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Background: Among all hematologic malignancies, B-cell chronic lymphocytic leukemia (BCLL) has the highest familial clustering (three- to sevenfold increase), strongly suggesting a genetic component to its etiology. Familial BCLL can be used as a model to study the early pathogenesis of this disease., Methods: We examined nine kindreds from the National Cancer Institute's Familial BCLL Registry, consisting of 19 affected members with BCLL and 33 clinically unaffected first-degree relatives. Flow cytometric immunophenotyping to detect a B-cell monoclonal lymphocytosis (BCML) was performed. Monoclonality was confirmed by polymerase chain reaction analysis of whole blood DNA. Cell cycle analysis for aneuploidy was conducted., Results: In all affected individuals, we observed the classic BCLL CD5/CD19/CD20/CD23 immunophenotypic patterns. Six of the 33 unaffected individuals (18%) had evidence of BCML. Additional individuals (13/33, 39%) showed some other abnormality, whereas 14 individuals (42%) were normal. Based on an estimated prevalence of 0.7% for BCML in the general population, the finding of six subjects (18%) with clonal abnormalities in this relatively modest sample was significantly greater than expected (i.e., 18% vs. 0.7%, P < 5.7 x 10(-9))., Conclusions: Individual components of BCML and other B-cell abnormalities were observed in almost half of the apparently unaffected individuals. Our findings suggested that BCML may be an early detectable abnormality in BCLL. The spectrum of some of these observed abnormalities suggested the involvement of different B-cell subpopulations or different pathways in clonal evolution. Population-based, longitudinal studies will be required to determine the incidence of BCML and other B-cell abnormalities and their relation to disease progression in BCLL and other closely related B-cell lymphoproliferative disorders., (Published 2003 Wiley-Liss, Inc.)

We have investigated the role of the double-stranded RNA-dependent protein kinase gene (pkr) in the regulation of the heat shock response. We show that the pkr gene is essential for efficient activation of the heat shock response and that pkr disruption profoundly inhibits heat shock protein 70 (HSP70) synthesis and blocks the development of thermotolerance. Despite these profound effects, pkr disruption did not markedly affect the activation of heat shock factor 1 by heat and did not reduce the rate of transcription of the HSP70 gene after heat shock. However, despite the lack of effect of pkr disruption on HSP70 gene transcription, we found a significant decrease in the expression of HSP70 mRNA in pkr-/- cells after heat shock. Kinetic studies of mRNA turnover suggested a block in the thermal stabilization of HSP70 mRNA in pkr-/- cells. As the thermal stabilization of HSP70 mRNA is thought to involve cis-acting A+U rich (ARE) elements in the 3'-untranslated region (UTR), we examined a potential role for pkr in this process. We found that a reporter beta-galactosidase mRNA destabilized by introduction of a functional ARE into the 3'-UTR became stabilized by heat but only in cells containing an intact pkr gene. Our studies suggest therefore that pkr plays a significant role in the stabilization of mRNA species containing ARE destruction sequences in the 3'-UTR and through this mechanism, contributes to the regulation of the heat shock response and other processes.

Multidrug-resistant opportunistic pathogens have become endemic to the veterinary hospital environment. Escherichia coli isolates resistant to 12 antibiotics were isolated from two dogs that were housed in the intensive care unit at The University of Georgia Veterinary Teaching Hospital within 48 h of each other. Review of 21 retrospective and prospective hospital-acquired E. coli infections revealed that the isolates had similar antibiotic resistance profiles, characterized by resistance to most cephalosporins, beta-lactams, and the beta-lactamase inhibitor clavulanic acid as well as resistance to tetracycline, spectinomycin, sulfonamides, chloramphenicol, and gentamicin. E. coli isolates with similar resistance profiles were also isolated from the environment in the intensive care unit and surgery wards. Multiple E. coli genetic types were endemic to the hospital environment, with the pulsed-field gel electrophoresis fingerprint identified among E. coli isolates from diseased animals and the hospital environment matching. The extended-spectrum cephalosporin resistance in these nosocomial E. coli isolates was attributed to the cephamycinase-encoding gene, bla(CMY2). Chloramphenicol resistance was due in part to the dissemination of the florfenicol resistance gene, flo, among these isolates. Resistance encoded by both genes was self-transmissible. Although bla(CMY2) and flo were common to the polyclonal, nosocomial E. coli isolates, there was considerable diversity in the genetic compositions of class 1 integrons, especially among isolates belonging to the same genetic type. Two or more integrons were generally present in these isolates. The gene cassettes present within each integron ranged in size from 0.6 to 2.4 kb, although a 1.7-kb gene cassette was the most prevalent. The 1.7-kb gene cassette contained spectinomycin resistance gene aadA5 and trimethoprim resistance gene dfrA17.

