Effect of ethanol intake on pancreatic exocrine secretion in mice.

Swiss mice were fed conventional lab chow and 10% ethanol or water as drinking fluid for 2 weeks. Pancreatic juice was obtained by cannulation of the bile pancreatic common duct of mice anesthetized with urethane. Isolated pancreatic lobules were also obtained. The flow rate and the amylase output were determined in pure pancreatic juice. The release of amylase was measured in pancreatic lobule preparations. The basal pancreatic juice flow rate and the amylase output were significantly increased by ethanol consumption. The magnitude of the pancreatic juice flow rate and the amylase output responses to increasing doses of bethanechol, a cholinergic agent, was significantly decreased in ethanol-fed mice. The amount of spontaneously released amylase was higher in pancreatic lobule preparations from ethanol-fed animals than that from control mice, and the difference was abolished by addition of atropine to the incubation media. The amylase release rate in response to increasing doses of bethanechol was significantly reduced in lobule preparations from the ethanol-fed group. These data indicate that ethanol intake in mice has a stimulating effect on the spontaneous pancreatic secretion and lends support to the hypothesis that ethanol consumption increases the intrapancreatic cholinergic tone.


Persistence of viremia in patients with type-C chronic hepatitis during long-term follow-up.

To clarify the evolution of antibody to hepatitis C virus (anti-HCV) and of liver histologic findings during the natural course of type-C chronic hepatitis, 111 patients with biopsy-proven chronic hepatitis type C were consecutively enrolled in this study and were followed up biochemically, serologically, and histologically for more than 5 years. All were positive for the first- and second-generation antibody to HCV (anti-HCV-1 and anti-HCV-2). None received antiviral therapy during the follow-up period. At the end of follow-up, all remained positive for anti-HCV-2, but four patients turned negative for anti-HCV-1. HCV RNA, detected by the polymerase chain reaction method, was tested serially in 20 patients who persisted positive anti-HCV-1 and in 4 patients who lost anti-HCV-1. HCV RNA disappeared from only two patients, who lost anti-HCV-1 during the follow-up period. A normalization of the serum transaminase level was found in only two patients, who lost both anti-HCV-1 and HCV RNA. A repeat liver biopsy was performed in 62 patients with chronic hepatitis who were persistently positive for all HCV markers and in 4 patients who lost anti-HCV-1. Of the 62 patients who retained all HCV markers, 16 progressed to hepatocellular carcinoma, 6 to cirrhosis of the liver, and 1 had a normal liver, whereas the remaining 39 had chronic hepatitis. Two patients who lost both anti-HCV-1 and HCV RNA showed a normal liver. Of two patients who lost only anti-HCV-1 one progressed to hepatocellular carcinoma and one to chronic hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)


Autoantibodies to the nuclear Sp100 protein in primary biliary cirrhosis and associated diseases: epitope specificity and immunoglobulin class distribution.

Sp100, a protein with a dot-like intranuclear localization in immunofluorescence microscopy, is a major target for patient autoantibodies in primary biliary cirrhosis (PBC) and occasionally in rheumatic disorders. The human Sp100 cDNA has recently been cloned, and the deduced amino acid sequence was found to contain sequence similarities with an MHC class I domain and several transacting regulatory proteins, including HIV-1 nef proteins. In this study, recombinant Sp100 fusion proteins were used to differentiate the immunoglobulin isotypes and to map the epitopes involved in the anti-Sp100 autoimmune response. PBC patients developed IgG as well as IgM and/or IgA class anti-Sp100 autoantibodies whereas most patients with rheumatic diseases developed IgG class autoantibodies only. For epitope mapping, truncated versions of the Sp100 protein were probed for immunoreactivity in ELISA and immunoblotting. With 55 sera, 17 different reaction patterns were obtained, and at least three non-overlapping major autoantigenic domains were recognized by the majority of sera. One domain, which contains the sequence similarity with HIV nef proteins, was recognized by all anti-Sp100 sera and harbours multiple, in part discontinuous, epitopes. These data demonstrate a heterogeneous and patient-specific anti-Sp100 autoimmune response which is antigen-driven and, at least in terms of isotype composition, different in PBC and non-PBC patients.


Interleukin 4 and tumour necrosis factor alpha induce different adhesion pathways in endothelial cells for the binding of peripheral blood lymphocytes.

We demonstrated that tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) increased endothelial cell (EC) adhesiveness for peripheral blood lymphocytes (PBL) by promoting transcription and protein synthesis. The different kinetics observed with TNF-alpha and IL-4 suggest the involvement of different adhesion molecules. Blocking adhesion assays and immunofluorescence analysis showed that PBL adhesion to endothelial cells involves different pair adhesion molecules. Whereas IL-4 promoted an LFA-1-dependent/ICAM-1-independent adhesion pathway on EC, TNF-alpha stimulated an LFA-1-dependent/ICAM-1-dependent adhesion pathway on EC. In contrast, VLA-4/VCAM-1 molecules were involved in PBL adhesion both to IL-4 and to TNF-alpha-stimulated EC. Finally, we found that a CD2-dependent/LFA-3-independent adhesion pathway was mainly involved in IL-4-stimulated EC.


Analysis of mature guinea pig T cells with a monoclonal antibody directed against a framework determinant of the T-cell receptor for antigen.

Rats were immunized with guinea pig T lymphocytes and the spleen cells were fused with cells of a mouse myeloma line. The resulting hybrids were screened for the production of antibodies selectively reacting with guinea pig T cells. The monoclonal antibody (MoAb) H159 was analysed in detail because it bound to T lymphocytes, but not to B lymphocytes or macrophages. Cellular ELISA, cytofluorometry and immunohistology revealed that the antigen detected by H159 is selectively expressed on the majority of peripheral mature T lymphocytes (about 95%). In contrast, it stained only a minor population of thymocytes in FACS analysis. H159 precipitated from NP40 lysate of T cells a protein with a molecular weight of about 90 kDa when separated under non-reducing conditions in SDS-PAGE. Under reducing conditions bands with molecular weights of about 50 kDa were found. After binding to anti-rat Ig coated beads, the MoAb H159 had a mitogenic effect for guinea pig T lymphocytes whereas soluble MoAb H159 in the presence or absence of macrophages was not mitogenic. The cellular expression and molecular characteristics of the H159 antigen together with the mitogenic activity of the antibody for T cells indicate that the MoAb H159 recognizes the guinea pig T-cell receptor for antigen via a constant region determinant.


Fresh, frozen, or decalcified bone grafts: a study of early vascularisation and mineralisation of allogeneic and syngeneic bone grafts in rats.

The incorporation of syngeneic and allogeneic bone grafts pretreated by freezing or demineralisation was studied in 10 rats. Fresh, decalcified, or frozen cancellous bone of syngeneic or allogeneic origin was transplanted to intramuscular pouches. Revascularisation was evaluated with radioactive microspheres; formation of new bone was assessed by incorporation of strontium, and resorption was assessed by measuring the reduction of graft weight. Three weeks after grafting, fresh syngeneic and allogeneic bone differed significantly in all three variables. Frozen syngeneic bone was revascularised significantly better than frozen allogeneic bone, but there was no difference in formation of new bone or resorption. There were no significant differences between syngeneic and allogeneic decalcified bone in any of the variables studied. We conclude that differences in incorporation between syngeneic and allogeneic bone grafts are reduced by pretreatment with deep-freezing or demineralisation. Both forms of pretreatment affect the incorporation of the grafts.


[Anti-arrhythmia treatment using L-carnitine in acute myocardial infarct].

Carnitine, a quaternary amine (3-hydroxy-4-N-trimethylaminobutyric acid), plays an important physiologic role in fatty acid transport and metabolism as well as in energy production of the myocardial cell. L-carnitine in high doses has been postulated to have an antiarrhythmic effect and this has also been clinically proven. We studied 20 patients with acute myocardial infarction (AMI), 4-12 hours after onset of pain. The patients were randomized and treated double-blind with 5 g L-carnitine (n = 12) or placebo (n = 8) at hours 0, 12, 24, 36, and with 2 x 3 g on days 3 to 7 by intravenous infusion over 2 hours. The two groups were similar for age, sex, infarct site, maximum CPK and conventional antiarrhythmic therapy. 24-hour Holter-ECG was performed on days 1, 2 and 7 and showed no significant difference between the two groups with respect to incidence of ventricular premature beats (VPB) per hour. On the second day following AMI, however, only 4 of 12 carnitine-treated patients showed high-grade VPB (Lown IVa and IVb), in comparison with 7 of 8 patients in the placebo group. The difference is significant: p = 0.028 (Fisher's Exact Test). Carnitine was well tolerated and the efficacy demonstrated on the second day following AMI must be interpreted with caution.


Kinetics of hydroxyethyl starch in horses.

In a controlled study, the distribution and elimination kinetics of hydroxyethyl starch as well as clinically relevant parameters were determined in horses. The half-life of the first phase was 5.59 hours, that of the second phase 122.22 hours. During the first phase, hydroxyethyl starch persisted almost exclusively intravascularly. The results of this study are largely in agreement with those in human beings. Thus, routes of elimination, duration of plasma-expanding action, distribution volume and redistribution kinetics in horses and human beings are very similar. However, the elimination kinetics of the second phase and the behavior of serum amylase appear to be equine-specific. Coagulation is barley influenced by the administration of hydroxyethyl starch. The results of this study confirm that hydroxyethyl starch is very suitable for use as a plasma-expander in horses.


Butenes and butadiene in urban air.

Samples of urban air hydrocarbons were taken on specifically made adsorbent cartridges and analysed by gas chromatography after thermal desorption. The four isomeric butenes and 1,3-butadiene were favourably resolved and separated from the abundant alkanes on an aluminium oxide PLOT column. The concentrations of butadiene, reflecting outdoor urban exposure, were in the range of 0.5-5 micrograms/m3. An approximate 1:4 ratio was observed between butadiene and propene which both originate predominantly from vehicle exhaust. The four butenes made up approximately 50% of the propene concentration in exhaust-polluted air, with methylpropene greater than 1-butene greater than trans-2-butene greater than cis-2-butene. Petrol vapour contributed less than exhaust but about five times more to the 2-butenes than to methylpropene and 1-butene. The highest exposure levels of butadiene and butenes were consistently observed in the vicinity of exhaust pipes and petrol-fuelled vehicles.


DDT and HCH residues in indoor air arising from their use in malaria control programmes.

