as recommended by the Bethesda System, but reporting rates vary. The results of this survey and associated surveys provide laboratories with useful benchmark figures for interlaboratory comparison of ASCUS practices.

Administration of p.g. 600 to sows at weaning and the time of ovulation as determined by transrectal ultrasound.

This study determined whether the interval from estrus to ovulation was altered by giving P.G. 600 to sows at weaning. Mixed-parity sows received P.G. 600 i.m. (n = 72) or no treatment (n = 65) at weaning (d 0). Beginning on d 0, sows were observed for estrus twice daily. At the onset of estrus and thereafter, ultrasound was performed twice daily to determine the average size of the largest follicles and time of ovulation. Weaning age (20.1+/-0.4 d) did not differ (P > 0.10) between treatments. More P.G. 600 sows expressed estrus within 8 d (P < 0.01) than controls (94.4% vs 78.4%, respectively). Parity was associated with expression of estrus (P < 0.02), with 78% of first-parity and 93% of later-parity sows exhibiting estrus. However, no treatment x parity effect was observed (P > 0.10). The interval from weaning to estrus was reduced (P < 0.0001) by P.G. 600 compared with controls (3.8+/-0.1 d vs 4.9+/-0.1 d). Follicle size at estrus was not affected by treatment (P > 0.10). The percentage of sows that ovulated did not differ (P > 0.10) for P.G. 600 and control sows (90.3% vs 81.5%, respectively). Time of ovulation after estrus was not affected by treatment and averaged 44.8 h. However, univariate analysis indicated that the interval from weaning to estrus influenced the interval from estrus to ovulation (r = 0.43, P < 0.0001). Further, multivariate analysis showed an effect of treatment on the intervals from weaning to estrus, weaning to ovulation (P < 0.0001), and estrus to ovulation (P < 0.04). Within 4 d after weaning, 81% of the P.G. 600 sows had expressed estrus compared with 33% of controls. However, this trend reversed for ovulation, with only 35% of P.G. 600 sows ovulating by 36 h after estrus compared with 40% of controls. The estrus-to-ovulation interval was also longer for control and P.G. 600 sows expressing estrus < or = 3 d of weaning (45 h and 58 h, respectively) than for sows expressing estrus after 5 d (39 h and 32 h, respectively). Farrowing rate and litter size were not influenced by treatment. However, the interval from last insemination to ovulation (P < 0.02) indicated that more sows farrowed (80%) when the last insemination occurred at < or = 23 to > or = 0 h before ovulation compared with insemination > or = 24 h before ovulation (55%). In summary, P.G. 600 enhanced the expression of estrus and ovulation in weaned sows but, breeding protocols may need to be optimized for time of ovulation based on the interval from weaning to estrus.

Effect of subcutaneous vs intramuscular administration of P.G. 600 on estrual and ovulatory responses of prepubertal gilts.

The effects of s.c. and i.m. administration of P.G. 600 on estrual and ovulatory responses of prepubertal gilts were investigated. One hundred eighty-four crossbred gilts between 159 and 174 d of age were assigned to receive P.G. 600 s.c. (s.c. P.G. 600) in the flank, P.G. 600 i.m. in the neck (i.m. P.G. 600), or no treatment (control). At the beginning of the study (d 0), animals were selected from a modified, open-front barn, regrouped, relocated to new pens, and exposed once daily to a mature boar to check for estrus. On d 17, ovaries were collected from all gilts and analyzed for the presence of corpora lutea (CL), cystic follicles, and cystic CL. A higher proportion of gilts expressed estrus with s.c. P.G. 600 (76%) than with i.m. P.G. 600 (52%, P < .01) or controls (15%, P < .01). The interval from initiation of treatment on d 0 to estrus was reduced (P < .01) by P.G. 600 (4.6 d) compared to controls (5.9 d), but there was no significant difference between P.G. 600 treatments. Both s.c. P.G. 600 (86%) and i.m. P.G. 600 (77%) induced more gilts to ovulate (P < .01) than controls (18%), but there was no significant difference between P.G. 600 treatments. No significant effect of treatment was detected on number of CL (17.9), number of cystic follicles (1.5), or number of cystic CL (2.1). Proportions of gilts that developed cystic follicles or cystic CL were not influenced by treatment. Results of this study indicated that s.c. administration of P.G. 600 significantly improved the induction of estrus in prepubertal gilts compared to i.m. administration.

Early scintigraphic findings of occult femoral and tibial fractures in infants.

Differentiation of occult skeletal injuries from early acute osteomyelitis in infants and young children is important clinically. The Tc-99m MDP scintigraphic findings in six patients who had occult femoral or tibial fractures were reviewed. The images obtained early (at 1-4 days from the onset of symptoms) shared the common characteristic finding of a subtle but definite, generalized increased uptake of the tracer along the entire length of the injured bone. This pattern of uptake was similar regardless of the type of fracture. These were different from the focal abnormalities that have been observed in early acute osteomyelitis. In the absence of an extensive cellulitis or a vascular occlusive disease, a bone image showing a mild diffuse uniform increased uptake along the entire length of the tibia or femur in infants and young children with lower extremity pain of less than 1 week's duration should suggest the diagnosis of occult skeletal fractures.