The excess risk of second malignancy after Hodgkin disease is an increasing problem. In light of the long-term data, guidelines for follow-up of survivors of Hodgkin disease need to be redefined. In this study we attempt to analyze the long-term risks and temporal trends, identify patient- and treatment-related risk factors, and determine the prognosis of patients who develop a second malignancy after radiation treatment with or without chemotherapy for Hodgkin disease. Among 1319 patients with clinical stage I-IV Hodgkin disease, 181 second malignancies and 18 third malignancies were observed. With a median follow-up of 12 years, the relative risk (RR) and absolute excess risk of second malignancy were 4.6 and 89.3/10 000 person-years. The RR was significantly higher with combined chemotherapy and radiation therapy (6.1) than with radiation therapy alone (4.0, P =.015). The risk increased with increasing radiation field size (P =.03) in patients who received combined modality therapy, and with time after Hodgkin disease. After 15 and 20 years, there was a 2.3% and 4.0% excess risk of second malignancy per person per year. The 5-year survival after development of a second malignancy was 38.1%, with the worst prognosis seen after acute leukemia and lung cancer. The excess risk of second malignancy after Hodgkin disease continues to be increased after 15 to 20 years, and there does not appear to be a plateau. Our analysis suggests that the risk may be reduced with smaller radiation fields, as are used in current trials of abbreviated chemotherapy and limited-field radiation therapy.

Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca(2+) flux, activate NF-kappaB, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-kappaB signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).

Heat shock factor (HSF) 1 is the major heat shock transcription factor that regulates stress-inducible synthesis of heat shock proteins and is also essential in protection against endotoxic shock. Following our previous study, which demonstrated the transcriptional repression of the IL-1beta gene by HSF1 (Cahill, C. M., Waterman, W. R., Xie, Y., Auron, P. E., and Calderwood, S. K. (1996) J. Biol. Chem. 271, 24874-24879), we have examined the mechanisms of transcriptional repression. Our studies show that HSF1 represses the lipopolyliposaccharide-induced transcription of the IL-1beta promoter through direct interaction with the nuclear factor of interleukin 6 (NF-IL6, also known as CCAAT enhancer binding protein (C/EBPbeta), an essential regulator in IL-1beta transcription. We show for the first time that HSF1 binds directly to NF-IL6 in vivo and antagonizes its activity. The HSF1/NF-IL6 interaction involves a sequence of HSF1 containing the trimerization and regulatory domains and the bZip region of NF-IL6. HSF1 has little effect on IL-1beta promoter activity stimulated by the essential monocytic transcription factor Spi.1 but is strongly inhibitory to transcriptional activation by NF-IL6 and to the synergistic activation by NF-IL6 and Spi.1. Because of its ability to bind to specific C/EBP elements in the promoters of multiple genes and its ability to interact with other transcription factors, NF-IL6 is involved in transcriptional regulation of a wide range of genes. Interaction between HSF1 and NF-IL6 could thus be an important mechanism in HSF1 regulation of general gene transcription during endotoxin stress.

We have examined the functional antagonism between the regulator of the heat shock response, HSF1, and NF-IL6, which plays a major role in control of the acute phase response (APR). Agents that activate HSF1 such as heat shock and sodium salicylate inhibit NF-IL6 induced transcription while NF-IL6 activators such as lipopolysaccharide (LPS) and interleukin 6 (IL-6) repressed the stress responsive HSP70B promoter. In transfection studies, the inhibitory effects of HSF1 and NF-IL6 on the c-fms promoter were shown to be highly dose-dependent. Furthermore, heat shock is inhibitory to differentiation-linked expression of macrophage colony stimulating factor (M-CSF) receptor, product of the c-fms gene, which is transcriptionally activated by NF-IL6 but repressed by HSF1. Our studies suggest a strong mutual antagonism between the heat shock response and APR, which may influence the sensitivity and duration of inflammatory responses.