Samples of air from rooms in a residential and rural environment, treated with either DDT or HCH at rates recommended for mosquito control under the National Malaria Eradication Programme of India, i.e. 2 and 3 g m-2, respectively, were analyzed for the residues of these insecticides. During the 8-month sampling period, DDT and HCH levels in indoor air ranged from 1.0 to 14.6 and 0.9 to 2000.1 micrograms m-3, respectively. After an initial fall, residues of DDT showed an increase again and were 5.9 micrograms m-3 in samples collected 240 days after the initial application. In contrast, HCH residues declined at a fast rate in a few days after application and remained low at the time of subsequent samplings. HCH residues were present chiefly in the vapour phase throughout the study. Thirty-four to seventy-eight percent of DDT residues were found in the particulate phase in the samples collected up to 64 days after its application and exclusively in the vapour phase at the time of later samplings. Apart from the contamination of food and feed commodities stored in premises treated for malaria control by absorption of insecticides present in the indoor atmosphere, the residues of these persistent compounds in air are also likely to result in low level pollution of the surrounding and distant environmental media by their dispersion in the global ecosystem.


Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase.

Entry into mitosis in Schizosaccharomyces pombe is negatively regulated by the wee1+ gene, which encodes a protein kinase with serine-, theonine-, and tyrosine-phosphorylating activities. The wee1+ kinase negatively regulates mitosis by phosphorylating p34cdc2 on tyrosine 15, thereby inactivating the p34cdc2-cyclin B complex. The human homolog of the wee1+ gene (WEE1Hu) was overproduced in bacteria and assayed in an in vitro system. Unlike its fission yeast homolog, the product of the WEE1Hu gene encoded a tyrosine-specific protein kinase. The human WEE1 kinase phosphorylated the p34cdc2-cyclin B complex on tyrosine 15 but not on threonine 14 in vitro and inactivated the p34cdc2-cyclin B kinase. This inhibition was reversed by the human Cdc25C protein, which catalyzed the dephosphorylation of p34cdc2. These results indicate that the product of the WEE1Hu gene directly regulates the p34cdc2-cyclin B complex in human cells and that a kinase other than that encoded by WEE1Hu phosphorylates p34cdc2 on threonine 14.


Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcineurin.

Migration of human polymorphonuclear neutrophils on vitronectin is dependent on repeated transient increases in the concentration of intracellular free calcium ([Ca2+]i). A specific peptide inhibitor of the Ca(2+)-calmodulin-dependent phosphatase calcineurin was introduced into the cytoplasm of neutrophils. The peptide inhibited neutrophil migration on vitronectin by interfering with the release of the cells from sites of attachment. A similar reduction in motility on vitronectin occurred when cells were treated with the immunosuppressant FK506, which also inhibits calcineurin when bound to its binding protein, FKBP. These results indicate that a rise in [Ca2+]i reduces integrin-mediated adhesion to vitronectin by a mechanism that requires calcineurin activity.


Acetylcholine receptor channel structure probed in cysteine-substitution mutants.

In order to understand the structural bases of ion conduction, ion selectivity, and gating in the nicotinic acetylcholine receptor, mutagenesis and covalent modification were combined to identify the amino acid residues that line the channel. The side chains of alternate residues--Ser248, Leu250, Ser252, and Thr254--in M2, a membrane-spanning segment of the alpha subunit, are exposed in the closed channel. Thus alpha 248-254 probably forms a beta strand, and the gate is closer to the cytoplasmic end of the channel than any of these residues. On channel opening, Leu251 is also exposed. These results lead to a revised view of the closed and open channel structures.


Effects of kinesin mutations on neuronal functions.

Kinesin is believed to generate force for the movement of organelles in anterograde axonal transport. The identification of genes that encode kinesin-like proteins suggests that other motors may provide anterograde force instead of or in addition to kinesin. To gain insight into the specific functions of kinesin, the effects of mutations in the kinesin heavy chain gene (khc) on the physiology and ultrastructure of Drosophila larval neurons were studied. Mutations in khc impair both action potential propagation in axons and neurotransmitter release at nerve terminals but have no apparent effect on the concentration of synaptic vesicles in nerve terminal cytoplasm. Thus kinesin is required in vivo for normal neuronal function and may be active in the transport of ion channels and components of the synaptic release machinery to their appropriate cellular locations. Kinesin appears not to be required for the anterograde transport of synaptic vesicles or their components.


Does hyaluronan have a role in endothelial cell proliferation of the synovium?

Hyaluranate (HA) is a major constituent of synovial fluid, but its concentration and molecular size differ in normal and inflamed joints. HA can induce or inhibit angiogenesis depending on both its size and its concentration. Endothelial-cell endocytose-labeled macromolecular HA and HA oligosaccharides and binding studies have identified an HA-specific receptor on the endothelial cell surface (KD, 10(-10) mol/L; approximately 2,000/cell). The molecular weight of HA-binding proteins was found to be 90 to 125, 78, and 46 kd.


Non-Hodgkin's lymphomas: a review of the M.D. Anderson experience.

The approach we have followed in developing treatment strategies for lymphoma consists of testing new combinations in patients with recurrent disease and, if these are found to be effective, advancing them to front-line therapy. Three series of salvage regimens tested at the M. D. Anderson Cancer Center (MDACC) will be described. These were initially based on ifosfamide/etoposide, later on high-dose cytarabine/cisplatin, and more recently on a novel strategy that first uses MINE (mesna/ifosfamide/Novantrone [mitoxantrone; Lederle International, Wayne, NJ]/etoposide) for induction of remission and, following the attainment of the maximum response to MINE, changes treatment to a non--cross-resistant combination known as ESHAP (etoposide/Solu-medrol ([methylprednisolone]/high-dose cytarabine/cisplatin). The front-line management of intermediate-grade lymphoma at MDACC currently is based on prognostic factors. The criteria for selecting categories of less favorable patients will be discussed. A new regimen, alternating triple therapy based on three non--cross-resistant drug regimens, currently is being explored for these patients. Already this regimen has shown an early advantage in the worst category of cases, those with M.D. Anderson stage D (high tumor burden, high levels of lactate dehydrogenase). The data, however, remain preliminary.


Etoposide, ifosfamide, and methotrexate combination chemotherapy for aggressive non-Hodgkin's lymphomas after failure of the LNH 84 regimen.

We assessed the efficacy and tolerability of VIM (etoposide/ifosfamide/methotrexate) combination therapy in 24 patients who were failing the treatment protocol of the Lymphomes Non Hodgkiniens (LNH) 84 study. Eight patients were refractory to the LNH 84 induction cycles, but ten achieved a partial response (PR). The six remaining patients attained complete response (CR) after LNH 84 induction, but relapsed either during consolidation therapy or after completing the whole program. Twenty-three patients are evaluable for response. The VIM regimen provided a CR rate of 43% and a PR rate of 17%. Treatment failed in nine cases (39%). The CR rate was particularly high (67%) in the group of patients who had PR with LNH 84 induction treatment. Of the ten who had attained CR, five relapsed after 4 to 42 months and five are still alive with no evidence of disease after 29 to 62 months. VIM therapy was well tolerated. A total of 101 VIM courses were given. Myelotoxicity was the most common side effect. Grade 3 or 4 cytopenia was recorded after 11% of the cycles. Among eight infectious episodes recorded, one was fatal. This study demonstrates that CR and long disease-free survival are obtainable with the VIM regimen in a small number of patients failing a high-dose doxorubicin-containing first-line treatment.


Carboplatin in small cell lung cancer: the Royal Marsden Hospital experience.

A series of studies has been carried out to evaluate and document the value of carboplatin in the treatment of patients with small cell lung cancer (SCLC). Encouraging response rates but disappointingly short response durations with both single-agent carboplatin and combination carboplatin/etoposide led to the use of more intensive study regimens. A six-course, dose-intensive study of combination carboplatin/etoposide/ifosfamide in good-prognosis patients resulted in a 97% objective response rate (31 of 32 patients), including 17 (53%) complete responses. However, the toxicity associated with this regimen was expectedly high. In a subsequent phase I dose-escalation study of carboplatin in SCLC, non-small cell lung cancer, and mesothelioma, patients received 800, 1,200, or 1,600 mg/m2 carboplatin given as a 1-hour intravenous infusion. Five of the seven SCLC patients achieved a complete or partial response, but no responses were seen among the other patients. The major toxicity noted was myelosuppression and, while treatment was well tolerated, toxicity was more pronounced at the higher doses. The role of carboplatin as palliative therapy for SCLC patients with advanced disease or poor performance status is currently under investigation with a regimen containing carboplatin/methotrexate/vinblastine. In light of the results of these several studies, it is apparent that carboplatin is an extremely active agent for treatment of SCLC and is not associated with the nephropathy, neuropathy, or severe nausea and vomiting induced by its parent compound, cisplatin. Further study of carboplatin as high-dose therapy, in dose-intensive combination regimens, and as palliative treatment is recommended.


Chemotherapy of male and female germ cell tumors.

Germ cell tumors of testicular origin are virtually always curable with modern therapy that appropriately integrates chemotherapy and surgery. Clinical trials of chemotherapy in patients with disseminated disease now separate those with limited metastatic disease and an excellent prognosis from those with bulky tumor and a less favorable prognosis. In the former group, it has been shown that three courses of BEP (bleomycin/etoposide/cisplatin) are therapeutically comparable to four courses. The next generation study compared BEP with a similar regimen with the bleomycin omitted. Early results of this study suggest that deletion of bleomycin worsens outcome. Other studies have addressed the same question but have used four courses of this agent. Ovarian germ cell tumors have been less well studied but are largely curable with surgery and chemotherapy. The specific regimens used are similar to their testis tumor counterparts. All patients with completely resected tumor should receive adjuvant chemotherapy; virtually all will remain cancer free. Of course, patients with advanced disease should also receive chemotherapy. Results are generally good in these patients, but probably less so than in testis tumor patients with similar tumor volume.


Treatment of recurrent and metastatic head and neck cancer with cisplatin/etoposide/bleomycin.

Cisplatin/etoposide/bleomycin (DEB) was given as an outpatient regimen in a novel weekly schedule to 27 patients with recurrent and/or widely metastatic cancer of the head and neck region. Six of these patients also received mitomycin (DEB/M) when their disease failed to respond after at least three weekly DEB doses. All but three patients had been treated previously with radiotherapy directed to the primary site and regional nodal disease; four had also received chemotherapy with cisplatin or carboplatin. Before treatment with DEB, 19 patients had distant metastases. Of an intended 12 doses per patient, a mean of 8.2 was achieved. Myelosuppression was the major toxicity, with neutropenia in 45% of patients and significant anemia in 26%. The overall response rate to DEB in 27 patients was 59%, increasing to 70% after the addition of mitomycin. There were two complete and 17 partial responses. The median duration of response was 12 weeks and median survival was 6 months, with 20% of patients surviving 1 year. We conclude that the relatively short survival time together with the significant toxicity of the DEB/M regimen does not warrant its routine use in clinical practice. However, this regimen, or one patterned on it, should be evaluated in combination with radiotherapy as the initial treatment for selected patients with previously untreated head and neck cancer.


