Opposing effects of anti-activation-inducible lymphocyte-immunomodulatory molecule/inducible costimulator antibody on the development of acute versus chronic graft-versus-host disease.

The functional role of inducible costimulator (ICOS)-mediated costimulation was examined in an in vivo model of alloantigen-driven Th1 or Th2 cytokine responses, the parent-into-F(1) model of acute or chronic graft-vs-host disease (GVHD), respectively. When the Ab specific for mouse ICOS was injected into chronic GVHD-induced mice, activation of B cells, production of autoantibody, and development of glomerulonephritis were strongly suppressed. In contrast, the same treatment enhanced donor T cell chimerism and host B cell depletion in acute GVHD induced host mice. Blocking of B7-CD28 interaction by injection of anti-B7-1 and anti-B7-2 Abs inhibited both acute and chronic GVHD. These observations clearly indicate that the costimulatory signal mediated by CD28 caused the initial allorecognition resulting in the clonal expansion of alloreactive T cells, whereas the costimulatory signal mediated by ICOS played a critical role in the functional differentiation and manifestation of alloreactive T cells. Furthermore, treatment with anti-ICOS Ab selectively suppresses Th2-dominant autoimmune disease.

A solid-phase assay for the detection of anti-sperm antibodies.

PROBLEM: ELISA is an ideal assay method for a large-scale screening of anti-sperm antibodies among a large number of infertile males. However, conventional ELISA with whole spermatozoa needs time-consuming steps of centrifugation. METHOD: A solid-phase assay used for detecting anti-sperm antibodies was established. This assay is suitable not only for detecting circulating anti-sperm antibodies of IgG, IgM, and IgA subclass simultaneously but also for screening hybridomas secreting anti-sperm monoclonal antibodies (mAbs). The microtiter plates, on which solubilized sperm antigens are fixed, can be stored at -80 degrees C for up to six months without losing reactivity with anti-sperm antibodies. RESULTS: Using this assay, 53 sera (13 were proven positive and 40 were proven negative for sperm agglutination antibody) were tested. Although the false-negative rate was 0%, the false-positive rate was 32%. One thousand one hundred sixty-five supernatants from hybridomas constructed with splenocytes of mice who were hyperimmunized with human sperm and nonsecreting myeloma cells were tested by this solid-phase assay and two anti-sperm mAb secreting clones were selected and established. CONCLUSIONS: It is recommended that for research work this assay could be used for the first screening of the hybridoma secreting anti-sperm mAb, and for clinical use this assay might be suitable for the first screening of sera of infertile patients. However, conventional bioassays should follow to confirm the biological meaning of the positivity.

Laser fluorescence spectroscopy from human spermatozoa.

The native fluorescence spectra, intensity dependence on sperm density, relaxation decay time, and polarization were investigated on spermatozoa and their components (sperm heads and sperm tails) under laser excitation.

Human prostatic cancer cell derived growth factor stimulating fibroblasts and its mechanisms.

A human prostatic cell line JCA-1 has been shown to release into culture medium an activity that promotes the proliferation of murine fibroblast 3T3 cells. The presence of additional serum-free conditioned culture supernatant from JCA-1 cells accelerates the 3T3 growth rate as determined by cell counts, [3H]thymidine uptake into DNA and colony formation in soft agar gel. The growth activity was determined not to derive from culture medium components. Treatment of JCA-1 cells with RNA synthesis inhibitor actinomycin D abrogated the activity, indicating that protein synthesis is required for the presence of this activity from JCA-1 cells. The molecular mass of this prostatic cell derived growth factor (PRGF) was larger than 10,000 KDa, and its interaction with fibroblasts resulted in an approximate 27 percent increase in replicating cell cycle phases S and G2M with a reciprocal decrease of G1 cells. These data indicated a mechanism causing a more rapid entry of cells into replication. The interaction between prostatic cancer cells and related tissues and their relation to pathogenesis is reviewed.

Application of microwave tissue coagulation in partial nephrectomy.

Microwave tissue coagulation was used during partial nephrectomy in 10 mongrel dogs, without clamping the renal artery. There were no major complications, such as retroperitoneal hematoma, abscess formation, or macroscopic infarction of the kidney tissue related to this new procedure. The advantages of microwave coagulation are reduced blood loss, shorter operative time, and minimal risk of vascular injury.

Establishment of new human prostatic cancer cell line (JCA-1).

