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An autoimmune kidney disease morphologically and functionally similar to Heymann nephritis (HN) was induced in mature male Sprague Dawley rats by repeated weekly IP injections of a chemically modified azo sonicated ultracentrifuged (u/c) rat kidney fraction 3 (rKF3) antigen in an aqueous medium. The experiment was terminated 15 weeks after the first injection of the chemically altered antigen. Serum samples collected and analysed by an indirect fluorescent antibody test on normal rat kidney sections during the course of the experiment showed a gradual rise in circulating pathogenic autoantibodies directed against the proximal tubular brush border regions. Proteinuria was present and significantly increased in the urine of two of eight rats. The arising immune-complex glomerulonephritis (ICGN) revealed typical HN kidney disease lesions in 70% of the rats in histological, direct fluorescent antibody and electron-microscopical examinations. Control rats injected similarly with the an unmodified version of the same antigen did not develop the HN-characteristic morphological and functional changes. To our knowledge, this is the first time that the autoimmune kidney disease designated as an active HN has been produced by the administration of a chemically altered renal antigen in an aqueous solution and not by the usual presentation of the nephritogenic renal antigen in an adjuvant.

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.

A slowly progressive autoimmune kidney disease was induced in Sprague Dawley rats by subcutaneous injection of a chemically modified kidney antigen (rKF3), incorporated into Alum and Distemper complex vaccine, followed by subcutaneous injections of an aqueous preparation of the same antigen. Pathogenic autoantibodies developed, which reacted with fixed glomerular nephritogenic antigen. Subsequently, immunopathological events lead to chronic progressive immune complex glomerulonephritis and proteinuria. The slowly developing disease was morphologically and functionally similar to Heymann nephritis (HN). The damage observed in the kidneys of experimental animals at 8 weeks and at the end of the experiment was examined by direct fluorescent antibody test, histology and electron microscopy. The changes were similar to the typical lesions found in HN rat kidneys, but less severe. Animals became proteinuric from 17 weeks onward (instead of the usual 4-8 weeks). By the end of the experiment, at 8 months, 100% of the rats were proteinuric. This new experimental model of autoimmune kidney disease, which is not complicated by intraperitoneal deposition and retention of Freund's complete adjuvant and renal tubular antigens, allowed us to investigate the pathogenesis of the disease processes from a different aspect, and promises to be a useful and improved model for the investigation of future treatment options.

Objective: To find out if the cost of health services was artificially increased because of a delay in surgery due to a lack of resources., Design: A retrospective cohort study., Setting: Three urban hospitals in Calgary, Alta., Patients: The study cohort comprised 4441 patients (1 index procedure for each patient)., Interventions: Cholecystectomy, discectomy, hysterectomy, total knee and total hip replacements., Outcome Measures: The costs for physician claims, use of home care and pharmaceutical prescriptions 1 year before and after the selected procedures, using 1997/98 administrative records and waiting times maintained by Alberta Health and Wellness and Calgary Regional Health Authority., Results: The median wait for joint surgery (88 d for knee replacements and 65 d for hip replacements) was longer than for the other selected procedures (29 d for cholecystectomies, 21 d for discectomies and 42 d for hysterectomies). Total per patient physician claim costs decreased after surgery (cholecystectomy--30%, discectomy--24%, hip replacement--6%, hysterectomy--23% and knee replacement--4%). Seeing the procedure specialist more than once preoperatively was associated with a greater decrease in postoperative physician claim costs. Longer waits were not associated with more physician claim costs or Blue Cross prescriptions claim costs for seniors (> or = 65 yr) in the year before or after surgery nor were they associated with more physician claim costs during the actual wait compared with a matched postoperative time period., Conclusions: No evidence was found to suggest that waiting for 1 of 5 common surgical procedures is correlated with greater health service expenditures pre- or postoperatively. In this study, wait time is not a proxy for health service use nor do health service costs decrease markedly after surgery.

Objective: Provincial air ambulance transports of injured patients were quality reviewed prospectively to determine utilization and appropriateness of care., Methods: All trauma air ambulance transports over a 2-month span were reviewed prospectively. Revised Trauma Score, Injury Severity Score, probability of survival, prehospital time, distance of transport, procedures performed, and outcome were determined. Quality control questions were asked of the sending and receiving physicians., Results: The majority of air ambulance transports reviewed (N = 97) were indicated for mechanism and severity of injury. Economics and requirement for advanced medical care were indications in only 15%. Physicians tended to perform more advanced procedures, likely related to higher patient Injury Severity Score (23 vs. 15, p = NS). Four problems with air ambulance access were identified. The overtriage rate was 5%. Inappropriate patient care was documented in six (6%) cases; a physician was present for only one of these., Conclusions: A low overtriage rate was documented, raising concerns that the undertriage rate may be too high. Injured patients air transported without physician accompaniment more often received inappropriate care, suggesting that physician accompaniment is beneficial.

A retrospective review of 332 needle localization biopsies for nonpalpable mammographic abnormalities was performed. Twenty-one invasive and 12 noninvasive carcinomas were identified in this population, for a true positive biopsy rate of 10 percent. A review of all needle localization mammograms performed on these patients by a radiologist specializing in mammography identified 225 mammograms with a low probability of malignancy. In this group, there were four in situ and six invasive carcinomas (true positive biopsy rate of 4 percent). In the remaining 107 mammograms with a high probability of malignancy, there were eight in situ and 15 invasive carcinomas (true positive biopsy rate of 21.4 percent). In the low probability group, fine needle aspiration or excisional biopsy were recommended for six of the ten neoplasms and follow-up mammograms at three to six months for the remaining four (two in situ and two invasive carcinomas). Using selectivity, 225 biopsies could have been avoided at a savings of $97,368 (Canadian dollars) in fees for physicians alone while still identifying 29 of 33 neoplasms (87 percent of all neoplasms) and possibly delaying diagnosis for three to six months or longer in four of 33 neoplasms (13 percent, two in situ and two invasive) for which follow-up mammograms were recommended.