Bleomycin pharmacology: mechanism of action and resistance, and clinical pharmacokinetics.

Bleomycin is a glycopeptide antibiotic with a unique mechanism of antitumor activity. The drug binds to guanosine-cytosine-rich portions of DNA via association of the "S" tripeptide and by partial intercalation of the bithiazole rings. A group of five nitrogen atoms arranged in a square-pyramidal conformation binds divalent metals including iron, the active ligand, and copper, an inactive ligand. Molecular oxygen, bound by the iron, can produce highly reactive free radicals and Fe(III). The free radicals produce DNA single-strand breaks at 3'-4' bonds in deoxyribose. This yields free base propenals, especially of thymine: cytotoxicity is cell-cycle-phase specific for G2 phase. In humans, bleomycin is rapidly eliminated primarily by renal excretion. This accounts for approximately half of a dose. In patients with renal compromise or extensive prior cisplatin therapy, the drug half-life can extend from 2 to 4 hours up to 21 hours. Thus, dose adjustments are needed when creatinine clearance is less than or equal to 3N mL/min. Finally, resistance to bleomycin in normal tissues can be correlated with the presence of a bleomycin hydrolase enzyme, which is in the cysteine proteinase family. The enzyme replaces a terminal amine with a hydroxyl, thereby inhibiting iron binding and cytotoxic activity. The low concentration of enzyme in the skin and lung may explain the unique sensitivity of these tissues to bleomycin toxicity. However, correlation of hydrolase levels with tumor cell sensitivity has thus far been negative.


Treatment of acquired immunodeficiency syndrome-related Kaposi's sarcoma using bleomycin-containing combination chemotherapy regimens.

Ninety-nine patients with advanced epidemic Kaposi's sarcoma were treated with bleomycin-containing regimens: 30 received bleomycin and vincristine (BV) and 69 received doxorubicin, bleomycin, and vincristine. Treatment regimens were well tolerated, with response rates ranging from 76% to 81%. However, neutropenia developed even with the relatively nonmyelotoxic BV regimen. Twenty-eight of the 99 patients (28%) were evaluated for pulmonary function prior to, during, and after completion of combination chemotherapy to assess pulmonary toxicity commonly associated with bleomycin. The carbon monoxide diffusion capacity (DLCO) was the only measurement that showed significant changes prior to and after completion of therapy (P = .0003). Moreover, patients receiving more than 100 cumulative units of bleomycin experienced significantly greater declines in DLCO measurements than those receiving lower cumulative doses (P = .0067). No patient, however, developed clinically significant pulmonary toxicity attributable to bleomycin, with individual cumulative bleomycin doses ranging from 10 to 313 U (median, 112 U). We conclude that bleomycin is active and safe in the treatment of Kaposi's sarcoma, and close monitoring of pulmonary function is warranted with cumulative doses exceeding 100 U.


ABVD in the treatment of Hodgkin's disease.

This paper summarizes the clinical results achieved at the Milan Cancer Institute in advanced Hodgkin's disease through successive randomized studies performed during the last two decades. Long-term results confirm the therapeutic activity of a regimen containing bleomycin and doxorubicin, such as ABVD (doxorubicin/bleomycin/vinblastine/dacarbazine), as salvage treatment and as primary chemotherapy, either when combined with radiation or cyclically alternated with MOPP (mechlorethamine/vincristine/procarbazine/prednisone). Delayed iatrogenic morbidity (namely, sterility and leukemogenesis) was less frequently documented in ABVD-treated patients compared with MOPP-treated patients. Nevertheless, bleomycin- and anthracycline-containing regimens can be refined in the attempt to further decrease iatrogenic toxicity.


Bleomycin in non-Hodgkin's lymphoma.

Bleomycin is a cell-cycle--specific chemotherapeutic agent, with several interesting properties, that lends itself to use in combination chemotherapeutic protocols, especially those for malignant lymphomas. In the following article, bleomycin's use as a single agent and subsequently in three generations of combination chemotherapeutic regimens is reviewed. Bleomycin's lack of myelosuppression and its tendency to concentrate in lymphoid tissue while maintaining tolerable toxicities has been the rationale for its incorporation into more aggressive treatment regimens.


The role of cisplatin/bleomycin-based chemotherapy in the treatment of poorly differentiated carcinoma of unknown primary site.

Between 1978 and 1990, 173 patients with poorly differentiated carcinoma or poorly differentiated adenocarcinoma of unknown primary site were treated with bleomycin-containing regimens at Vanderbilt University Medical Center. Patients were prospectively identified based on light microscopy findings. The median age of patients was 39 years; 78% were male and 76% had metastatic tumors in two or more sites. Dominant tumor location was the mediastinum, retroperitoneum, or peripheral lymph nodes in 82 patients (47%). Between 1978 and 1985, patients received cisplatin 20 mg/m2 intravenously (IV) days 1 through 5, vinblastine 0.15 mg/kg IV days 1 and 2, and bleomycin 30 U IV weekly. In 1986, etoposide 100 mg/m2 IV days 1 through 5 replaced vinblastine in the regimen. Responding patients received four courses of therapy at 3-week intervals. One hundred thirteen patients (65%) had a major response to therapy, including 47 (27%) complete responses. At present, 27 patients are disease free at a median of 78 months posttherapy (range, 11 to 142 months); an additional patient was lost to follow-up while in complete response. Median survival of the entire group was 11 months, with a 12-year actuarial disease-free survival of 16%. Combination chemotherapy with cisplatin/bleomycin and either vinblastine or etoposide is highly active in patients with poorly differentiated carcinoma of unknown primary site and is curative in a minority of these patients. A trial of this therapy should be considered in all such patients.


Bleomycin and tetracycline in malignant pleural effusions: a review.

Pleural effusions remain a distressing and symptomatic problem in cancer patients. Once a malignant pleural effusion is diagnosed, appropriate therapy may provide symptomatic relief and improved quality of life. In this paper, we review previous data on chest tube drainage and pleurodesis in patients with malignant pleural effusions. A comparison is made of the various sclerosing agents. Specifically, recent data from a study comparing bleomycin and tetracycline as sclerosing agents are discussed. These data show a clear advantage with bleomycin at both 30-day and 90-day end points--30-day recurrence, 36% versus 67%; 90-day recurrence, 30% versus 53%.


Bleomycin pulmonary toxicity: current status and future directions.

Bleomycin is a polypeptide antibiotic that has been used in clinical cancer chemotherapy for over 20 years. Risk factors associated with bleomycin include total dose of drug, age of patient, high-dose oxygen therapy during surgery, and prior and concomitant radiation therapy. Attempts at developing predictive and precise monitoring systems have not been completely successful; however, the combined use of clinical and laboratory tests, such as the DLCO test, can be used to permit safe administration of the drug. Recent work has emphasized the development of new analogues, such as liblomycin, that appear to possess less pulmonary toxicity than does bleomycin, and has focused on understanding the pathogenesis of pulmonary toxicity, particularly as it relates to the immune system.


Experience with bleomycin, ifosfamide, and cisplatin in primary and recurrent cervical cancer.

The unfavorable prognosis for patients with advanced and bulky early stage cancer of the cervix may be improved by initial neoadjuvant cytoreductive chemotherapy. In a phase II study using ifosfamide in combination with cisplatin/bleomycin (BIP) in advanced and recurrent cervical cancer, we demonstrated a response rate of 69%. To determine whether this high response rate was a result of patient selection, and to assess the influence of combination chemotherapy on survival in patients with recurrent disease, a retrospective analysis of five phase II studies of bleomycin, ifosfamide, and cisplatin combinations was performed. The type of chemotherapy regimen (BIP v others) was the most significant factor in determining the likelihood of response. Combination chemotherapy did not appear to confer a survival advantage in patients with recurrent disease. The BIP regimen produced rapid responses with acceptable toxicity, and had potential for use as neoadjuvant therapy prior to radical radiotherapy in patients with advanced and bulky early stage disease. In an initial pilot study of this approach, 13 of 19 patients (68%) with primary inoperable disease had significant tumor regression prior to radical local radiotherapy. Interim analysis of the first 66 patients entered into a randomized study evaluating this approach has shown complete clinical tumor resolution after radical radiotherapy in 24 of 32 patients (75%) treated with up to three cycles of BIP prior to radiotherapy compared with 19 of 34 patients (56%) treated with radiotherapy alone. There has been no evidence that neoadjuvant chemotherapy enhances the acute toxic effects of pelvic radiotherapy. Therefore, this approach has potential to improve therapeutic outcome in patients with poor-prognosis primary disease.


Hypersensitivity reactions.

All cancer chemotherapeutic agents, except altretamine, the nitrosoureas, and dactinomycin, have produced at least an isolated instance of a HSR. Certain drugs, such as L-asparaginase and mitomycin (administered intravesically), cause HSRs of significant degree in approximately 10% of patients. All four types of HSRs are represented in the reactions produced by antitumor drugs, although Type I is the most common. Some of the Type I reactions are IgE-mediated, and others are probably mediated by nonspecific release of vasoactive substances from targets such as mast cells. It is possible to continue therapy with some drugs, despite a prior HSR, if the prophylactic measures outlined in Table 2 are taken. An example of this is provided by taxol in which the lengthening of the infusion time and the administration of preventive medication allowed some patients to continue taxol therapy. The mechanisms of the HSRs have been carefully assessed in only a minority of patients who sustained such toxicity. Such evaluation would increase our understanding of this form of drug toxicity and perhaps lead to means of effectively reducing the risk and severity.


Patient classification, a key to evaluate pain treatment: a psychological study in chronic low back pain patients.

It has been proposed that pain treatment evaluation is hindered by heterogeneous properties of patient samples. Therefore, to facilitate pain treatment evaluation in this psychological study, a heterogeneous group of chronic low back pain patients was classified into more homogeneous subgroups. Two designs were used to compare the outcome by the "functioning activation" and the "spa resort" type of rehabilitation. In the first design, the outcome was compared in groups, clinically homogenized by sociodemographic variables and in respect to contraindications for heavy physical training. In the second design, the pain patient subgroups, homogenized by cluster analysis technique in accordance with the psychological profiles of functioning, were compared in their response to treatments. The results indicated that the outcome evaluation was facilitated by the latter design releasing more specific information about the effects of the program quality, the patient characteristics, and their interaction on the improvement by rehabilitation. It was concluded that in treatment outcome analysis, the subgroup's homogeneity must be considered.


Combined isodose curves of high-dose rate interstitial brachytherapy with external-beam radiation therapy in pancreatic carcinoma.