This study reports the subjective, psychomotor and physiological properties of subanaesthetic concentrations of xenon. Ten healthy male volunteers received either xenon or nitrous oxide in a randomised crossover study design. The subjects breathed either xenon (Xe) or nitrous oxide (N2O) from a closed circuit breathing system, according to a randomised, double-blind protocol. The concentration of xenon required to produce sedation, ranged between 27 and 45% (median 35%). All subjects completed the xenon protocol. Subjects were tested using the Critical Flicker Fusion test and derived electroencephalogram parameters, however, neither test was found to reliably predict sedation. The respiratory rate decreased markedly during sedation with xenon. The subjects did not experience any airway irritability (coughing, breath-holding or laryngospasm) during administration of either gas. One subject required anti-emetic treatment in the N2O group compared to none in the Xe group. Eight subjects reported that they found sedation with xenon pleasant and preferable to nitrous oxide. Xenon sedation was well tolerated and was not associated with any adverse physiological effects, however, it was reported to be subjectively dissimilar to nitrous oxide.

A unilateral intravesical ureterocele was diagnosed by ultrasonography in a 5-year-old female Pekingese that was referred for evaluation of increased hepatic enzymes. Ureteroceles are cystic dilatations of the submucosal portion of the distal ureter. They are frequently reported in humans but are uncommonly reported in dogs. This report describes surgical resection of the ureterocele and reduction of ipsilateral hydroureter in a dog that also had bilateral renal dysfunction and suffered progressive mild azotemia postoperatively. This report demonstrates that canine ureteroceles can occur concurrently with bilateral renal dysfunction and offers a classification system designed to encourage thorough urinary tract evaluation for determining prognosis.

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G(0/1) doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G(2) + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G(0/1) doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G(0/1) doublets and G(2) + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult., (Published 2001 Wiley-Liss, Inc.)

Objective: To determine whether a group of 3 genetic differences in the nonstructural protein (NS1) or 1 genetic difference in the structural protein (VP2) of Aleutian disease parvovirus (ADV) is responsible for an increase in the in vivo replication and pathogenicity of G/U-8, a chimera of ADV-G (nonpathogenic) and ADV-Utah (pathogenic), compared with G/U-10., Animals: 32 eight-month-old female sapphire mink (Mustela vison)., Procedure: Chimeric viruses were constructed, propagated in vitro, and used to inoculate mink. Antiviral antibody responses, presence of serum viral nucleic acid, and serum gamma globulin concentrations were monitored for 120 days following inoculation. Histologic examination of the liver, kidneys, spleen, and mesenteric lymph nodes was performed after necropsy., Results: A chimera containing only the 3 amino acid substitutions in NS1 did not elicit measurable responses indicative of replication or pathogenicity in inoculated mink. Serum antiviral antibody responses, frequency of detection of viral nucleic acid in serum, gamma globulin response, and histologic changes in mink inoculated with chimeras containing a valine residue at codon 352 (352V) of VP2 capsid were increased, compared with values from mink inoculated with chimeric viruses that did not contain 352V., Conclusions and Clinical Relevance: A valine residue at codon 352 in the VP2 capsid protein of ADV affects in vivo viral replication and pathogenicity. This amino acid may be part of an incompletely defined pathogenic determinant of ADV. Further characterization of the pathogenic determinant may allow future development of focused preventive and therapeutic interventions for Aleutian disease of mink.

It has previously been demonstrated that hyperthermia can activate prostaglandin synthesis and that prostaglandins are protective against hyperthermia. This study examined the use of inhibitors of prostaglandin synthesis on the response of prostate tumours to hyperthermia. The non-steroidal anti-inflammatory drugs (NSAID) ibuprofen and sulindac, known cyclooxygenase inhibitors that inhibit prostaglandin production, were effective hyperthermia sensitizers and augmented growth delay of DU-145 and PC-3 prostate tumours to combined radiation and hyperthermia treatment protocols. Pre-treatment of mice with ibuprofen and sulindac at hyperthermia sensitizing doses resulted in significant (p < 0.01) inhibition of hyperthemia-induced serum prostaglandin E2. These findings indicate that NSAID may have both sensitizing effects on prostate tumour growth and may function by inhibiting prostaglandin synthesis.