The molecular basis of androgen insensitivity was investigated in a family with the complete form of the syndrome. Polymerase chain reaction amplification and Southern blot analysis of genomic DNA revealed a deletion of the entire androgen receptor (AR) gene in affected individuals. The carrier status of female members of this family was examined using a HindIII restriction fragment length polymorphism associated with the AR gene. Obligate carriers were hemizygous for one of the two alleles at this locus, while heterozygosity for the polymorphic alleles, implying the presence of two copies of the AR gene, indicated noncarrier status. This conclusion was supported by gene dosage studies using comparative densitometric analysis of Southern blots hybridized simultaneously with an AR cDNA probe and a control cDNA probe from an unrelated gene. Finally, the pattern of inheritance of another X-linked DNA polymorphism allowed us to conclude that the original mutation had occurred in the germ line of the maternal great-grandfather of the index patient. Although rare, complete deletion of the AR gene is of particular importance in terms of correlation between molecular defect and phenotype, as it represents the quintessential form of complete androgen insensitivity, the null phenotype.

A skin hook flange was created to ease and distribute the forces involved in skin hook retraction, shifting these forces from the fingertips to the shaft of the fingers of the person performing the retraction. It will allow for easier retraction and can be used on most standard skin hooks.

Percutaneously inserted indwelling subclavian vein silastic catheters have revolutionized the administration of chemotherapeutic agents. Complications associated with insertion of such lines have always included bleeding, pneumothorax, haemothorax, arterial cannulation, and catheter displacement. Recently a patient receiving 5-fluorouracil and folinic acid for 11 months for small bowel carcinoma experienced a catheter fracture with distal segment embolization in the right atrium. A literature survey revealed 13 cases where mechanical shearing forces on the catheter caused by compression of the catheter between the clavicle and first rib were thought to be the cause for this complication. Interestingly all cases involved patients receiving chemotherapy. Radiologically, potential candidates for catheter fracture can be identified by the pinched-off sign with bending and pinching of the catheter at the thoracic inlet. Recommendations are for more lateral insertions of such percutaneously placed catheters and if the pinched-off sign is seen, then said catheters should be followed radiologically and probably should not remain in situ for longer than 6 months.

The adoptive transfer of tumor infiltrating lymphocytes (TIL) in conjunction with recombinant interleukin-2 (rIL-2) for the treatment of advanced cancer has recently been under intense investigation. Despite extensive research, the precise surface phenotype of TIL remains to be fully defined. To elucidate this unsolved problem, we established 11 TIL clones derived from rIL-2 expanded TIL obtained from B16-F10 murine melanoma tumors. These clones could be divided phenotypically into four groups: CD8 (+) T-cell clones, natural killer (NK)-cell clones, NK-like CD8 (+) T-cell clones, and double negative T-cell clones. Functionally, CD8 (+) T-cell clones demonstrated specific cytotoxic activity against B16-F10 melanoma cells, whereas NK-cell clones and double negative T-cell clones demonstrated only non-specific cytotoxic activity against NK-sensitive YAC-1 cells. NK-like CD8 (+) T-cell clones showed dual cytotoxic activity. Clones T1 [a CD8 (+) T-cell clone] and T2 [an NK-like CD8 (+) T-cell clone] which had cytotoxic activity against B16-F10 melanoma cells, demonstrated a proliferative response against immunoblotted B16-F10 melanoma antigens, whereas clones T7 (an NK-cell clone) and T10 (a double negative T-cell clone), which had no cytotoxic activity against B16-F10 cells, demonstrated no proliferative response against them. Winn assays revealed that only the CD8 (+) T-cell clone (T1) had an antitumor effect in vivo, whereas the double negative T-cell clone (T10) and NK-like CD8 (+) T-cell clone (T2) stimulated tumor growth in vivo. Adoptive immunotherapy using tumor-specific, highly cytotoxic TIL clones may represent a useful future immunotherapeutic option for the treatment of human tumors.

Cytotoxic T lymphocytes (CTL) specific for autologous human melanoma have been successfully generated in vitro from tumor bearing lymph nodes without any stimulation by the autologous tumor. Tumor-involved lymph node cells (LNC) were cultured in serum free medium (AIM-V) containing 1,000 U/ml of recombinant interleukin-2. The best expansion and specific cytotoxicity of CTL were achieved in 4 to 6 weeks of culture. The predominant populations in cultured LNC-derived CTL were CD2+, CD3+, CD4-, CD8+, CD56-, and HLA-DR+ T cells. These data suggested that tumor-involved LNC may provide an alternative source for the generation of tumor-specific CTL in adoptive immunotherapy.

Non-parenchymal liver cells (NPCs) have been implicated in murine host resistance to hepatic metastases. We have examined the relative cell number, morphology, phenotype, and cytotoxic potential of Percoll fractionated C57BL/6 murine liver NPCs. Low density (Percoll fractions 2 and 3) cells showed a large granular lymphocyte morphology and made up 76% of all NPCs recoverable, while high density (fractions 5 and 6) showed a small lymphocyte morphology and made up 10% of all NPCs. Low density cells demonstrated the following phenotype: 14% of the cells demonstrated the Thy 1.2 marker; 12%, the Lyt-2 marker; 67%, the L3T4 marker; 74%, the asialo GM1 marker; 30%, the 49H.8 marker; and 65%, the F4/80 marker. The high density cells expressed the same markers on 71%, 21%, 33%, 68%, 37%, and 19% of their cell surface, respectively. There were no differences phenotypically between high density NPCs and splenocytes except for the F4/80 expression (fractions 5 and 6 NPCs, F4/80 expression 19%, fresh splenocytes 60%). Dual color analysis of L3T4+ NPCs documented that fractions 2 and 3 cells also expressed the F4/80 marker on 85% of their cell surface and the Thy 1.2 marker on 11% of their cell surface. The high density fractions 5 and 6 L3T4+ cells expressed the F4/80 marker on 16% of their cell surface, and the Thy 1.2 marker on 89% of their cell surface. Cytotoxicity against YAC-1 [a natural killer (NK) sensitive target], MCA-102 (a NK resistant target), and WEHI-164 (a natural cytotoxicity target) were similar for fractions 2 and 3, and 5 and 6 cells. Based upon the expression of the F4/80 marker on L3T4+ cells that are Thy 1.2 negative and appear to be similar to LGLs morphologically (fractions 2 and 3 NPCs), we propose that these cells are monocyte precursors while fractions 5 and 6 cells are small lymphocytes. These findings with liver LGLs support the need for the evaluation of monocyte directed biological response modifiers in therapeutic models of murine hepatic metastases.