From September 1989 until March 1992 nine patients with unresectable, though localized carcinoma of the pancreas were treated by a multimodality therapy consisting of palliative surgery, interstitial conformal brachytherapy in high-dose rate mode (HDRBT) with iridium-192 up to 30 Gy and external-beam radiation therapy (EBRT) of about 52 Gy. Four patients simultaneously received two cycles of chemotherapy consisting of 5-FU and Leucovorin. Since high radiation doses are applied which are not tolerated in adjacent healthy tissues, doses to tumor and critical areas need to be known precisely and are to be adjusted before treatment. A three-dimensional imaging system is required. A self developed method combines the data of simulation radiographs and those of CT scans. The prescribed minimum target absorbed dose in HDRBT is adjusted to the target volume sparing organs at risk. The specialized quality assurance is adapted to this method. Differences between measured and calculated doses do not exceed 5%. The addition of isodoses of HDRBT and EBRT on CT scans is demonstrated. Due to patients' selection the treatment concept did not reveal any positive effects on survival. However, excellent palliative results were obtained without severe side-effects.


Maternal zinc and fetal neural tube defects.

Among the factors implicated in the heterogeneous etiology of neural tube defects (NTDs) is the trace element zinc (Zn). In a case-control study, we collected midtrimester maternal toenail samples for multiple trace element analyses, including Zn, which were assayed by neutron activation analysis. We studied 17 women with NTD offspring and 1,787 controls. The crude OR for NTD comparing Zn values greater than normal range to normal Zn values was 3.2 (95% CI 1.1,9.7). These results were not materially affected when adjustment was made for folic acid supplementation. An overall increased risk for NTD associated with increasing toenail Zn was also evident. A matched subset of 17 cases and 73 controls yielded a crude OR of 3.1 (95% CI 0.9,10.3) when cases with elevated Zn (greater than or equal to 120 ppm) were compared to those with normal Zn. Matched analyses controlling for folic acid supplements, family history of NTD, assay batch, age of mother and year of delivery yielded an OR of 5.0 (95% CI 1.1,21.6). This study reveals an association between increased toenail Zn in the second trimester of pregnancy and the risk of having a child with an NTD. Whether Zn sequestration has resulted in relative Zn deficiency at the site of neural tube closure remains uncertain.


An unusual mosaic karyotype detected through prenatal diagnosis with duplication of 1q and 19p and associated teratoma development.

A 40-year-old white woman underwent amniocentesis for advanced maternal age at 15.4 weeks gestation. Fetal chromosome analysis demonstrated two distinct cell lines: [46,XX,t(1;19)(p11;p11)]--10%; and [47,XX,t(1;19)(p11;p11) + der(1)t(1;19)(p11;q11)]--90%. The latter karyotype was trisomic for both 1q and 19p. The mother carried the balanced translocation; the father had a normal karyotype. Amniotic fluid alpha-fetoprotein level was elevated and an acetylcholinesterase band was detected. Level II ultrasonography at 17 and 24 weeks revealed several abnormalities, including a large facial cleft and a probable facial teratoma and intracranial tumor. Autopsy following pregnancy termination confirmed the presence of both. Chromosome evaluation of 172 metaphases of both the epignathus and the intracranial teratoma demonstrated a predominance of the cell line with 47 chromosomes (166/172 = 96.5%), while from nonteratoma tissue (lung, liver, skin, and brain) only the balanced karyotype was detected. These observations suggest that the chromosomal imbalance is instrumental in the etiology of the teratoma.


Seroepidemiologic study of hepatitis C virus in sexually transmitted disease risk groups.

To identify the importance of heterosexual activity as a possible route for the transmission of the hepatitis C virus (HCV), a screening of antibodies against HCV (anti-HCV) was performed in 200 sexually transmitted disease patients with different risks for incurring genital infections as well as in 100 registered prostitutes. Out of all 300 persons tested, 14 cases of HCV infection were detected. Anti-HCV was present in 3 of the prostitutes and in 11 of the STD patients. Evaluating known risk factors, such as intravenous drug use or blood transfusion, 6 out of the 11 STD patients and all of the prostitutes in whom anti-HCV was present were intravenous drug users and exhibited highly promiscuous behavior. Intravenous drug use was the probable means of acquisition in 9 of the 14 subjects in whom anti-HCV was present, and homosexual promiscuous behavior was assumed to be the means of acquisition in another 2 subjects. In heterosexual patients engaging in high-risk behavior (high number of sexual partners and genital infections), the exclusion of intravenous drug use decreased the prevalence of anti-HCV from 12.1% to 4.1%, demonstrating no significant increase from the prevalence among low-risk persons. Most of the patients were screened for STDs, such as syphilis, Neisseria gonorrhoeae, Chlamydia trachomatis, human immunodeficiency virus (HIV), hepatitis B virus (HBV), trichomoniasis, and yeast infections. The highest rate of coinfection with anti-HCV was found in patients with serologic evidence of an HIV infection (50%).(ABSTRACT TRUNCATED AT 250 WORDS)


Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis.

The native fibrin gel structure formed in vitro from plasma samples was examined by liquid permeation of the hydrated fibrin gel networks in 18 men who had suffered a myocardial infarction before the age of 45 years and in 20 control subjects. Patients with an elevated plasma fibrinogen concentration had a considerably lower fibrin gel porosity (permeability coefficient, Ks) compared with patients with a normal plasma fibrinogen level and with controls. The calculated fiber mass-length ratio of the fibrin gel networks was decreased in both patient groups. Gel porosity differed markedly between individuals at a given plasma fibrinogen concentration. Fairly strong inverse correlations were found between plasma orosomucoid level on the one hand and Ks (r = -0.617, p less than 0.01) or fiber mass-length ratio (r = -0.499, p less than 0.05) on the other. The low density lipoprotein (LDL) cholesterol concentration also correlated inversely with Ks (r = -0.471, p less than 0.05) and fiber mass-length ratio (r = -0.522, p less than 0.05). Significant inverse relations, which were independent of plasma fibrinogen and lipoprotein concentrations, were detected between Ks (r = -0.519, p less than 0.05) and calculated fiber mass-length ratio (r = -0.723, p less than 0.001) and number and severity of coronary artery stenoses determined by angiography. A proneness to formation of tight, rigid and space-filling fibrin network structures with small pores thus appears to be associated with premature coronary artery disease.


[Incidental carcinoma of the prostate. Study of 683 patients operated upon for benign prostatic hypertrophy].

During the period 1988-1990, 683 patients with diagnosis of benign prostatic hyperplasia underwent transvesical prostatectomy. The purpose of the study was to establish the possible presence of incidental carcinoma of the prostate (ICP). Prostatic tissue was studied with sections taken every 0.5 cm. The incidence of ICP was 12.00%. The mean age of patients was 73.5 years. 60.98% of tumors classified T1a, 39.02% T1b. No case of G3 tumor was observed while 92.68% of neoplasms was classified G1 and 7.32% G2. 74.95% of tumoral nests into the prostatic tissue had a breadth about one millimetre.


The ex vivo effect of preadsorbed vitronectin on platelet activation.

The activation of ex vivo canine platelets by preadsorbed vitronectin (VN) was sensitive not only to the polymer substrate utilized but also to the adsorption conditions employed. Lower levels of maximal platelet deposition were obtained for VN-coated silicone rubber (SR) than for other VN-coated substrates with comparable levels of adsorbed VN, but this effect was diminished with increased residence time of VN on the SR surface. Submonolayer and monolayer surface concentrations of VN elicited similar maximal levels of platelet deposition at both short (less than 3 h) and long (greater than 12 h) residence times, but thrombi were larger and more dense for the submonolayer surface concentrations. VN was also more effective in forming thrombi when adsorbed sequentially before albumin instead of after albumin. To further examine these differences in the nature of adsorbed VN between substrates and adsorption conditions, sodium dodecyl sulfate (SDS) elutability measurements and Fourier transform infrared spectroscopy with attenuated total reflectance optics (FTIR-ATR) evaluations of the adsorbed protein were performed. An SDS solution was able to remove a greater percentage of the VN which was adsorbed to a submonolayer than a monolayer surface concentration when SDS displacement was initiated immediately after adsorption was terminated. However, if the adsorbed protein was allowed to reside on the surface for a length of time before the introduction of the SDS displacing media, a greater percentage of the monolayer surface concentration was removed. The submonolayer surface concentration may be better able to increase its strength of contact with the surface during the added residence time than the monolayer surface concentration.(ABSTRACT TRUNCATED AT 250 WORDS)


Progesterone regulation of plasminogen activator inhibitor 1 (PAI-1) antigen and mRNA levels in human endometrial stromal cells.

Plasminogen activator activity decreases in the endometrium in the secretory phase of the menstrual cycle. This is partly due to decreased release of urokinase plasminogen activator in response to progesterone. Plasminogen activator inhibitor type 1 (PAI-1) is an efficient inhibitor of both tissue-type and urokinase-type plasminogen activators, and may therefore be instrumental for the control of plasminogen activation. In this study we examined the effects of steroid hormones on PAI-1 release and PAI-1 mRNA levels in primary cultures of human endometrial stromal cells. In these cells the secretion of PAI-1 was increased by progesterone in a dose and time dependent way, but was not affected by estradiol. The progesterone induction of PAI-1 secretion was preceded by a 7-8 fold increase of the steady state level of PAI-1 mRNA in the cells, suggesting that progesterone activates PAI-1 gene expression. Cultured endometrial glandular epithelial cells were found to release only insignificant amounts of PAI-1 with or without hormone treatment. The effect of progesterone on endometrial stromal cells was mimicked by DH-testosterone. However, while the response to progesterone was completely blocked by ZK112993, a potent antagonist of the progesterone receptor, the response to DH-testosterone was partially blocked by ZK112993, and partially by OH-flutamide, a potent antagonist of the androgen receptor. This suggests that a secretory response on PAI-1 expression is mediated via androgen receptors in endometrial tissue.


Intercellular transport through a partially denuded arterial endothelial monolayer. Effect of platelets and PGI2.

Fluorescein Dextran (FD) was shown to be transported at increased rates through partially denuded endothelial monolayer. Platelet binding to the partially denuded monolayer lowered transport rates to those comparable with intact endothelium. Inhibition of transport by platelet binding was not affected by the addition of isocarbacyclin (a stable derivative of PGI2). This result suggests that adherent platelets at the partial denudation site are sufficient to suppress transport of FD.


A monoclonal antibody (LK-4) which differentiates PLA1 from PLA2 platelet extracts but not intact platelets.