A spatial simulation model of foot-and-mouth disease was used in March and early April 2001 to evaluate alternative control policies for the 2001 epidemic in Great Britain. Control policies were those in operation from March 20, 2001, and comprised a ban on all animal movements from February 23, 2001, and a stamping-out policy. Each simulation commenced with the known population of infected farms on April 10, 2001, and ran for 200 days. For the control policy which best approximated that actually implemented from late March, the model predicted an epidemic of approximately 1800 to 1900 affected farms, and estimated that the epidemic would be eradicated between July and October 2001, with a low probability of continuing beyond October 2001. This policy included the slaughter-out of infected farms within 24 hours, slaughter of about 1.3 of the surrounding farms per infected farm within a further 48 hours, and minimal interfarm movements of susceptible animals. Delays in the slaughter of animals on infected farms beyond 24 hours after diagnosis slightly increased the epidemic size, and failure to achieve pre-emptive slaughter on an adequate number of at-risk farms substantially increased the expected size of the epidemic. Vaccination of up to three of the most outbreak-dense areas carried out in conjunction with the adopted control policy reduced the predicted size of the epidemic by less than 100 farms. Vaccination of buffer zones (designed to apply available vaccine and manpower as effectively as possible) carried out in place of the adopted control policy allowed the disease to spread out of control, producing an epidemic involving over 6000 farms by October 2001, with no prospect of immediate eradication.

Tumour hyperthermia, although potentially a powerful therapeutic agent and radiation sensitizer, is hindered by a number of considerations including inhomogeneous heating of deep seated tumours due to energy deposition and perfusion issues. One solution is to design hyperthermia sensitizers to amplify the effects of hyperthermia, particularly at cold spots within the tumour undergoing treatment. This study examined the use of Quercetin, a flavonoid drug shown previously to antagonize the expression of HSP72 and induce apoptosis as a sensitizer of prostate cancer growth in vivo. Quercetin dose-dependently suppressed PC-3 tumour growth in vitro and in vivo. When combined in a treatment protocol with hyperthermia, quercetin drastically inhibited tumour growth and potently amplified the effects of hyperthermia on two prostate tumour types, PC-3 and DU-145 in vivo. These experiments, thus, suggest the use of Quercetin as a hyperthermia sensitizer in the treatment of prostate carcinoma.

Objective: To compare in vitro replication kinetics and nucleoside analog susceptibilities of a natural feline immunodeficiency virus (FIV) isolate (FIV-Maxam), a molecular clone of FIV (FIV-pPPR), and two (-)-beta-L-2',3'-dideoxy-3'-thiacytidine- (3TC-) resistant mutants of FIV-pPPR., Sample Population: Peripheral blood mononuclear cells (PBMC) from 4 specific-pathogenfree cats., Procedure: Two point mutations corresponding to mutations of human immunodeficiency virus type 1 (HIV-1) were engineered into the highly conserved YMDD motif of the reverse transcriptase- (RT-) encoding region of the FIV-pPPR pol gene. Replication kinetics and nucleoside analog susceptibilities of FIV-Maxam, FIV-pPPR, and the 2 mutant viruses were measured in vitro, using feline PBMC., Results: Replication kinetics and nucleoside analog susceptibilities were similar between FIV-Maxam and FIV-pPPR. However, FIV-Maxam was significantly more susceptible to 3TC. A methionine-to-valine mutation at codon 183 (M183V) of the RT-encoding region of the pol gene of FIV-pPPR conferred high-level phenotypic resistance to 3TC and cross-resistance to the related compound (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine., Conclusions and Clinical Relevance: Similarities between FIV-Maxam and FIV-pPPR suggest that results of studies performed using FIV-pPPR will have relevance to natural FIV infection in cats. In vitro evaluation of nucleoside analog susceptibilities of FIV-Maxam may help determine concentrations of nucleoside analogs required for effective treatment of FIV-infected cats., Impact for Human Medicine: 3TC resistance of FIV-pPPR M183V was similar in magnitude to that of HIV-1 M184V, a mutant described in infected humans treated with 3TC. Thus, FIV-pPPR M183V may be a useful model for studying the in vivo effects of 3TC resistance on lentivirus pathogenesis.