Using a panel of human/rodent somatic cell hybrids segregating human X/autosome translocations and deletions, we have refined the localization of the X-linked sequences homologous to ornithine-delta-aminotransferase (OAT), the structural locus for which (OAT) maps to chromosome 10. OAT-related ("-like") (OATL) sequences mapped to two nonadjacent intervals: OATL1 mapped to Xp11.3-p11.23, while OATL2 mapped to Xp11.22-p11.21. X-linked OATL1 sequences polymorphic for ScaI and StuI map to the more distal interval in Xp11.3-p11.23. These results should help guide long-range cloning and mapping studies, as well as refine the genetic linkage map in this region of the X chromosome.

The growth, in vitro cytolytic activity and phenotype of murine MC-38 adenocarcinoma tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 monoclonal antibody (mAb) and recombinant interleukin-2 (RIL-2) as compared to RIL-2 alone was investigated. When assayed for growth, anti-CD3 mAb + RIL-2 MC-38 TILs demonstrated an enhanced proliferative activity compared to RIL-2 alone (fold expansion, 16,228 and 365,713 compared to 112 and 5594, culture times: 55 and 118 days, experiments 1 and 2, respectively). TILs cultured with anti-CD3 mAb alone demonstrated little expansion (fold expansion 6 and 3, experiments 1 and 2, respectively). Early during culture, the anti-CD3 mAb + RIL-2 MC-38 expanded TILs demonstrated broad cytolytic activity (LU: day 17, against MCA-102: greater than 125, YAC-1: greater than 125, MC-38, greater than 125). This lytic picture reversed with time with increasing specificity demonstrated against MC-38 (LU: day 53, MCA-102: less than 1, YAC-1: less than 1, MC-38: 8). TILs expanded with RIL-2 alone demonstrated more lysis of the YAC-1 target and little lysis of the other targets.(ABSTRACT TRUNCATED AT 250 WORDS)

X-chromosome inactivation results in the cis-limited dosage compensation of genes on one of the pair of X chromosomes in mammalian females. Although most X-linked genes are believed to be subject to inactivation, several are known to be expressed from both active and inactive X chromosomes. Here we describe an X-linked gene with a novel expression pattern--transcripts are detected only from the inactive X chromosome (Xi) and not from the active X chromosome (Xa). This gene, called XIST (for Xi-specific transcripts), is a candidate for a gene either involved in or uniquely influenced by the process of X inactivation.

X-chromosome inactivation results in the strictly cis-limited inactivation of many but not all genes on one of the two X chromosomes during early development in somatic cells of mammalian females. One feature of virtually all models of X inactivation is the existence of an X-inactivation centre (XIC) required in cis for inactivation to occur. This concept predicts that all structurally abnormal X chromosomes capable of being inactivated have in common a defineable region of the X chromosome. Here we report an analysis of several such rearranged human X chromosomes and define a minimal region of overlap. The results are consistent with models invoking a single XIC and provide a molecular foothold for cloning and analysing the XIC region. One of the markers that defines this region is the XIST gene, which is expressed specifically from inactive, but not active, X chromosomes. The localization of the XIST gene to the XIC region on the human X chromosome implicates XIST in some aspect of X inactivation.

A recent case of a biliary granular cell tumor of the cystic duct prompted a literature review and an extensive pathological examination of the tumor in question. A total of 44 cases have been described mostly in black females. Most cases present with biliary symptoms, and simple surgical resection allows complete control of this benign condition. Granular cell tumors are most likely derived from neural crest cells.

The human gene for histidase (histidine ammonia-lyase; HAL), the enzyme deficient in histidinemia, was assigned to human chromosome 12 by Southern blot analysis of human X mouse somatic cell hybrid DNA. The gene was sublocalized to region 12q22----q24.1 by in situ hybridization, using a human histidase cDNA. The homologous locus in the mouse (Hal) was mapped to region 10C2----D1 by in situ hybridization, using a cell line from a mouse homozygous for a 1.10 Robertsonian translocation. These assignments extend the conserved syntenic region between human chromosome 12 and mouse chromosome 10 that includes the genes for phenylalanine hydroxylase, gamma interferon, peptidase, and citrate synthase. The localization of histidase to mouse chromosome 10 suggests that the histidase regulatory locus (Hsd) and the histidinemia mutation (his), which are both known to be on chromosome 10, may be alleles of the histidase structural gene locus.

The syndrome of multiple endocrine neoplasia type 2B (MEN 2B) resembles that of MEN 2A in that both include medullary carcinoma of the thyroid, pheochromocytoma, and autosomal dominant inheritance, but is distinct in that MEN 2B patients have neuromas of the mucous membranes. MEN2A has been linked to RBP3, D10S5, FNRB, D10S15, and D10Z1 near the centromere of chromosome 10. We examined linkage between MEN2B and RFLPs on chromosome 10 in all available members in two or three generations of 14 kindreds. The centromere marker D10Z1 was linked to MEN2B with a peak lod score of 5.42 at theta = 0.02. One possible recombinant was observed between D10Z1 and MEN2B. Multipoint analysis of RFLPs at FNRB, D10Z1, RBP3, and D10S15 gave a peak lod score of 7.12 at the midpoint between D10Z1 and RBP3 on the long arm (band q11). The most likely gene order FNRB-D10Z1-MEN2B was 27 times more likely than MEN2B-FNRB-D10Z1 and 31/2 times more likely than FNRB-MEN2B-D10Z1. Additional data will be required to establish the order of these loci with confidence.