A unique murine monoclonal antibody (LK-4) is described which differentiates PLA1/PLA1 platelet extracts from PLA2/PLA2 and PLA1/PLA2 platelet extracts on solid phase ELISA and immunoblot at the 100kD GPIIIa location, but not on intact platelets. LK-4 reacts equally with intact PLA1/PLA2 and PLA2/PLA2 platelets. Adsorbtion of LK-4 with PLA1/PLA1 platelets results in loss of reactivity for intact platelets as well as platelet extracts on ELISA or immunoblot. LK-4 inhibits platelet aggregation induced by ADP, epinephrine, collagen and thrombin, suggesting reactivity at or near the fibrinogen binding site on GPIIIa. It is suggested the LK-4 reacts with a conformation-induced common epitope for PLA1 and PLA2 on GPIIIa, with loss of this conformation for PLA2 GPIIIa following solubilization with Triton X-100.


Okadaic acid and vanadate inhibit collagen-induced platelet aggregation; the functional relation of phosphatases on platelet aggregation.

The different specific inhibitors for phosphoserine/threonine and phosphotyrosine protein phosphatases were used to study the role of these protein phosphatases in collagen-platelet interaction. The collagen-induced platelet aggregation and the release reaction as measured ATP release were inhibited in a dose-dependent fashion by the addition of okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatase 1 and 2A. The inhibition was also observed by the addition of a phosphotyrosine protein phosphatase inhibitor, vanadate. Suboptimal concentrations of both inhibitors together also inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent fashion. These results suggest that collagen-platelet interaction is modulated by both protein phosphatases.


[From endothelium-derived relaxing factor to L-arginine and nitrogen monoxide].

Endothelium-derived relaxing factor has been identified as nitric oxide. It was originally found to be released from endothelial cells, mediating vascular relaxation and platelet inhibitory action. Nitric oxide is formed enzymatically from L-arginine by nitric oxide synthase, and this enzyme has up to the present been demonstrated in endothelial cells, platelets, neutrophils, macrophages, neurons and smooth muscle cells. Both a constitutive and an inducible nitric oxide synthase have been identified, which may serve different physiological/pathophysiological purposes. The biological implications of nitric oxide have now been manifested, and the number of reports on its physiological role is rapidly increasing. Compounds which inhibit the formation of nitric oxide have become available, and this expanding field has created great interest amongst clinicians.


Omentopexy for revascularization of free tracheal grafts in rats.

In rats, we examined the effect of an omentum wrapping on the vascularization of the trachea and on regeneration of the mucosal epithelium in the very early stage after free tracheal grafting. Two pieces of trachea were obtained from each donor rat. One piece was transplanted into the peritoneal cavity of the recipient rat and wrapped with omentum. The other piece was transplanted subcutaneously into the abdominal wall, without omentopexy. Recipients were sacrificed at random on days 1, 2, 3, and 4 after operation (5 rats/day). The increase of blood vessels rose significantly earlier in the omentopexy tracheas than in those without omentopexy (p less than 0.01), with all the former showing a normal ciliated, columnar epithelium in the membranous region of the trachea on day 4, accompanied by excellent epithelial regeneration in the cartilaginous region. These results suggest that omentopexy causes revascularization to occur earlier and to be completed in a shorter time, and that it also promotes epithelial regeneration following free tracheal grafting.


The molecular basis for reactivity of anti-Cw1 and anti-Cw3 alloantisera with HLA-B46 haplotypes.

HLA haplotypes containing the HLA-B46 allele react with both anti-Cw1 and anti-Cw3 alloantisera, a pattern of reactivity defined as the Cw11 antigen and postulated to involve either a distinctive Cw11 allele or a duplicated HLA-C locus. From serological characterization of CIR cells transfected with B46 cDNA we now demonstrate that the anti-Cw3 reactivity with these haplotypes is solely due to the B46 molecule and not to an HLA-C molecule. Furthermore, isolation and characterization of HLA-C mRNA from cells expressing B46 strongly suggest that anti-Cw1 reactions are directed against the product of a conventional Cw1 allele. The antigenic cross-reactivities of B46 with B62 and Cw3 correlate with its chimaeric primary structure, which is identical to that of B62, except in the alpha 1 helix where it is identical to both Cw3 and Cw1. The structure, distribution and genetic linkage of B46 indicate it is of recent, Asian origin and is the result of a gene conversion, involving Cw1 as the donor gene and B62 as the recipient. These results demonstrate that the Cw11 antigen neither corresponds to a novel HLA-C allele nor a duplicated HLA-C locus, but to a combination of epitopes contributed by linked Cw1 and B46 alleles. The nucleotide sequence we previously and erroneously attributed to a distinct Cw11 allele is now demonstrated to encode Cw8. Isolation of the cDNA clone with this sequence from a library made from a cell homozygous for the B46 haplotype was probably an artefact of contamination.


A cytotoxic monoclonal human hybridoma antibody (TrJ3) against HLA-B44(12) and -B45(12).

We have generated a human monoclonal cytotoxic IgM lambda antibody (TrJ3) that reacted specifically with all lymphoblastoid B-cell lines expressing HLA-B44(12) and B45(12). TrJ3 hybridoma supernatant was suitable for HLA-B12 typing of freshly isolated blood mononuclear cells. Analysis of available amino acid sequences of HLA-B molecules indicated that the alpha 1 domain does not contain the TrJ3 serological epitope. Since HLA-B44 is associated with a unique serine residue at position 167 that points towards the peptide binding groove, we propose that S167 of the alpha 2 domain helix is a critical part of the TrJ3 epitope.


Mechanism of cell swelling of HeLa cells in an isosmotic Na(+)-free, high-K+ medium: study on the K+ transport pathways.

The K+ content and the free Ca2+ concentration ([Ca2+]c) in HeLa cells began to increase after a latent period following medium change to an isosmotic Na(+)-free, high-K+ medium in the presence of 10-100 microM ouabain. These increases were accompanied by cell swelling, and stimulated by the addition of 1 microM ionomycin, whereas treatment with 1 mM quinine significantly inhibited the net K+ uptake. [Ca2+]c was slightly decreased, but the K+ uptake was unaffected in the Ca(2+)-free, high-K+ medium. However, when [Ca2+]c was extremely reduced by preloading 5 microM BAPTA-AM, the K+ uptake decreased to a level attained in the presence of quinine. Addition of DIDS and substitution with NO3- or SCN- for medium Cl- inhibited the K+ uptake to similar extents, and their effects were less strong than that of quinine. Substitution of medium Cl- with an impermeable anion gluconate completely inhibited the K+ uptake. Therefore, we conclude that cell swelling in the high-K+ medium is associated with K+ uptake mediated in greater part by quinine-sensitive and in smaller part by -insensitive pathway. The former pathway depends on cellular Ca2+ and its major component is Cl(-)-dependent, whereas the latter is independent of the Ca2+ and unselective for anions.


Modulation of ganglioside expression in human melanoma cell lines; increased resistance to chemo- and radiation treatment.

Cell surface gangliosides in human melanoma cell lines were modulated by pretreatment and adaptation to 6-thioguanine and 5-bromo-deoxyuridine. Chemo- and radiation sensitivities were compared in original cell lines and modulated cells by the human tumor colony-forming assay. Modulated cells showed decreased expression of cell surface GM2 and GD2 gangliosides. This reduction was correlated with increased resistance to bleomycin, vincristine, cisplatin and radiation treatment. These results suggest that cell surface GM2 and GD2 ganglioside expression in human melanoma cells is intimately associated with several cellular biological properties, such as drug or radiation sensitivity and cellular differentiation.


Alpha 2u-globulin is the only member of the lipocalin protein superfamily that binds to hyaline droplet inducing agents.

The rate-limiting step in chemically induced, male rat-specific hyaline droplet nephropathy is the reversible binding of a xenobiotic to alpha 2u-globulin. In this study, equilibrium saturation binding experiments were conducted to evaluate the in vitro binding of d-limonene-1,2-oxide (dLO) and 2,4,4-trimethyl-2-pentanol (TMP-OH) to alpha 2u-globulin and members of the alpha 2u-globulin protein superfamily. Both dLO and TMP-OH bound to alpha 2u-globulin, with Scatchard analysis yielding dissociation constants of 5.6 and 6.4 x 10(-7) M, respectively. The Bmax for binding (nmol bound/mg protein) was 50.7 and 61.1 for dLO and TMP-OH, respectively, yielding a molar ratio of approximately 1 for both ligands. The ability of dLO and TMP-OH to bind to human-derived alpha 1-acid glycoprotein, rat-derived retinol-binding protein, human protein-1, and bovine beta-lactoglobulin was also studied. These superfamily proteins are generally abundant in plasma, are freely filtered across the glomerulus, and can bind a wide range of ligands. However, neither dLO nor TMP-OH bound to any of the superfamily proteins. In contrast, under identical experimental conditions, alpha 1-acid glycoprotein did bind progesterone (Kd = 10(-6) M), whereas both beta-lactoglobulin and retinol-binding protein bound retinol (Kd = 10(-8) M for both proteins). These results indicate that, under conditions where alpha 2u-globulin superfamily proteins bind to established ligands, the proteins do not interact with hyaline droplet inducing agents. Thus, the interaction between male rat-specific nephrotoxicants and alpha 2u-globulin is unique to this protein. More importantly, these results provide direct evidence that the presence of the alpha 2u-globulin superfamily proteins does not predispose humans to develop hyaline droplet nephropathy and renal cancer from this class of chemicals.


Quantification of metallothionein isoforms using an enzyme-linked immunosorbent assay (ELISA) with two specific antisera.

The specificity of three populations of antibodies to the two metallothionein (MT) isoforms has been characterized using a competitive enzyme-linked immunosorbent assay (ELISA). A polyclonal sheep anti-MT-1 antibody (PAb 1) showed higher affinity to MT-1 than to MT-2 with I50 (concentrations required to achieve 50% inhibition) being 18 and 480 ng for MT-1 and MT-2, respectively. Conversely, a polyclonal rabbit anti-MT-2 antibody (PAb 2) showed higher affinity to MT-2 than to MT-1 (I50 being 250 and 15 ng for MT-1 and MT-2, respectively). A third antibody (PAb 3), isolated by removal of anti-MT-1 antibodies in PAB 2 by immunoaffinity purification, showed no cross-reactivity with MT-1. This result suggests that there are two populations of antibodies which cross-react specifically with the MT isoforms. Rat tissue MT concentrations were quantified by ELISA using the three different antibodies and silver-saturation method. The sum of MT isoform concentrations (ELISA estimation using antibodies PAb 1 and PAb 3) were not significantly different (p < 0.05) from the total MT concentrations obtained by silver-saturation method. Therefore, the ELISA can be used to estimate the relative quantities of the two MT isoforms in rat tissues. The predominant isoform in rat tissues was MT-2 and the proportion of MT-1 increased with induced synthesis of MT by metal injection.


A comparison of European High Test gasoline and PS-6 unleaded gasoline in their abilities to induce alpha 2u-globulin nephropathy and renal cell proliferation.