LA7 rat mammary tumor cells stimulate the proliferation, in culture, of three normal epithelial cell types, namely mouse mammary, rat mammary, and mouse thymic cells. Gap-junctional communication between LA7 feeders and mouse mammary cells was demonstrated by microinjection of lucifer yellow, which traveled from LA7 to the surrounding mouse mammary cells. The amount of 3H-uridine exchange between feeder and recipient mouse mammary, rat mammary, and mouse thymus cells correlated with the growth rate induced by the feeders. Cells of the Madin Darby canine kidney (MDCK) line, which do not appreciably stimulate mouse mammary cell growth when used as feeder cells, also exchange little 3H-uridine with them. Expression of connexins Cx43, 32, and 26 was studied in all these cell lines and strains by immunocytochemistry. Mouse mammary cells expressed Cx26, and a few mouse thymic cells expressed Cx32. LA7, mouse mammary, mouse thymic, and rat mammary cells all expressed easily detectable amounts of the gap-junction protein Cx43, in contrast to MDCK cells, which expressed only a hint of the protein. These results suggest that gap junctions composed of Cx43 are those by which the normal epithelial cells communicate with the LA feeders. Thus, the ability of feeder cells to stimulate proliferation in recipients correlates with the expression of Cx43 in both members of the feeder/recipient pair and the capacity to form functional gap junctions between these cells.

We have studied mechanisms of HSP70 gene regulation at 37 degrees C by the cellular factors NF-IL6 and Ku70. As both factors repress HSF1, we first examined whether phosphorylation on serine 303 and 307 of HSF1 by MAPK and GSK3, which has known to inhibit HSF1, was involved in the repression. However, repression by NF-IL6 was found using HSF1 mutants S303G and S307G refractory to the effects of MAPK and GSK3. We then examined whether NF-IL6 repressed HSP70B by a mechanism resembling Ku proteins. However, in Ku-deficient cells, NF-IL6 was still able to displace HSF1 from heat shock element (HSE) and repressed HSF1 activation. In addition, activation of the HSP 70B promoter by wild type, S303G, or S307G HSF1 was observed to be much more pronounced in Ku-deficient cells. In vitro translated Ku70 interacted with HSF1 by binding to and displacing it from HSE. These data indicate that the repression of the HSP70B promoter by NF-IL6, Ku70, and MAPK occurs independently of each other and involves three complementary mechanisms., (Copyright 2001 Academic Press.)

HSF-1 is regulated at multiple molecular levels through intra- and intermolecular protein-protein interactions as well as by post-translational modification through phosphorylation. We have found that elevating intracellular calcium ion levels by exposure to the ionophore A23187 or thapsigargin inhibits the conversion of HSF-1 from a latent cytoplasmic form to its nuclear/DNA binding form. To examine a role for calcium/calmodulin regulated enzymes in this process, we examined the ability of specific inhibitors to abrogate the effects of calcium elevation. While the inhibitor of calmodulin dependent kinase II, KCN62 enhanced activation of HSF-1 during heat shock, it failed to block the inhibitory effects of calcium increase. By contrast, the immunosuppresant drugs cyclosporin A and FK506 abolished the effects of calcium elevation on HSF-1 activation. As the biological effects of the drugs are effected through inhibition of the calcium/calmodulin regulated phosphatase calcineurin, this suggests a role for calcineurin in antagonizing HSF-1 activity. The experiments suggest the existence of phosphorylated residue(s) in HSF-1 important in one or more of the processes that lead to activation (trimerization, nuclear localization, DNA binding) and which becomes dephosphorylated due to the activation of a calcium/calmodulin/calcineurin complex.

Heat shock transcription factor 1(HSF1) activation is a multistep process. The conversion of a latent cytoplasmic form to a nuclear, DNA binding state appears to be activated by nonsteroidal anti-inflammatory drugs. In previous studies, we showed that HSF 1 is phosphorylated by the protein kinase RSK2 in vitro and that this effect is inhibited by nonsteroidal anti-inflammatory drugs at the concentration that leads to the activation of HSF1 in vivo (Stevenson et al 1999). In the present study, using cells from a patient with Coffin-Lowry syndrome (deficient in RSK2), we demonstrate that RSK2 slightly represses activation of HSF1 in vivo at 37 degrees C. In Coffin-Lowry syndrome cells, HSF1-HSE DNA binding activity after treatment with sodium salicylate was slightly higher than that in untreated cells, indicating that although RSK2 is involved in HSF1 regulation, it is not the unique protein kinase that suppresses HSF1-HSE binding activity at 37 degrees C. However, heat shock treatment resulted in significantly higher HSF1-HSE binding activity in Coffin-Lowry syndrome cells as compared with normal controls, suggesting that RSK2 represses HSF1-HSE binding activity during heat shock.