Synaptophysin is an integral membrane protein of small synaptic vesicles in brain and endocrine cells. We have determined the structure and organization of the human synaptophysin gene and have established the chromosome localizations in man and mouse. Analysis of a cosmid clone containing the human synaptophysin gene (SYP) revealed seven exons distributed over approximately 20 kb, when compared with the previously published cDNA sequence. The exon-intron boundaries have been identified and do not correlate with functional domains. One intron interrupts the 3' untranslated region. Chromosomal localization of the human and murine genes for synaptophysin established the human SYP locus on the X chromosome in subbands Xp11.22-p11.23 and the mouse synaptophysin gene locus (Syp) on the X chromosome in region A-D. In addition, an Eco0109 RFLP has been identified and used in genetic mapping of the human SYP locus and supports the order TIMP-SYP-DXS14 within a span of approximately 4-7 centimorgans.

The in vivo administration of recombinant interleukin-2 (rIL-2) in humans has led to anemia, thrombocytopenia, and eosinophilia. In an attempt to evaluate the effects of the in vivo administration of rIL-2 on murine bone marrow, we administered rIL-2 to C57BL/6 female mice i.p. three times a day at doses ranging from 10,000 to 100,000 U for 10 days; we then harvested blood and bone marrow from these animals every other day and performed the following analyses: White blood cell count, red blood cell count, hemoglobin concentration, histogram analysis of nucleated cell volume, manual differential counts, and in vitro colony-forming assays for granulocytes and monocytes (CFU-GM). The administration of rIL-2 induced an overall increase in the total white blood cell count that was dose-dependent for its appearance and overall number. This increase was secondary to an increase in monocytes and granulocytes but not to a change in lymphocyte number. Myeloid proliferative activity measured by CFU-GM revealed a biphasic pattern of activity. An early proliferation at 2 days was not followed by lymphocytosis. However, a second peak of proliferation at 6 days was associated with peripheral blood granulocytosis and monocytosis. After rIL-2 was discontinued on day 10, the CFU-GM activity returned to normal by days 16-18. These results suggest that the in vivo administration of rIL-2 may play an important role in the regulation of hematopoiesis.

Five cases of bloody nipple discharge during pregnancy without associated breast masses were seen over the past 3 years by the author. Because of the reported association of breast cancer with bloody nipple discharge, close follow-up of these women at monthly intervals during pregnancy and trimonthly during the postpartum period was carried out. In all instances, the discharge appeared late during the second trimester or during the third trimester of pregnancy. It was unilateral and spontaneous and arose from multiple ducts, and it was associated with an increase in breast size and always with the larger breast of the two. The discharge cytologic study done on all cases was negative for neoplastic cells and the discharges resolved spontaneously within 2 months of onset. Postpartum follow-up ranging from 6 months to 3 years has revealed no evidence of neoplastic changes thus far. Mammograms ordered before these patients were referred were not helpful due to the increase in density of the breast tissue secondary to the pregnancy. Because a few cases of breast cancer during pregnancy have presented solely with a bloody nipple discharge, I recommend extremely close follow-up of these women and no surgical intervention unless a mass is discovered or the nipple discharge cytology is either suspicious or positive at the initial visit or during follow-up.

The systemic administration of high dose recombinant interleukin 2 (RIL-2) can mediate significant reductions in the number of hepatic metastases in a murine system. This effect is sensitive to host irradiation. Both large granular (LGLs) and small (SLs) lymphocytes have been implicated as the cells mediating the antitumor effect. Utilizing selective Percoll fractionation of liver nonparenchymal lymphoid cells, we have attempted to determine the cell types involved in tumor immunotherapy of murine liver metastases during RIL-2 administration. At a RIL-2 dose of 25,000 units given i.p. three times a day, the total number of lymphoid cells seen in murine livers reached a peak on day 6 after the onset of RIL-2 therapy, lasting until day 10 and ranging from 25 to 29 times baseline values. Both LGLs and SLs were identified and SLs made up over one-half the cells present in murine livers. Phenotypic analysis of LGLs and SLs revealed that during exposure to RIL-2, bands 5 + 6 SLs expressed the Thy-1.2, Lyt-2, and Lyt-1 antigens to a greater degree than LGLs. LGLs exposed to RIL-2 demonstrated a decrease in the expression of the asialo GM1 antigen during exposure to RIL-2; however, the 49H.8 antigen normally expressed on natural killer cells and not on circulating T-cells was found only on LGLs. The role of murine liver LGLs and SLs needs to be further characterized.

Tumor-infiltrating lymphocytes (TILs) comprise a subpopulation of lymphoid cells that infiltrate into growing tumors. These cells can be activated in vitro with recombinant interleukin-2 (rIL-2) to become highly cytotoxic against fresh tumor targets in vitro and against a variety of systemic metastases in vivo. OK-432 is a well-known inducer of NK cells and immune effector T cells. This study was designed to evaluate the effects of OK-432 on (a) the generation and (b) the cytotoxic potential of rIL-2-induced TILs. When TILs obtained from a murine colon adenocarcinoma (the MC-38 tumor) were cultured in complete media supplemented with 100 U of rIL-2/ml and 1.0 microgram of OK-432/ml, the number of TILs generated was greater than that seen with rIL-2 or OK-432 alone (number of TILs on day 15 of culture: 100 U of rIL-2/ml: 268 x 10(5) TILs; 1.0 microgram of OK-432/ml: 30 x 10(5) TILs; 100 U of rIL-2/ml + 1.0 microgram of OK-432/ml: 528 x 10(5) TILs). Higher concentrations of OK-432 had deleterious effects on TIL growth characteristics. TILs generated in 100 U of rIL-2 and 1.0 microgram of OK-432/ml of complete media demonstrated greater tumor lysis compared to rIL-2 alone (% lysis against MCA-102 target; 100 U of rIL-2/ml: 12%; 100 U of rIL-2/ml and 1.0 microgram of OK-432/ml: 50%; effector target ratio 20:1; p less than 0.001). Similar results were seen against the NK-sensitive YAC-1 lymphoma target.(ABSTRACT TRUNCATED AT 250 WORDS)