Male Fischer-344 rats were administered European High Test gasoline (EHT) (50-500 mg/kg), PS-6 unleaded gasoline (UG) (16-500 mg/kg) or 2,2,4-trimethylpentane (TMP) (0.95-30 mg/kg) by gavage for ten consecutive days. To measure cell replication, rats were exposed to [3H]thymidine continuously over the last 7 days of the exposure period. Twenty-four hours after the final dose, protein droplet (PD) accumulation, alpha 2u-globulin (alpha 2u) concentration and the nuclear labeling index (LI), as a measure of cell replication, were measured in the kidneys of control and treated rats. Dose-related increases in PD, alpha 2u and cell replication were detected in the kidneys of rats treated with either gasoline mixture or TMP. The accumulation of PD and the increase in alpha 2u was greater in the kidneys of UG- and TMP-treated rats than in the kidneys of rats treated with EHT. These differences were attributed to the higher composition of branched hydrocarbons in UG, which have been shown to be the biologically active components for these endpoints. The extent of renal cell proliferation was similar in both EHT-, UG- and TMP-treated rats. This suggests that other components besides the branched hydrocarbons are responsible for the increased renal cell replication in EHT-treated rats.


Radioimmunoassays for spirapril and its active metabolite spiraprilate: performance and application.

Radioimmunoassays for a nonsulfhydryl angiotensin converting enzyme inhibitor prodrug--spirapril--and its active metabolite--spiraprilate--are described. Nonextraction equilibrium assays using antibodies with a high specificity for spirapril or spiraprilate were used, with charcoal separation of bound and free tracer. Within-assay reproducibility (CV%) was less than 20% in the concentration range 0.5-40 micrograms/L for both analytes and the comparable value for between-assay reproducibility was less than 25%. Results for external quality control samples were in good agreement with the expected values of 0-250 micrograms/L (spirapril, r = 0.997) and 0-300 micrograms/L (spiraprilate, r = 0.999). Overall, samples circulated to four laboratories gave good agreement for measured values, including one center using gas chromatography-mass spectrometry analysis for the two compounds. Data are presented to show the suitability of these two assays to the measurement of spirapril and spiraprilate in clinical samples from assays to the measurement of spirapril and spiraprilate in clinical samples from dose-ranging and bioequivalence studies. Results are also shown relating drug plasma concentration data to a measurement of the pharmacodynamic effects of spiraprilate, namely inhibition of angiotensin converting enzyme activity. It is concluded that these assays have the sensitivity for use in studies to model the relationship between the pharmacokinetics and pharmacodynamics of the two compounds.


In vitro genetics.

Understanding the basis of specificity in an intermolecular interaction is a common if difficult task; designing a specific intermolecular interaction is much more challenging. A new technique is described that has applications to both problems, at least with regard to nucleic acids. The power of this method lies in its ability to isolate extremely rare sequences with precisely specified properties from very large pools of random sequences.


The 'ins' and 'outs' of mitochondrial membrane channels.

The outer membrane of the mitochondrion contains thousands of copies of a pore-forming protein called VDAC or porin. Considerable progress has been made towards elucidating the molecular structure of this channel. Moreover, mounting evidence that the permeability of VDAC may be regulated is challenging the textbook notion of the outer membrane as a simple sieve. Numerous other channel activities have been detected by electrophysiol approaches in both the outer and inner mitochondrial membranes. The inner-membrane channels do not appear to be open under normal physiological conditions and so should not dissipate energy-transducing ion gradients. The biological functions of the different classes of mitochondrial channels are uncertain, but several possibilities (including protein translocation) are being explored.


Exploiting the chemical synthesis of RNA.

A number of nucleotide phosphoramidites are now available that permit the chemical synthesis of RNA, modified RNA and RNA-DNA chimeric oligonucleotides. Since the chemical strategy allows the introduction of a particular modification at any given site in a nucleotide polymer, very subtle and specific questions regarding structure-function relationships in RNA may be addressed.


Induction of vascular adhesion molecules during rejection of human cardiac allografts.

Adhesion of leukocytes to vascular endothelium is a necessary step leading to the migration of cells into underlying tissues. Vascular adhesion molecules regulate this process and may play an important role in graft rejection. Immunocytochemical studies have been used to investigate the expression of vascular adhesion molecules (ICAM-1, PECAM, VCAM-1, and ELAM-1) in normal donor heart (n = 15) and myocardial biopsies from heart transplant patients with acute rejection (n = 15). Sections were also stained with antibodies against endothelium, leukocytes, MHC antigens, and markers of cell activation. In donor heart EN4, vWF, ICAM-1, PECAM, MHC class I--and, to a lesser extent, VCAM-1 and DR antigen--are expressed on arterioles and venules, whereas ELAM-1 and Pal-E are restricted to venules. Expression of Pal-E, VCAM-1, ICAM-1, and DR antigen was increased during rejection. Capillary endothelium normally expresses EN4, ICAM-1, PECAM, MHC class I, and DR antigen but little, if any, VCAM-1 or ELAM-1. During rejection, however, there is an increased expression of all adhesion molecules. This is paralleled by an increased expression of vWF by capillary endothelium. In addition, ICAM-1 like MHC class I antigen is induced on the myocardial membrane and intercalating discs. Endocardium from donor heart expresses EN4, vWF, PECAM, MHC class I, and sometimes Pal-E and ICAM-1, but very little VCAM-1, ELAM-1 or DR antigen. There is an increased expression of Pal-E, ICAM-1, VCAM-1, and DR antigen on endocardium from rejecting heart biopsies. Proliferating Ki-67+ cells and activated T cells expressing the receptor for IL-2 were also found in biopsies during rejection episodes.


Comparison of DNA amplification and immunofluorescence for detecting Pneumocystis carinii in patients receiving immunosuppressive therapy.

Two studies were performed to compare the sensitivity of DNA amplification with immunofluorescence for the detection of Pneumocystis carinii in asymptomatic normal and immunosuppressed subjects receiving no anti-Pneumocystis chemoprophylaxis. In the first study, immunofluorescence and silver stains were used to examine 12 induced sputa and 12 bronchoalveolar lavage specimens from 24 normal control subjects; induced sputa from 20 renal transplant recipients; and induced sputa from 11 patients with fibrosing alveolitis. All specimens were negative for P carinii using both stains, apart from one renal patient in whom 2 P carinii cysts were seen by immunofluorescence alone. In the second study, DNA amplification and immunofluorescence were used to examine induced sputa from 3 groups of 10 control, renal, and heart/lung transplant recipients. All 30 specimens were negative for P carinii by immunofluorescence. However, 3 renal and 2 heart/lung patients were positive for P carinii by DNA amplification alone. One of these patients developed P carinii pneumonia 6 weeks after sputum induction. DNA amplification is a more sensitive technique than immunofluorescence for detecting P carinii. P carinii colonization occurs in asymptomatic organ transplant recipients, but not in normal individuals.


Evidence that the systematic analysis of bile cytology permits monitoring of hepatic allograft rejection.

Cytologic analysis was performed on 128 bile specimens collected by schedule from 12 liver transplant recipients over a 4-month period. Clinical diagnoses at the time of specimen collection were determined retrospectively, as follows: clinically stable, 75; acute rejection, 15; CMV hepatitis, 1; systemic infection, 8; ischemic injury, 24 (all within the first 4 days postop); nonclassifiable, 5. Bile analysis was done by a blinded investigator. Specimens contained ductal epithelial cells (EC) and inflammatory cells (IC), which were counted using Cytospin slide preparations. Greater than 10 cells/slide were seen in 93.3% of rejections, 91.7% of ischemic injuries, 100% of systemic infections, and 14.6% of stable patients. In samples collected after POD 4, IC were seen in 86.7% of rejections, yielding a specificity of 94.4% (P less than 0.001). If lymphoblastic cells were also seen, the specificity increased to 96.6%. Five specimens were obtained the day before the clinical diagnosis of rejection; all demonstrated IC. Seven specimens were obtained 3 days after beginning therapy for rejection. In 5 the bile contained no IC, and clinical improvement occurred; in the 2 in whom IC were found, further therapy was subsequently required. IC were seen in 5 of 8 specimens taken when systemic infection was present; the clinical setting allowed differentiation from rejection. Only 1 case of CMV hepatitis was included, thus no conclusions can be drawn for this entity. Cytoplasmic vacuolization of EC was observed in 30% of cases, in these, cyclosporine levels were significantly higher (989.9 +/- 356.9 vs. 672.8 +/- 421.2, P = 0.02). In summary, bile cytology analysis aides in the monitoring of the onset and duration of rejection. It may be an indicator of persistent rejection, and it may help prevent overimmunosuppression in those cases with normal cytological findings.


Induction of transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody.

The T-cell-mediated immune response usually results in the rapid destruction of organ allografts transplanted between murine strains incompatible for major and minor histocompatibility antigens. This response may be modified by pretreatment with either donor-specific antigen or anti-CD4 monoclonal antibody. Previous work by others has shown that combined treatment of mice with soluble protein antigens and anti-CD4 monoclonal antibody can produce antigen-specific B cell unresponsiveness that continues long after the nonspecific immunosuppressive effect of the mAb treatment has resolved. Following this principle we have shown that adult C3H/He mice can be made specifically unresponsive to vascularized C57BL/10 cardiac allografts by pretreating the recipient with donor alloantigen under the cover of a brief course of mAb against CD4. A full-dose response analysis shows that the dose of mAb is critically important for the successful induction of tolerance. Tolerance induction using this protocol is dependent on treatment with donor major histocompatibility complex antigens and occurs in the presence of marked depletion but not complete elimination of the CD4+ T cell subset. The unresponsiveness to alloantigen is antigen specific, as determined by the ineffectiveness of third-party (C57BL/10) alloantigen when combined with anti-CD4 mAb to induce long-term survival of BALB/c allografts in C3H/He recipients. The tolerant state is specific and effective in the long-term as indicated by the specific acceptance of C57BL/10 skin grafts in recipients with surviving C57BL/10 cardiac allografts. This study provides a simple method for the successful induction of specific transplantation tolerance in the adult across a full H-2 major and minor antigen mismatch strain combination. The results illustrate the important role of the CD4 molecule in the T cell response to alloantigen in vivo and suggest possibilities for the therapeutic manipulation of complex immune reactions.


Evidence that long-term cardiac allograft survival induced by anti-CD4 monoclonal antibody does not require depletion of CD4+ T cells.