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-kappaB activity and subsequent augmentation of proinflammatory cytokine TNF-alpha, IL-1beta, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

This was a spatial analysis of the epidemic of bovine spongiform encephalopathy (BSE) in Great Britain, based on agricultural census data collected between 1986 and 1996 and BSE case data collected up to June 1997. Kernel smoothing techniques were used to plot the distribution of BSE-positive cattle holdings per 100 holdings per square kilometre and the distribution of confirmed BSE cases per 100 head of cattle per square kilometre. In the early stages of the epidemic reported BSE cases were scattered widely throughout Great Britain, with no clearly identifiable focus. By June 1997, a statistically significant cluster of BSE-positive holdings was identifiable in the eastern part of the South west region of England. During the epidemic the highest densities of confirmed BSE cases per 100 cattle per square kilometre occurred in the greater part of the South west region of England and within Dyfed in the south west of Wales. In Wales, a small number of holdings experienced large numbers of confirmed BSE cases. In the South west region of England a large number of holdings experienced small numbers of confirmed cases. By June 1997, the distribution of BSE-positive holdings across Great Britain was largely determined by factors that influenced the amount of recycled infectious material they were exposed to.

The objectives of this study were first to determine the cumulative incidence of bovine spongiform encephalopathy (BSE) in the British cattle population from July 1986 to June 1997, secondly, to identify individual animal-associated risk factors that influenced the age of onset of clinical signs in confirmed BSE cases, and, thirdly, to assess the effectiveness of the measures introduced to control BSE during the epidemic. The analyses were based on the population of British cattle at risk, derived from agricultural census data collected between 1986 and 1996, and BSE case data collected up to June 30, 1997. The unit of interest was individual adult cattle recorded on annual agricultural censuses between June 1986 and June 1996. Univariate and multivariate survival analysis techniques were used to characterise the age of onset of clinical signs. In total 167,366 cases of BSE were diagnosed in Great Britain up to June 30, 1997. The cumulative incidence of BSE between July 1986 and June 1997 was 1.10 (95 per cent confidence interval [CI] 1.09 to 1.10) cases per 100 adult cattle at risk. Cattle from the South east, South west and Eastern regions of England had 4.26 to 5.96 (95 per cent CI 4.15 to 6.14) times as great a monthly hazard of being confirmed with BSE as cattle from Scotland. Compared with cattle born before June 1985, those born between July 1987 and June 1988 had 22.5 (95 per cent CI 22.1 to 22.8) times the monthly hazard of being confirmed with BSE, whereas those born in the 12 months after July 1988 had only 7.39 (95 per cent CI 7.24 to 7.54) times the monthly hazard of being confirmed with BSE. This reduction in hazard was directly attributable to the ban on the use of ruminant protein as a feed instituted in July 1988. Successive cohorts from 1989 to 1991 experienced further reductions in the hazard of experiencing BSE. The additional decrease in hazard observed for the 1990 cohort may be attributed to the effect of the Specified Bovine Offal ban instituted in September 1990.

The objectives of this study were first to describe the pattern of the epidemic of bovine spongiform encephalopathy (BSE) in Great Britain in terms of the temporal change in the proportion of all cattle holdings that had experienced at least one confirmed case of BSE to June 30, 1997, and secondly to identify risk factors that influenced the date of onset of a holding's first confirmed BSE case. The analyses were based on the population of British cattle at risk, derived from agricultural census data collected between 1986 and 1996, and the BSE case data collected up to June 30, 1997. The unit of interest was the cattle holding and included all those recorded at least once on annual agricultural censuses conducted between June 30, 1986, and June 30, 1996. The outcome of interest was the date on which clinical signs were recorded in a holding's first confirmed case of BSE, termed the BSE onset date. Univariate and multivariate survival analysis techniques were used to describe the temporal pattern of the epidemic. The BSE epidemic in Great Britain started in November 1986, with the majority of affected holdings having their BSE onset date after February 1992. After adjusting for the effect of the size and type of holding, holdings in the south of England (specifically those in the Eastern, South east and South west regions) had 2.22 to 2.43 (95 per cent confidence interval [CI] 2.07 to 2.58) times as great a monthly hazard of having a BSE index case as holdings in Scotland. After adjusting for the effect of region and type of holding, holdings with more than 53 adult cattle had 5.91 (95 per cent CI 5.62 to 6.21) times as great a monthly hazard of having a BSE index case as holdings with seven to 21 adult cattle. Dairy holdings had 3.06 (95 per cent CI 2.96 to 3.16) times as great a monthly hazard of having a BSE index case as beef suckler holdings. These analyses show that there were different rates of onset in different regions and in holdings of different sizes and types, that the epidemic was propagated most strongly in the south of the country, and that the growth of the epidemic followed essentially the same pattern in each region of the country, with modest temporal lags between them. The control measures imposed in 1988 and 1990 brought the expansion of the epidemic under control, although the rate of progress was slowed by those regions where the effectiveness of the control methods took some time to take full effect.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