The establishment of a new human prostatic cancer cell line is described. This cell line was derived from a poorly to moderately differentiated prostatic adenocarcinoma. It has been maintained in tissue culture for fourteen months and has been passed fifty-two times. This cell line has an ability to form colonies in soft agar suspension cultures, and also is transplantable to nude mice. Tumors grown in nude mice revealed a poorly differentiated adenocarcinoma with positive PSA staining. Acid phosphatase activity was detected in freeze-thawed cells by enzymatic assay. A karyotype analysis demonstrated aneuploidy with a model chromosomal number of 69 and six marker chromosomes.

Immunosuppressive factor derived from renal cancer cells.

An immunosuppressive factor derived from conditioned medium of a human renal cancer cell line, KU2, was analyzed, using mitogen-activated normal peripheral blood lymphocytes. Addition of conditioned medium to lymphocyte cultures resulted in suppression of [3H]-thymidine incorporation to DNA of lymphocytes in a dose-dependent manner. This effect was cytostatic and reversible. This factor inhibited lymphocyte activation stimulated by mitogen at an early stage, and it also suppressed Interleukin-2 production by activated lymphocytes. This factor was non-dialyzable and heat sensitive at 56 degrees C. DNA replication of the cells was necessary for the production of this factor.

A solid-phase immunoassay to screen anti-HLA monoclonal antibodies.

A solid-phase assay to detect anti-HLA monoclonal antibodies was developed. In this assay microtiter plates are coated with antigens solubilized from cultured lymphoid cells by sonication and then incubated with anti-HLA monoclonal antibodies. The antigen-antibody interaction is indicated by the development of color following the addition of peroxidase-conjugated anti-mouse Ig xenoantibodies and its substrate. The assay is rapid since it does not require centrifugations during the washing steps. Furthermore the assay is simple, reproducible and suitable to screen large numbers of samples and to detect antibodies recognizing determinants not exposed on the membrane of viable cells. The sensitivity of the assay is influenced by the pH of the buffer used to coat plates with antigens, by the number of cells used to prepare soluble antigens, by the incubation time of antigen preparations with plates and by the incubation time of antibody preparations with antigen-coated plates. Titration of anti-HLA monoclonal antibodies with known specificity and screening of hybridomas generated with splenocytes from mice immunized with cultured human lymphoid cells indicate that the sensitivity of the solid-phase assay is similar to that of the ELISA with lymphoid cells.

Correlation of heterogeneity index score (HIS) of DNA content in bladder cancer recurrence.

Automated flow cytometry (FCM) has been used to monitor the effects of therapy and progression of human bladder carcinoma. We have previously reported a computer-based model which has shown a correlation to relative mean DNA content in cell populations analyzed by FCM. Two patients with superficial transitional cell carcinoma of the bladder were followed for several months. Using FCM the patients' tissue samples were examined and heterogeneity index scores (HIS) were determined. Both cases had recurrence. The first patient has increased in grade with a respective increase in HIS within four months (grade I----II, HIS 27.2----80.8). The second patient also has recurrence in three months both times with a grade I tumor and a score of 106.2. High scores reflect large aneuploid populations which have shown to recur. Since such a correlation exists HIS may not only offer a more objective technique with quantitative results to monitor patients but possibly can distinguish the degree of tumor malignancy.

Monocyte cytolytic factor in promoting monocyte-mediated lysis of bladder cancer cells by bacillus Calmette-Guerin.

The role of monocytes in cell-mediated cytolysis of bladder cancer cells was investigated. Human peripheral monocytes released a cytolytic factor which lysed T24 bladder cancer cells and a number of human tumor cells, but not normal lymphocytes or fibroblasts. After incubation of monocytes with bacillus Calmette-Guerin for 48 hr. in vitro, cytolysis of T24 cells was increased up to 56.7 +/- 4.1%. Treatment of monocytes with actinomycin D (an inhibitor of RNA transcription) reduced release of cytolytic factor from 27.3 +/- 5.7% cytolysis to 4.5 +/- 1.4% (p less than 0.05). The response to mitomycin C was different between lymphokines and monocyte cytolytic factor. The mouse monoclonal antibody against human recombinant tumor necrosis factor did not neutralize monocyte cytolytic factor. These results show that this monocyte cytolytic factor is distinct from lymphokines and tumor necrosis factor. The evidence that bacillus Calmette-Guerin increases release of monocyte cytolytic factor may be associated with anti-tumor activity of bacillus Calmette-Guerin in intravesical therapy for treatment of bladder cancer.

Flow cytometric analysis of relative mean DNA content of urogenital cancer cells in fresh and paraffin-embedded materials.