TILs can be isolated and expanded in vitro in the presence of RIL-2. The resulting cells are highly cytotoxic in vitro and in vivo against a variety of tumor targets. Although most of the TILs bear T cell antigens on their cell surface, recent evidence suggests that natural killer (NK) cells may be part of the overall population of infiltrating cells. Based upon this evidence, we have evaluated the effects of Picibanil (OK-432), a well-known inducer of NK cell and T cell cytotoxicity, on TILs. OK-432 was administered intravenously at a dose of 100 micrograms, previously determined to be optimal for NK stimulation, to tumor-bearing animals. Two days later, control and experimental animals had their tumors harvested and processed in order to grow their TILs in vitro in complete medium containing RIL-2 at a final concentration of 1,000 U/ml. The following observations were made: 1) a greater than 300% increase in overall TIL number compared to controls on day 10 of culture returning to normal by day 30; 2) a marked increase in the percent of cells expressing cytotoxic and differentiation antigens in the experimental group compared to controls, such increase seen mostly from day 7 to day 10; 3) a marked increase in the cytotoxic activity of the experimental TILs against an NK-sensitive tumor target, the YAC-1 lymphoma, throughout the period of growth of the TILs (3-4 times controls) and to a lesser extent against an NK-resistant tumor target. These findings may have potential application, in immunotherapeutic trials against human tumors and may help to understand the reasons for the effectiveness of OK-432 in vivo against selected murine tumors.

Tumor infiltrating lymphocytes (TILs) are capable of mediating significant tumor regressions in vitro and in vivo in animal systems. In humans, however, many TIL cell lines are not cytotoxic in vitro, and clinical trials thus far have been less than encouraging. We attempted to correlate TIL cytotoxicity with time of tumor harvest and TIL cell surface antigenic expression. TILs harvested from early MC-38 adenocarcinoma tumors (days 9 and 20 post-tumor implantation), demonstrated significantly higher cytotoxicity against a variety of tumor targets compared to older TILs (days 31 and 37). The younger TILs had a higher expression of the Lyt-1 (Helper T cells), asialo GM1 (NK and T cells), and 49H.8 (NK cells) antigens. Comparison with the MCA-102 sarcoma, a tumor that does not lead to cytotoxic TILs, revealed a low expression of the Lyt-1 antigen on their cell surface. We conclude that TILs cytotoxicity is time-dependent and may be dependent on the presence of Lyt-1+ cells in the overall TIL population of cells.

Lymphokine-activated killer cells appear to arise from precursor cells bearing natural killer (NK) cell antigens. Cyclosporin (CsA) is a well-known immunosuppressive agent that can down-regulate NK cell cytotoxicity. Studies were initiated to evaluate the effects of CsA on splenocytes before and after exposure to recombinant interleukin-2 (rIL-2). Normal C57BL/6 mice receiving CsA at a dose of 100 mg/kg demonstrated a decrease in NK cell lysis against the YAC-1 lymphoma target in a 4-h chromium-release assay. When splenocytes obtained from CsA-treated mice were cultured for 3 days in complete medium containing 1000 U rIL-2/ml, they demonstrated a return of NK cell lysis to normal (mean cytotoxicity = 65 LU versus 60 LU for control and CsA-exposed splenocytes respectively; P, NS, five consecutive experiments) but revealed a decrease in the lysis of a NK-resistant target: the MCA-102 sarcoma (mean cytotoxicity = 20 LU vs 12 LU for control and CsA-exposed splenocytes respectively; P less than 0.02, five consecutive experiments). Fresh splenocytes cultured in media containing rIL-2 and CsA demonstrated a decrease in proliferation, cell-cycle S-phase fraction and cell yields compared to splenocytes cultured in media containing rIL-2 alone. In addition, a decrease in tumor cell lysis for NK-cell sensitive (mean percentage lysis = 98% vs 60%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) and resistant targets (mean percentage lysis = 68% vs 28%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) was also seen. CsA had no effects on the phenotypic antigenic expression of splenocytes cultured with high-dose rIL-2 although activated T cell antigens were down-regulated when fresh splenocytes were evaluated after in vivo exposure to CsA. These studies support the down-regulating effects of CsA on NK cell lysis and suggest that the rIL-2-activated cell population is heterogeneous as demonstrated by the differential down-regulation and recovery of NK-resistant cell lysis versus NK-sensitive cell lysis.

Due to the recent surgical and technical developments in aesthetic surgery and occasional dissatisfaction of women with their external contour, augmentation mammaplasty has become a common occurrence in the surgical practice of the aesthetic surgeon. Breast carcinomas, incidentally associated with augmentation mammaplasty, have been sporadically reported in the literature and have all been treated with a mastectomy when resection was possible. We present a case of breast carcinoma occurring 5 years after augmentation mammaplasty performed with silicone-gel-filled implants. Therapy of this carcinoma was achieved through a segmental mastectomy followed by postoperative radiation. The patient remains well 15 months postoperatively. A large number of breast implants are performed each year and the likelihood of a higher incidence of incidental breast carcinomas seen with implants is certainly possible as these patients become older; thought should be given to the notion that possibly not all of these cases need to be managed with radical surgery.

Gestational macromastia is a rare entity. Causes are many and include excess hormonal production, hormonal imbalance, and decreased hormonal catabolism. Documentation of elevated serum prolactin levels and tissue hormonal receptor levels brings new light to this pathologic condition. Pharmacologic measures are not recommended because of lack of effectiveness and possible teratogenic side effects. Primary therapy should consist of local measures, such as breast support, bed rest, and analgesics. If this fails and progression is inevitable, we recommend total mastectomy with provision made for reconstruction and nipple banking. Skin flaps must be thin, and all breast tissue must be removed, otherwise the pathologic condition will continue during the ensuing months of the pregnancy and will recur with each successive pregnancy. Abortion is feasible, but this leaves the patient in need of a reduction or total mastectomy at a later date, and thus is not recommended as a primary line of therapy. If the patient progresses to delivery without complication, a reduction mammoplasty can be considered but only if no future pregnancies are planned.