The purpose of this study was to determine clinical disease incidence in eight non-seasonally calving, pasture-fed dairy herds in the southern highlands district of New South Wales. This was a longitudinal population study. The study included all cows that calved between January 1994 and December 1995 and consisted of 2111 lactation records from 1430 cows. The incidence of the more common diseases were: calving-associated disorders, 18.0 cases per 100 calvings (95% CI 16.4-19.8 cases per 100 calvings); metabolic disorders, 5.5 cases per 100 cow-yr at risk (95% CI 4.5-6.6 cases per 100 cow-yr at risk); reproductive-tract disorders, 22.3 cases per 100 cow-yr at risk (95% CI 19.2-25.8 cases per 100 cow-yr at risk); udder disorders, 17.6 cases per 100 cow-yr at risk (95% CI 15.9-19.5 cow-yr at risk) and lameness, 3.7 cases per 100 cow-yr at risk (95% CI 2.9-4.7 cow-yr at risk). In agreement with dairy-cow disease-incidence studies conducted elsewhere, disorders of the reproductive-tract and udder were the most frequent clinical conditions encountered. These findings confirm that dairy herd-health programs should emphasise the control of these two groups of disorders.

Sodium salicylate (NaSal) and other nonsteroidal anti-inflammatory drugs (NSAIDs) coordinately inhibit the activity of NF-kappa B, activate heat shock transcription factor 1 and suppress cytokine gene expression in activated monocytes and macrophages. Because our preliminary studies indicated that these effects could be mimicked by inhibitors of signal transduction, we have studied the effects of NSAIDs on signaling molecules potentially downstream of LPS receptors in activated macrophages. Our findings indicate that ribosomal S6 kinase 2 (RSK2), a 90-kDa ribosomal S6 kinase with a critical role as an effector of the RAS-mitogen-activated protein kinase pathway and a regulator of immediate early gene transcription is a target for inhibition by the NSAIDs. NSAIDs inhibited the activity of purified RSK2 kinase in vitro and of RSK2 in mammalian cells and suppressed the phosphorylation of RSK2 substrates cAMP response element binding protein (CREB) and I-kappa B alpha in vivo. Additionally, NaSal inhibited the phosphorylation by RSK2 of CREB and I-kappa B alpha on residues crucial for their transcriptional activity in vivo and thus repressed CREB and NF-kappa B-dependent transcription. These experiments suggest that RSK2 is a target for NSAIDs in the inhibition of monocyte-specific gene expression and indicate the importance of RSK2 and related kinases in cell regulation, indicating a new area for anti-inflammatory drug discovery.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.

Recent studies have shown that the non-steroidal anti-inflammatory drugs (NSAIDs) activate heat shock transcription factor (HSF1) from a latent cytoplasmic form to a nuclear, DNA binding state. As HSF1 can function as both an activator of heat shock genes and a repressor of non-heat shock genes such as IL1B and c- fos, we have examined the potential role of HSF1 in the effects of NSAIDs on gene expression in a human monocytic cell line THP-1. We found that two members of the NSAIDs, sodium salicylate and sulindac repress the IL1B promoter to similar degree to heat shock or HSF1 overexpression. In addition, sodium salicylate and additional NSAIDs used at concentrations that activate HSF1 also inhibited the expression of other monocytic genes (TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, ICAM-1) activated by exposure to a pro-inflammatory stimulus (lipopolysaccharide, LPS). At least in the case of the IL1B promoter, repression did not seem to involve another factor whose activity is affected by the NSAIDs, NFkappaB as the IL1B promoter fragment used in our studies is not NFkappaB responsive and binds specifically to HSF1. Exposure to NSAIDs had a complex effect on HSP gene expression and while sulindac activated the stress responsive HSP70B promoter, sodium salicylate did not. In addition, only a subset of the NSAIDs induced HSP70 mRNA species. These findings reflect the properties of HSF1 which can be activated to at least two DNA binding forms only one of which activates heat shock promoters and suggest that individual NSAID family members may differentially induce one or other of these forms. Overall therefore, exposure to NSAIDs leads to a profound switch in gene expression in monocytic cells, with suppression of genes involved in macrophage activation and induction of stress genes and HSF1 appears to play a regulatory role in these effects., (Copyright 1999 Academic Press.)