Monoclonal antibodies that deplete cells carrying their target antigen are being used increasingly for immunosuppression in clinical and experimental transplantation. We have characterized a panel of rat antimouse CD4 monoclonal antibodies with the aim of establishing, in a vascularized organ transplant model, whether prolonged graft survival can be induced without recipient T cell depletion. The spatial relationship of the epitopes recognized by the anti-CD4 mabs was established. Mabs of the IgG2b isotype were found to profoundly deplete CD4+ T cells in vivo, whereas IgG2a mabs did not. The IgG2b anti-CD4 mab YTS191 and the IgG2a mab KT6 both blocked proliferation of C3H/He leukocytes in mixed leukocyte culture. Potent suppression of rejection and indefinite survival of cardiac allografts, mismatched for both major and multiple minor histocompatibility antigens (C57BL/10, H-2b into C3H/He, H-2k), was achieved with the IgG2b anti-CD4 mab YTS191 that depleted CD4+ T cells, (n = 9, median survival time (MST) greater than 100 days, P less than 0.001). The non-depleting IgG2a anti-CD4 mab, KT6, which had been shown to recognize and epitope on the CD4 molecule closely related to that recognized by YTS191 and to block comparably in MLC, was also shown to be capable of producing long-term cardiac graft survival in this strain combination (n = 6, MST greater than 100 days P less than 0.001). The kinetics of the KT6 therapy on the blocking of the CD4 molecule in vivo were investigated and shown to correlate with the effectiveness of the mab in prolonging graft survival.


Evidence that indefinite survival of small bowel allografts achieved by a brief course of cyclosporine or FK506 is not due to systemic hyporesponsiveness.

The immunological status of Lewis (LEW) recipients of indefinitely surviving (greater than 400 days) orthotopic Brown-Norway (BN) small bowel allografts was investigated 1 to 1 1/2 years after cessation of immunosuppressive therapy with either cyclosporine or FK506 and compared with recipients of syngeneic grafts. A normal proliferative response (as measured by a mixed lymphocyte culture) of recipient peripheral lymph node lymphocytes in response to the donor-specific (BN) and the third-party (ACI) antigen, was observed in all experimental groups. Cytolytic T cell generation (as measured by a standard 51Cr-release cytotoxicity assay) in response to the donor-specific (BN) and the third-party (ACI) antigen was observed also in all groups. A FACS analysis of allograft-recipient splenocytes showed no evidence for systemic lymphoid chimerism. BN or ACI skin grafts transplanted onto recipients of allogeneic and syngeneic small bowel grafts were rejected completely in 12-17 days, while the intestinal grafts remained functional. Immunohistologic evaluation of the allografts, using anti-BN class I and anti-Lewis class II monoclonal antibodies showed anti-BN staining on the epithelial and endothelial structures, whereas the mononuclear cells in the lamina propria stained positively with the anti-LEW monoclonal antibody. However, lymphoid depletion and scarring of Peyer's patches and mesenteric lymph nodes as well as focal obliterative mesenteric arteriopathy, indicative of an indolent chronic rejection, were observed. These data demonstrate that recipients of indefinitely surviving small bowel allografts remain immune competent and do not retain the intestinal graft on the basis of specific hyporesponsiveness to the donor antigens.


The effects of immunosuppressive drugs on the regulation of activation-induced apoptotic cell death in thymocytes.

This study investigated the effects of immunosuppressive drugs on the regulation of thymocyte sensitivity to clonal deletion via programmed cell death, or apoptosis. We have previously shown that TcR/CD3 cross-linking and intracellular stimuli that mimic TcR/CD3 cross-linking induce apoptosis in many immature thymocytes in the presence, but not in the absence, of cyclosporine (CsA). We have interpreted those results to suggest that TcR/CD3-associated signals induce a CsA-sensitive mechanism that protects the cells from activation-induced apoptosis. In the present study, we compared the effects of CsA, FK506, and rapamycin (RAP) on the regulation of thymocyte apoptosis. Optimal concentrations of CsA and FK506 augmented apoptosis to similar levels. However, FK506 was approximately 100-fold more potent than CsA in thymocytes, which parallels the relative potencies of these drugs in inhibiting mitogen-induced proliferation of mature T cells. In contrast to CsA and FK506, RAP did not exhibit substantial apoptosis-augmenting activity. However, RAP interfered with the activity of FK506. This pattern mirrors that of RAP in TcR/CD3-mediated signaling pathways in mature T cells. Together these results provide evidence (1) that CsA, FK506, and RAP can act on immature thymocytes, (2) that the mechanisms by which the drugs affect mature and immature T cell responses are similar, and (3) that immunosuppressive drug therapy may affect not only mature peripheral T cells but also developing immature thymic T cells.


The salutary effect of FK506 in ischemia-reperfusion injury of the canine liver.

The present study was designed to elucidate the effect of FK506 on 90 min of warm ischemia of the liver and reperfusion in 30 dogs. Three groups of animals were studied. Group 1 animals received FK (0.15 mg/kg/day) for three days prior to the ischemia and group 2 animals got 2 ml of saline solution for three days instead of FK and were considered controls. In group 3 FK (0.15 mg/kg/day) was injected immediately upon reperfusion and two days thereafter. Evaluation of the effectiveness of the drug was monitored by measuring the serum activities of AST, ALT, LDH, serum total bilirubin, malondialdehyde, and by histopathological examinations of the liver specimens and survival of the animals for 7 days after reperfusion. The 7 day survival of the animals in group 1 (80%) was significantly (P < 0.05) improved compared with those in group 2 (30%) and group 3 (20%). The serum activities of AST, ALT, and LDH and total bilirubin were significantly lower in group 1 than in group 2 and group 3. FK pretreatment significantly prevented hepatocellular necrosis and neutrophilic infiltration in group 1 in comparison with those in group 2 and group 3. Although the malondialdehyde level in hepatic venous blood was relatively lower in group 1, this difference was not statistically significant. Three days FK pretreatment prevented hepatocellular injury and enzyme leakage after 90 min of hepatic ischemia, whereas FK treatment immediately upon reperfusion failed to do so. In conclusion, donor organ pretreatment with FK may become a promising strategy for improved allograft survival in liver transplantation.


FK506-induced impairment of glucose metabolism in the primate--studies in pancreatic transplant recipients and in nontransplanted animals.

The effect of FK506 on glucose metabolism was studied in five cynomolgus monkeys after pancreatic transplantation and in 10 nontransplanted cynomolgus monkeys. We have clearly demonstrated that FK506 can induce hyperglycemia in these animals. In the orally treated nontransplanted animals the hyperglycemia was usually very mild (4.5-6.0 mmol/L). In one of the five transplanted animals, hyperglycemia was induced by the FK506 treatment, since histological signs of rejection were absent and since plasma glucose levels normalized on dose reduction. The glucose disappearance rates, as indicated by the K-values, decreased from a mean of 3.0 +/- 0.5%/min before FK506 treatment to 2.4 +/- 0.6%/min at one month and 1.5 +/- 0.4%/min at three months in the nontransplanted animals. In the transplant group, the K values decreased significantly from 4.2 +/- 0.6%/min in the donor animals to 1.4 +/- 0.4%/min at day 10 posttransplantation (P < 0.02). At one and three months postoperatively, the mean K-values were 1.4 +/- 0.2%/min and 1.2 +/- 0.6%/min, respectively. We conclude that FK506 is diabetogenic in the cynomolgus monkey. This side effect, however, was found to be reversible on dose reduction.


The alloantigen-specific immunosuppressive activity of estradiol-chlorambucil conjugate (KM2210) and its beneficial effect on allogeneic bone marrow transplantation in mice.

KM2210, a conjugate of estradiol and chlorambucil (CBL), which was originally developed as an anti-breast cancer agent, inhibits proliferative response of human mononuclear cells to alloantigens in mixed lymphocyte culture in a dose-dependent manner, but has no effect on their response to phytohemagglutinin. Neither estradiol benzoate nor CBL alone showed these unique actions. The suppressive effect of KM2210 on MLC was abrogated by adding of anti-transforming growth factor-beta (TGF-beta) antibody to the culture, but was not affected by the addition of interleukin-2, suggesting that KM2210, unlike CBL, displays its actions via TGF-beta. In experimental allogeneic bone marrow transplantation using mice, daily oral administration of KM2210 (2 mg/kg/day) for 30 days posttransplant significantly inhibited the alloantigen-specific immune reactions. Furthermore, the survival rate of the KM2210-treated mice was significantly higher than that of the cyclosporine-treated (2 mg/kg/day, p.o.) mice, and no adverse effect of KM2210 on hematopoietic recovery was found. These results strongly suggest possible clinical benefits of KM2210 as a new immunosuppressive agent for the prevention and treatment of graft-versus-host disease and other allospecific immune reactions.


Reduction of leukocyte adherence and emigration by cyclosporine and L683,590 (FK506) in postcapillary venules.

Although the actions of immunosuppressive agents on lymphocytes are well characterized, their influence on leukocyte-endothelial cell adhesive interactions has not been defined. The objective of this study was to determine whether cyclosporine and L-683,590 (FK520) could modify the adhesion and emigration of leukocytes in postcapillary venules that are exposed to inflammatory mediators such as platelet activating factor (PAF) and leukotriene B4 (LTB4). The rat mesentery was prepared for in vivo microscopic observation. Venules with internal diameters ranging between 25 microns and 35 microns were selected for study. Erythrocyte velocity, vessel diameter, leukocyte rolling velocity, and the number of adherent (stationary for > 30 sec) and emigrated leukocytes were measured during superfusion of the mesentery with bicarbonate-buffered saline. Repeat measurements of adhesive and hemodynamic parameters were obtained between 50 and 60 min of superfusion with either 100 nM PAF or 20 nM LTB4 added to bicarbonate-buffered saline. In some experiments, animals were treated with either cyclosporine or L-683,590. Both PAF and LTB4 caused increases in leukocyte adherence and emigration, and reductions in leukocyte rolling velocity and venular shear rate. Cyclosporine prevented all of the adhesive and hemodynamic alterations induced by PAF, but not LTB4. L-683,590 was effective in preventing the responses elicited by both PAF and LTB4. These results indicate that modulation of leukocyte-endothelial cell adhesive interactions may represent a novel mechanism of action for cyclosporine and L-683,590.


Role of the central auditory system in hearing: the new direction.

The mammalian central auditory system contains a large number of subcortical auditory nuclei, which were once thought to form a simple relay system, taking signals from the ear to the cortex where all information processing would have occurred. Now it appears that these subcortical nuclei are themselves responsible for the extraction and analysis of the dimensions of sounds. Not only do the nuclei encode dimensions defining the nature of the sound, but also they extract features of sound location. Three major nuclei in the superior olivary complex of mammals extract the horizontal direction of a sound source, and it seems likely that other nuclei in the auditory system encode elevation and distance. This shift in viewpoint away from the attributes of sound to the attributes of sound sources is an important new step in the investigation of the role of the central auditory system in hearing.


Neural lesioning with ribosome-inactivating proteins: suicide transport and immunolesioning.