The relative mean DNA content calculation was performed by flow cytometry on single cell suspensions prepared from fresh and paraffin-embedded specimens of 10 patients with surgically resected urogenital cancer. Samples were processed by a modified method of Hedley et al. including two hours of pepsinizing time, ribonuclease digestion, and propidium iodide staining. The mean DNA content which is a quantitative description of flow cytometric characteristics was significantly correlated between the fresh and paraffin-embedded materials (n = 10, r = 0.869, p less than 0.01). This method allows for the objective, retrospective analysis of DNA content in relation to diagnosis and prognosis of urogenital cancer.

Heterogeneity index score (HIS). New computerized method for classification of human bladder carcinomas using flow cytometry.

The rationale for studying nuclear DNA may be its direct relationship to the aggressiveness of cancer. Recent flow cytometric studies (FCM) of cancer cells show the limitation of the current methods for the accurate determination of the degree of aneuploidy or proliferative characteristics of a tumor cell. Here we report a new methodology for a computerized determination which is well correlated with relative mean DNA content in cell populations analyzed by FCM (heterogeneity index, HI). A total of seventy-six tissue samples were examined. Twenty-two specimens were benign tissue while fifty-four were histologically malignant bladder tumors. Forty tumors were grade (G)I-II, ten G-III, and four carcinoma in situ. The samples were mechanically minced into a single cell suspension and stained with propidium iodide. An Ortho system 50-H multiparameter flow cytometer equipped with an Ortho 2150 computer was used to determine DNA content and cell number. HI was calculated using the following formulas: (formula; see text) The mean HIS of twenty-two normal and benign tissues was 9.805 +/- 5.6. The forty G-II tumors had a mean HIS of 23.576 +/- 26.519. Statistical differences were observed between benign tissue and G-I-II tumors (P = 0.0196). G-III tumors had a marked increase in HIS of 160.965 +/- 63.404. The limited study of four carcinoma in situ tumors showed a mean HIS of 45.4 +/- 9.5. Our computer extrapolation of flow cytometric DNA analysis quantifies an objective description of FCM characteristics and histochemical index which may distinguish the degree of tumor malignancy.

Ureteral replacement using collagen sponge tube grafts.

The use of a collagen sponge tube graft as a material for segmental ureteral replacement was investigated. The structural design of the collagen sponge graft was achieved by cell culture on the matrix. MGH-U1 cells, derived from bladder cell carcinoma, were grown in vitro on the collagen sponge matrix with excellent biocompatibility and without evidence of cytotoxicity. The collagen sponge demonstrated biodegradability when implanted subcutaneously in dogs. However, a urine exposure test of collagen sponge in rat bladders revealed extensive salt deposits on its surface in some rats, as observed by crystallographic examination. Segmental ureteral replacements by collagen sponge tube grafts, accompanied by ureteral splint catheters, were performed in dogs. There was extensive uro-epithelial cell regeneration on the inner surface of the collagen grafts, without evidence of severe hydronephrosis, 5 to 12 weeks following the procedure. The results indicate the potential for ureteral replacement by collagen sponge tube grafts, which would act as non-toxic, biodegradable scaffolds inducing the regenerative activity of the ureter.

Depressed myocardial function after transurethral resection of prostate.

Cardiovascular physiologic monitoring was undertaken in 12 patients undergoing transurethral resection of the prostate with the aid of flow-directed Swan-Ganz catheter and the Automated Physiologic Profile. Cardiac and pulmonary pressures and physiologic parameters were derived pre- and postoperatively. Resecting time, body temperature, intravenous fluid administered, serum hemoglobin, and sodium also were recorded. Of the 12 patients studied, 66 per cent experienced a drop in their cardiac index as well as their left ventricular function after surgery. Myocardial function curves revealed that 7 patients (58 per cent) had decreased cardiac function, 2 had no change, and 3 had increased function. Four patients with preoperative pulmonary wedge pressures (PAW) over 9 mm. Hg experienced depressed cardiac function. Three patients were resected for over sixty minutes, and all experienced depressed cardiac function. Vital signs, serum hemoglobin, or serum sodium did not reflect this change. We believe that relative hypervolemia, undetected elevation of pulmonary wedge pressure. We believe that relative hypervolemia, undetected elevation of pulmonary wedge pressure, and prolonged resection are factors that depress cardiac function and increase the risk of cardiovascular complication in transurethral surgery.

Ureteral diverticulosis in a case of dermatomyositis.

We present the third case of ureteral diverticulosis discovered by excretory urography alone and the twenty-sixth case reported in the world literature. Unlike previously reported cases our patient had dermatomyositis and is urologically asymptomatic.