Lymphokine-activated killer (LAK) cells are generated in vitro by the incubation of normal murine splenocytes in interleukin 2. We have shown previously that the systemic injection of LAK cells in conjunction with recombinant interleukin 2 can reduce the number of established pulmonary metastases in mice. In an attempt to study this approach in the treatment of hepatic metastases, we developed a technique for the induction of hepatic metastases in mice based on the intrasplenic injection of tumor cells and have tested the effects of LAK cells and recombinant interleukin 2 produced in Escherichia coli (RIL-2) therapy on these metastases. Treatment with LAK cells alone in 14 consecutive experiments rarely produced significant reduction in metastases over control (mean percentage reduction, 12%). Therapy with RIL-2 alone produced a dose-dependent reduction in the number of liver metastases. In 20 consecutive experiments when RIL-2 was administered i.p. three times a day at doses varying from 1,000 to 5,000, 10,000 to 15,000, and 25,000 units, a statistically significant (P less than 0.05) reduction in liver metastases was seen in 2 of 12, 2 of 4, and 8 of 12 determinations, respectively (percentage reduction, 0 to 97; mean, 42%). At doses greater than 25,000 units, the reduction in metastases was highly reproducible (percentage reduction, 66 to 95; mean, 83%) and was statistically significant in 14 of 14 determinations. When LAK cells were given i.v. in addition to RIL-2 administration in 16 consecutive experiments, the percentage reduction in liver metastases was markedly increased over that seen with RIL-2 alone (mean percentage reduction, 77% at doses of 5,000 to 25,000 units of RIL-2 and mean reduction, 97% for doses greater than 25,000 units of RIL-2). At doses of 5,000, 10,000, 25,000, and greater than 25,000 units of RIL-2 plus LAK cells, significant reduction of liver metastases (P less than 0.05) was achieved in 3 of 7, 2 of 2, 8 of 8, and 6 of 6 determinations, respectively. When animals were given fresh splenocytes or splenocytes cultured in complete medium without RIL-2 instead of LAK cells, no reduction in liver metastases was seen except for that attributable to the administration of RIL-2 alone. Sublethal total body irradiation of the mice prior to therapy abrogated the therapeutic effects of RIL-2, but the effects of treatment with LAK cells plus RIL-2 were maintained. Thus, treatment with RIL-2 alone or in combination with LAK cells is effective in reducing the number of established hepatic micrometastases in a murine model.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclophosphamide, combined with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), is known to mediate regression of tumors, but the effects of cyclophosphamide on the subsequent generation of LAK cells are unclear. It was the aim of the experiments in this paper to determine whether fresh splenocytes cultured with rIL-2 would maintain or regain their cytotoxicity in vitro after being exposed to the cytotoxic agent cyclophosphamide in vivo. Functional monitoring of splenocytes after in vitro incubation with rIL-2 was performed at various times through chromium-release assays, thymidine assays and cell-cycle analysis. Chromium-release assays determined that the cytotoxicity of cultured splenocytes returned to normal after 12 days of in vitro culture with rIL-2. The thymidine assays indicated a normal rate of uptake of thymidine after 7 days in culture, while the cell cycle was still abnormal by day 12 of culture. The growth and expansion of rIL-2-activated splenocytes after different times of in vitro culture indicated a return to normal compared to control animals after 7 days of continuous in vitro exposure to rIL-2. It is concluded that murine splenocytes can demonstrate cytotoxicity after exposure to cyclophosphamide, through prolonged continuous in vitro culture with rIL-2. Since cyclophosphamide did not jeopardize the production of splenocyte cytotoxic effectors generated with rIL-2, it appears to be a strong contender for use in chemoimmunotherapy protocols.

Pathological curiosities have always fascinated the surgical community, and management of breast cancer by the physician has, at times, revealed such rarities. A recent case of squamous cell carcinoma in a clear fluid breast cyst has prompted a review of our experience with this rare pathology, and allowed us to make the following statements: We propose that squamous cell carcinoma of the breast arises from ductal metaplasia. When the T.N.M. system is applied to the squamous cell lesion, the apparent poor prognosis that it is believed to carry may not be so apparent. If no other primaries are identified after extensive metastatic work-up, surgical therapy consisting of total mastectomy with complete axillary dissection, is very effective in local control of tumor progress. Nodal status will indicate the need for additional modalities of therapy.

Because of the problems associated with the large, bulky bridges presently used for construction of loop ostomies, particularly complications of leaks and skin excoriation, we have studied the use of the Penrose drain as an alternative. This method has been time-tested on 45 patients and has been found to be safe, reliable, and inexpensive and has gained popularity among patients and ostomy nurses. The bulky colostomy bridge should no longer be accepted as a standard of care.

The adoptive transfer of tumor-infiltrating lymphocytes (TIL) expanded in interleukin-2 (IL-2) to mice bearing micrometastases from various types of tumors showed that TIL are 50 to 100 times more effective in their therapeutic potency than are lymphokine-activated killer (LAK) cells. Therefore the use of TIL was explored for the treatment of mice with large pulmonary and hepatic metastatic tumors that do not respond to LAK cell therapy. Although treatment of animals with TIL alone or cyclophosphamide alone had little impact, these two modalities together mediated the elimination of large metastatic cancer deposits in the liver and lung. The combination of TIL and cyclophosphamide was further potentiated by the simultaneous administration of IL-2. With the combination of cyclophosphamide, TIL, and IL-2, 100% of mice (n = 12) bearing the MC-38 colon adenocarcinoma were cured of advanced hepatic metastases, and up to 50% of mice were cured of advanced pulmonary metastases. Techniques have been developed to isolate TIL from human tumors. These experiments provide a rationale for the use of TIL in the treatment of humans with advanced cancer.