Toxic lectins, plant proteins that inactivate ribosomes, irreversibly inhibit protein synthesis with high efficiency. After intraneural (subepineurial) microinjection, these agents are taken up by axons and are retrogradely transported to the perikarya, where they result in cell death. These 'suicide transport' toxins can produce pathway-specific lesions that are useful in several types of experiment, including cellular localization of neurotransmitter receptors. The toxins can be coupled to monoclonal antibodies to produce immunotoxins: reagents that can make highly selective lesions of specific types of neurons. Central or peripheral neurons that express the low-affinity NGF receptor are selectively destroyed by the immunotoxin 192 IgG-saporin. Development of other anti-neuronal immunotoxins should provide a variety of powerful selective lesioning tools.


G-protein cascades: gain and kinetics.

G-protein cascades provide amplification in a wide variety of biological signal transducers--from hormonal and synaptic systems to the receptor cells of vision and olfaction. Through recent understanding of the molecular mechanisms involved, it is possible to construct a quantitative description of the amplification and speed of response of the cascade. The gain and kinetics can now be described in terms of physical parameters, such as enzyme activities and the densities and lateral diffusion coefficients of the proteins involved.


Spatial memory and adaptive specialization of the hippocampus.

The hippocampus plays an important role in spatial memory and spatial cognition in birds and mammals. Natural selection, sexual selection and artificial selection have resulted in an increase in the size of the hippocampus in a remarkably diverse group of animals that rely on spatial abilities to solve ecologically important problems. Food-storing birds remember the locations of large numbers of scattered caches. Polygynous male voles traverse large home ranges in search of mates. Kangaroo rats both cache food and exhibit a sex difference in home range size. In all of these species, an increase in the size of the hippocampus is associated with superior spatial ability. Artificial selection for homing ability has produced a comparable increase in the size of the hippocampus in homing pigeons, compared with other strains of domestic pigeon. Despite differences among these animals in their histories of selection and the genetic backgrounds on which selection has acted, there is a common relationship between relative hippocampal size and spatial ability.


Calcium-binding proteins in the nervous system.

Among the many calcium-binding proteins in the nervous system, parvalbumin, calbindin-D28K and calretinin are particularly striking in their abundance and in the specificity of their distribution. They can be found in different subsets of neurons in many brain regions. Although it is not yet known whether they play a 'triggering' role like calmodulin, or merely act as buffers to modulate cytosolic calcium transients, they are valuable markers of neuronal subpopulations for anatomical and developmental studies.


Direct regulation of cardiac Ca2+ channels by G proteins: neither proven nor necessary?

The cardiac L-type Ca2+ channel has served as a model for ion channel regulation for over a decade. The Ca2+ current is increased by beta-adrenoceptor stimulation and this effect is inhibited by muscarinic acetylcholine receptor stimulation. It is well established that beta-adrenoceptor stimulation increases this current largely by cAMP-dependent phosphorylation but recently data have been presented that suggest that this channel may also be regulated directly by G proteins. This review by Criss Hartzell and Rodolphe Fischmeister evaluates evidence for this second regulatory pathway and concludes that, although G proteins affect cardiac Ca2+ channels in bilayers and excised patches, there is little evidence that this pathway is physiologically significant.


Galanin and galanin antagonists: molecular and biochemical perspectives.

The neuropeptide galanin potently inhibits insulin release, hippocampal acetylcholine release and firing of locus coeruleus cells, and stimulates feeding and release of growth hormone. Galanin regulates K+ channels, adenylyl cyclase and phospholipase C by acting at Gi/Go protein-coupled high-affinity receptors. Galanin receptor agonists such as the N-terminal fragment galanin1-16 act synergistically with morphine in the somatosensory system and have potential analgetic application. Galanin antagonists may be useful therapeutic agents in endocrinology, neurology and psychiatry. The enhancing effect of such agents on hippocampal cholinergic function would be useful in treatment of Alzheimer's disease. Recent synthesis of a series of high-affinity galanin antagonists, reviewed, along with galanin's actions, by Tamas Bartfai and colleagues, opens the possibility of examining the functions of endogenous galanin and test the pharmacological usefulness of antagonism of galanin function in the endocrine, somatosensory and central nervous systems.


[Loop ileostomy--the first choice for relief of colon and rectum].

In numerous comparative investigations, loop-ileostomy has proved superior to transversostomy. The operative technique is described and experience from 27 loop-ileostomies carried out in a department for organ surgery are reviewed. The qualities of the method as compared with transversostomy are discussed on the basis of the literature and the authors' own results. It is concluded that loop-ileostomy may, in general, be preferred for temporary and palliative relief of the colon.


[Diagnosis and therapy of metastasis-induced pathologic fractures].

Improved survival rates of cancer patients have led to an increase in the incidence of metastatic disease of the bone. Normal load and minimal trauma may result in pathological fractures. The malignant diseases most commonly diagnosed were breast cancer, bronchial carcinoma and hypernephroma. The majority of the patients treated were female. The average interval observed between diagnosis of primary malignant disease and occurrence of the pathological fracture was 2.8 years. The purpose of the surgical procedure is to achieve immediate and lasting stability and ultimately to increase and restore the quality of life. Immediate postoperative mobilization and early functional treatment are an indispensable part of the management of pathologic fractures. If possible extensive bone destructions involving the risk of fracture should be stabilized prophylactically. Specific techniques of composite osteosyntheses of fractures in metastatic disease of the bone are presented.


Temperature steering in prostate by simultaneous transurethral and transrectal hyperthermia.

Localized hyperthermia (HT) is presently under investigation as a treatment for benign prostatic hyperplasia and carcinoma of the prostate (CaP). One popular approach employs a transrectal (TR) device, a directional microwave (MW) applicator inserted into the rectum and aimed at the prostate. Alternatively, in the transurethral (TU) technique, a symmetrically radiating MW antenna is placed directly within the prostatic urethra. Used individually, TR applicators are capable of effectively heating (> 42 degrees C) the prostate up to 2 cm from the rectum, whereas TU applicators selectively heat the periurethral tissue with effective radial penetration of about 0.6 cm. Neither technique is of much value in heating the anterior prostate. In general, the highest temperatures are produced in the tissue immediately adjacent to the surface of intracavitary microwave devices. However, when MW antennas are used in arrays, the resulting heating pattern can differ significantly from that of the individual antennas. Heating at depth can be selectively enhanced and "steered" by adjusting the phase relationship between the devices. Prostatic temperature profiles were measured in 6 patients treated with TR alone, TU alone, and simultaneous TR and TU heating. In the combined treatments different phase relationships between the antennas were applied. We found that a higher temperature could be produced in the center of the prostate than on the surface of either applicator for certain phase relationships, and that the temperature profiles could be changed by shifting phase. The results of these measurements are in agreement with those of a computer simulation. Based on the above data we feel the combined use of TU and TR hyperthermia may be justified in Phase I-II trials for patients with locally advanced CaP.


Local staging of prostate cancer by tumor volume, prostate-specific antigen, and transrectal ultrasound.

Conventional methods of staging prostate tumors are highly inaccurate. To improve clinical staging, prostate-specific antigen (PSA) levels (> 10 ng/mL), sonographic tumor volume (> 3 cc), maximum tumor diameter, length of capsular tumor abutment, and overall impression of capsular irregularity suggesting periprostatic tumor spread were assessed in 29 men prior to undergoing radical prostatectomy for clinically localized tumor. After surgery, 18 men had tumor confined to the prostate, while 11 men had histologic evidence of extracapsular disease. Analysis of the parameters measured showed these were the most helpful factors in predicting the presence of extracapsular disease. However, the positive and negative predictive values were only 70 to 90 percent. Therefore, the clinical usefulness of any one measurement alone in determining treatment for the individual patient is limited. However, combining these parameters yields an improved prediction of extracapsular disease. All 6 patients with PSA < 10 ng/mL, tumor volume < 3 cc, and no capsular irregularity on ultrasound had localized disease (neg. predictive value = 100%), while all 7 patients who had more than one of these parameters had extracapsular disease (pos. predictive value = 100%). Thus, using the factors in combination may provide more accurate staging and thereby help in counseling patients regarding therapy.


Relationship of response to transurethral hyperthermia and prostate volume in BPH patients.

A response to transurethral microwave hyperthermia (TUHT) at 915 MHz and its relationship to prostate volume was examined in 63 poor surgical risk benign prostatic hyperplasia (BPH) patients. All patients had moderate-to-severe obstructive signs and symptoms, and received > or = 5 TUHT one-hour sessions. Treatment temperature was controlled on the urethral surface at 45 degrees C +/- 1 degree C. Follow-up ranged from twelve to forty-four months (mean 18 months). The mean prostate volume was 57 cc (range 10-301 cc). There were 40 patients (63%) with prostate volume < or = 50 cc and 23 (37%) with a volume > 50 cc. Treatment failure was seen in 6 patients (10%). It was 10 percent in 40 patients with smaller glands and 9 percent for those 23 with larger prostates, N.S. at p = 0.49. Subjective treatment response was seen in 58 patients (92%). It was 90 percent for the 40 patients with < or = 50 cc prostates vs. 96 percent for the 23 with > 50 cc prostates, N.S. at p = 0.75. This study suggests that the initial prostate volume is not an important parameter predicting response to TUHT.


Distribution of cytokeratins, vimentin and desmoplakins in normal renal tissue, renal cell carcinomas and oncocytoma as revealed by immunofluorescence microscopy.

Forty-two renal cell carcinomas, one oncocytoma and normal renal tissue were studied for the presence of cytokeratins and vimentin. The investigations were performed by immunofluorescence microscopy applying a panel of mono- and polyclonal antibodies to intermediate filament proteins. In all tumours except chromophobic renal cell carcinoma (CRCC) and oncocytoma a co-expression of cytokeratins and vimentin could be shown. The intermediate filament expression was often, however, very heterogeneous particularly with respect to the distribution of cytokeratins and vimentin, to the clonality of the antibodies used and to the tumour areas studied. The latter could be impressively demonstrated by examining a whole tumour. In CRCC and oncocytoma all tumour cells expressed cytokeratins and, in addition, single tumour cells also expressed vimentin. In normal renal tissue we could show vimentin-positive epithelia of proximal and distal tubules, which is reported for the first time.


[The effect of renal artery embolization on the results of treating kidney cancer patients].

To analyze the prognosis, 401 renal cancer cases were examined retrospectively and prospectively. There were 157 females and 244 males. Preoperative embolization of the renal artery resulted in longer survival: in stage T3 it increased from 44 to 60%, in stage T4 from 7 to 50%. After palliative nephrectomy the patients' mean survival reached 10.6 months against 16 months following palliative embolization of the kidney. Palliative embolization is thought the most effective intervention in inoperable cases of renal carcinoma.