Dissatisfaction with time-consuming double layer anastomoses in intestinal surgery and with occasionally questionable stapled anastomoses prompted a review of available techniques for hand sewn suture lines. Single layer anastomoses have been used extensively and their integrity and safety have often been questioned. In 1951, Gambee described a method of single layer anastomosis which, in practice, is a double layer technique. His results at 10 years were comparable to those obtained with other techniques. We have sought a modification to decrease anastomotic complications and improve ease of suture insertion. Along with a description of technique, a review of 170 anastomoses performed from July 1982 to June 1984 is presented without a single clinically apparent leak.

The systemic administration of lymphokine activated killer (LAK) cells and recombinant interleukin-2 (RIL-2) is effective in reducing the number of established pulmonary and hepatic metastases from multiple murine tumors and has recently been shown to be effective in mediating the regression of metastatic cancer in humans as well. The generation of sufficient numbers of LAK cells for the effective therapy of human tumors remains a major obstacle to the widespread application of this immunotherapeutic approach. We have thus studied methods for the in vitro expansion of LAK cells effective in immunotherapy. Our previous studies used LAK cells generated in culture with RIL-2 for 3 days. LAK cells cultured in RIL-2 for 5 or 7 days were not significantly different from cells cultured for 3 days either in the number of cells obtained, their in vivo cytotoxicity or their in vivo therapeutic effectiveness. When day 3 LAK cells were transferred to fresh culture medium containing 1000 U/ml of RIL-2, a highly reproducible expansion of these cells was obtained. By day 5, cell numbers expanded 9.6 +/- 0.8-fold (mean +/- SEM; n = 36) and by day 8, cells expanded 15.1 +/- 1.0-fold (n = 19). In 4 h 51Cr release assays against fresh tumor target cells, day 3 LAK cells had a mean of 13 lytic units/10(6) cells in 24 experiments. Day 5 expanded LAK cells had a mean of 30 lytic units/10(6) cells in 13 experiments (P less than 0.05 compared to day 3 LAK cells) and day 8 expanded LAK cells had a mean of 11 lytic units/10(6) cells in 6 experiments (P = NS compared to day 3 LAK cells) When day 5 and day 8 expanded LAK cells were infused in vivo with RIL-2, they were found to significantly reduce the number of experimentally induced pulmonary metastases as effectively as non-expanded conventional day 3 LAK cells. Similar findings were documented in experiments against hepatic metastases. These experiments demonstrate that LAK cells could expand a mean of 15-fold in vitro in RIL-2 and maintain their anti-tumor therapeutic effectiveness when adoptively transferred. These experiments suggest methods for generating increased numbers of cells for use in the adoptive immunotherapy of human cancers and may substantially reduce the need for repeated leukophereses of cancer patients undergoing this therapy.

We examined the antitumor efficacy of rTNF-alpha administration on established tumor at two visceral sites, lungs and liver. Treatment of B6 mice harboring multiple (greater than 100 foci of less than or equal to 0.5 mm diameter) 10-day pulmonary macrometastases from the MCA-106 sarcoma, with dosages of rTNF-alpha (5-10 micrograms, single dose i.v.) that caused hemorrhagic necrosis and regression of a 6 mm MCA-106 s.c. tumor, had no impact on the number (or size) of lung nodules. Similarly, rTNF-alpha failed to show an antitumor effect in B6 mice with advanced day 8 or 10 multiple (greater than 100 foci of less than or equal to 0.5 mm diameter) hepatic metastases at single i.v. doses up to 20 micrograms, as measured by either enumeration of residual liver nodules or survival. B6 mice injected s.c. with MCA-106 sarcoma and treated with rTNF-alpha as a single i.v. dose on day 0, 3, 5, or 7 experienced marked tumor regression only after the day 7 rTNF-alpha injection, when the tumor had achieved a size of 5-6 mm in diameter. Since tumor size appeared important for rTNF-alpha susceptibility in vivo, we next induced a single hepatic tumor of the MCA-106 sarcoma by the direct injection of cells into the left lobe of the liver and treated these mice at day 10 when the nodule had achieved a size of 5-6 mm in diameter. Increasing doses of rTNF-alpha (up to 8 micrograms) given as a single i.v. injection resulted in increasingly greater reductions in hepatic tumor as well as significant survival benefit of the treated mice. Sites of regressing hepatic tumor exhibited central necrosis accompanied by polymorphonuclear leukocytes and lymphocytes. Collectively, these results show that rTNF-alpha administration can mediate a significant antitumor effect on visceral tumor and suggest that tumor size is an important factor in rTNF-alpha susceptibility not only for tumors growing at s.c. sites but also for those established at visceral sites.

Tumor-infiltrating lymphocytes (TILs) have potent antitumor effects in murine models of advanced disease. Although these cells are 50-100 times more potent than lymphokine-activated killer cells against micrometastases, their antitumor benefits are virtually nonexistent against large tumor burdens unless cyclophosphamide is added to the immunotherapy regimen. In an effort to determine the effects of cyclophosphamide on TILs obtained from tumor-bearing animals, we harvested tumors from animals having received either 0, 50, or 100 mg/kg cyclophosphamide intravenously and investigated the expansion, cytotoxicity, and phenotypic expression of these TILs co-cultured in vitro with recombinant interleukin-2. TILs obtained from animals given cyclophosphamide, demonstrated a greater fold expansion than TILs obtained from normal animals (mean fold expansion on day 59 of culture: 85, 400, and 150 for TILs obtained from animals given 0, 50, and 100 mg/kg cyclophosphamide, n = 3 consecutive experiments). In addition, these TILs demonstrated enhanced cytotoxicity compared to controls [effector to target ratio 4:1, day 16 TILs, % lysis: 24, 35, and 45%, cyclophosphamide 0, 50, and 100 mg/kg, respectively, against the MCA-102 sarcoma, natural killer (NK) insensitive tumor; 29, 38, and 56% against the YAC-1 lymphoma, NK sensitive tumor]. Similar results were seen with day 34 and day 59 TILs. When phenotypic analysis was performed, TILs obtained from animals given cyclophosphamide consistently demonstrated a greater percent expression of the Thy1.2 and Lyt-2 antigens up to day 59 of culture when the experiments were terminated. The NK cell marker 49H.8 was expressed on the majority of TILs and its expression did not change with respect to the cyclophosphamide concentration. The increase in TIL number and cytotoxicity seen with cyclophosphamide given intravenously before tumor harvest could have important ramifications for human immunotherapy with TILs.

Transillumination and Infrared Light Scanning (I.L.S.) of the breast were evaluated in a high referral breast clinic over a 15-month period. Seven hundred (700) patients were examined and blindly transilluminated; 101 were biopsied, all had mammography. The study was conducted in two phases: Phase I evaluated transillumination without I.L.S. This included 22 biopsied patients out of 101, with eight carcinomas identified histologically, demonstrating 87 per cent sensitivity and 64 per cent specificity for transillumination, versus 87 per cent and 71 per cent for mammography, and 87 per cent and 57 per cent for examination. Phase II evaluated simple transillumination combined with I.L.S. This included 79 biopsied patients out of 101 with 26 carcinomas identified histologically, demonstrating 96 per cent sensitivity and 74 per cent specificity for transillumination combined with I.L.S. compared to 85 per cent and 72 per cent for mammography and 81 per cent and 73 per cent for examination. Of the 26 Phase II carcinomas identified, two were not felt by examination, and two were neither felt nor read correctly by mammography. I.L.S. of the breast has proven effective in the hands of trained personnel and should be used with routine breast examination or mammography to increase yield of breast pathology.

A syngeneic transplantable sarcoma induced in C57BL/6 mice, MCA 105, was used in studies to examine host suppression on the adoptive immunotherapy of established intradermal and experimentally induced pulmonary and hepatic metastases. Fresh immune splenocytes were generated from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum. The adoptive immunotherapy of intradermal MCA 105 tumor with immune cells required prior immunosuppression of the recipient by sublethal irradiation with 500 R or T-cell depletion. The effect of whole-body sublethal irradiation appeared to eliminate a systemic host suppression mechanism, since partialbody irradiation involving the tumor-bearing area did not permit successful immunotherapy. Host irradiation was not required to achieve successful immunotherapy of experimentally induced pulmonary or hepatic metastases. In nonirradiated recipients bearing both intradermal and pulmonary tumors, host suppression did not affect the function of transferred immune cells to induce regression of pulmonary metastases. Thus, suppression of adoptive immunotherapy appears to be relevant to tumors confined to the skin and subcutaneous tissue but not to tumor in visceral sites, such as the lung and liver.

A patient receiving chemotherapy for multiple myeloma suddenly developed an acute abdomen, fever, and neutrophil leukocytosis. At laparotomy, the distal two-thirds of the duodenum was swollen and hemorrhagic and was surgically excised. The specimen displayed an acute phlegmonous (suppurative) duodenitis with submucosal and transmural acute inflammation and edema despite an intact mucosa. Blood cultures grew Group B, beta-hemolytic streptococci and gram-positive cocci were present histologically. The patient recovered uneventfully following the surgery and a course of broad spectrum antibiotic therapy. This case illustrates that localized suppurative intestinal infection should be considered when immunosuppressed patients present with an acute abdomen, and that aggressive surgical and antibiotic therapy is warranted.

Since 1921, when Henry Hartmann first described his procedure for rectal carcinoma, multiple modifications of the original technique have been proposed. However, the basic principle of a rectal pouch has always been retained. Two cases of carcinoma developing in such a pouch are described; both occurred years after creation of the pouch. One was managed by local resection through a transsacral approach; the other required an abdominoperineal resection. Consideration must be given to careful examination and sigmoidoscopy of these pouches as they tend to be forgotten by the physicians due to their hidden location.

Patients with coronary artery disease (CAD) may undergo periods of reversible myocardial ischemia without experiencing angina. To study the prognostic implications of "silent" myocardial ischemia induced by exercise, exercise electrocardiography and radionuclide angiography were performed in 131 consecutive patients with CAD, preserved left ventricular (LV) function at rest and mild or no symptoms during medical therapy. All patients who died during medical therapy were in the subgroup of patients with 3-vessel CAD in whom exercise-induced ischemia developed, which was characterized by both a decrease in LV ejection fraction and ST-segment depression. Patients in whom angina pectoris developed during exercise (54% of all patients) had a greater prevalence of this combined ischemic response to exercise than patients without angina (61% vs 27%, p less than 0.001) and also a greater prevalence of left main or 3-vessel CAD (59% vs 25%, p less than 0.001). However, when inducible ischemia was demonstrated, risk stratification and prognosis were the same whether the ischemic episode was symptomatic or silent. Among patients having both a reduction in ejection fraction and a positive ST-segment response, the likelihood of significant left main narrowing (13% vs 26%), 3-vessel CAD (56% vs 51%) and death during subsequent medical therapy (16% vs 9%) was similar in patients with silent compared to those with symptomatic ischemia. These data indicate that patients in whom angina develops during exercise have a greater prevalence of high-risk coronary anatomy and of inducible ischemia than patients without angina. However, once inducible ischemia is documented, the symptomatic response to exercise appears irrelevant for prognostic or risk stratification considerations.

A reproducible model for the selective generation of liver metastases has been developed. The spleen is exteriorized by means of a small subcostal incision and is directly injected with a 1-ml suspension of tumor cells. Tumor cells flow out of the splenic vein into the portal vein and lodge in the liver. Splenectomy is performed approximately 1 minute after tumor cell injection. The procedure is simple, requires a single operative procedure, and is applicable to a wide variety of tumors. Over a 6-month period, more than 3,000 such procedures with the use of 5 different tumors were performed in C57BL/6 mice, resulting in a 1.9% mortality. For reliable enumeration of the number of hepatic metastatic deposits, a suspension of india ink was injected iv, and the liver was removed and bleached with Fekete's solution. Tumor nodules appeared as discrete white nodules against the black background of normal liver parenchyma. This model provides a useful tool for the study of the experimental therapy of hepatic metastases in mice.