Aim: To determine the effect of calcium supplementation of dairy cattle after calving on serum concentrations of calcium, magnesium, and phosphorous after calving, and on reproductive and productive performance in the following lactation., Methods: This was a double-blind randomised clinical trial using 356 dairy cows of parity 3 or greater from six seasonally calving commercial dairy herds. Cows were randomly assigned to a treatment and control group. The treatment group received two doses of 380 ml of calcium chloride in oil (49 g of elemental calcium); the first dose was given as soon as practical after calving and the second 12 hours later. Control cows received 380 ml of a placebo using the same treatment regime. Treatment and control cows had blood samples taken during the first 7 days after calving to determine serum calcium, magnesium and phosphorous levels. Treatment and control cows were monitored throughout the lactation to measure production and reproductive performance., Results: Treatment had no effect on PSM to first service intervals. Treatment significantly reduced PSM to conception intervals in only one of the four herds with data available for analysis (p = 0.09). There was no difference between treatment groups for adjusted corrected milk yield calculated from the first herd test milk yield (p = 0.93) or 305-day milk solids production (p = 0.19)., Conclusion: Further studies are required to identify reasons for the between-herd effectiveness of calcium chloride treatment in improving fertility.

The heat shock transcription factor (HSF) genes encode proteins that bind to the heat shock elements (HSE) of stress-inducible genes. We have observed the induction of HSF1, the ubiquitous member of the HSF family from a latent cytoplasmic state to a form competent to bind HSE during early G(1)in HeLa cells in the absence of stress. The induction of DNA-binding HSF1 coincided with a burst in cellular protein synthesis in early G(1)and inhibition of this translational peak prevented the formation of DNA binding-activated HSF1. A potential role for HSF1 in cell cycle regulation was suggested by the finding that cell lines stably overexpressing HSF1 showed an increased proportion of G(1)cells relative to other cell cycle phases. However, in contrast to the effects of heat shock, entry into G(1)did not lead to HSF1 hyperphosphorylation or increased activity of a heat shock promoter-reporter gene and did not cause the induction of heat shock protein 70 expression. Thus HSF1, previously implicated in the heat shock response is activated to a DNA binding from in G(1)under non-stress conditions and may play a role in G(1)regulation that does not involve the transcription of heat shock genes., (Copyright 1999 Harcourt Brace & Co. Ltd.)

Cells of the LA7 rat mammary tumor line stimulate proliferation of normal mouse mammary epithelial cells in culture when in direct physical contact with them. We examined junctional connections between these LA7 feeders and the recipient mouse mammary epithelial cells in order to study the role these junctions may play in growth signaling. Tight junctions and desmosomes between LA7 feeders and mouse mammary cells were detected by immunocytochemistry. These junctions connected every cell of either type with each of its neighbors. Adherens junctions, although evident between mouse mammary cells, could not be detected between LA7 and mouse mammary cells or between LA7 cells themselves. However, E-cadherin, the transmembrane protein of adherens junctions, was present in LA7 cell lysates. beta-catenin, which normally binds cadherins, was detected at the borders of LA7 cells. Presence of gap junctions between LA7 and mouse mammary cells was determined by traverse of lucifer yellow from an injected LA7 cell to surrounding mouse mammary epithelial cells. The experiments thus indicate that three types of intercellular junctions occur between cells active in direct cell-cell-stimulated proliferation signaling., (Copyright 1998 Academic Press.)

Heat shock factor 1 (HSF1) is the key transcriptional regulator of the heat shock genes that protect cells from environmental stress. However, because heat shock gene expression is deleterious to growth and development, we have examined mechanisms for HSF1 repression at growth temperatures, focusing on the role of phosphorylation. Mitogen-activated protein kinases (MAPKs) of the ERK family phosphorylate HSF1 and represses transcriptional function. The mechanism of repression involves initial phosphorylation by MAP kinase on serine 307, which primes HSF1 for secondary phosphorylation by glycogen synthase kinase 3 on a key residue in repression (serine 303). In vivo expression of glycogen synthase kinase 3 alpha or beta thus represses HSF1 through phosphorylation of serine 303. HSF1 is also phosphorylated by MAPK in vitro on a second residue (serine 363) adjacent to activation domain 1, and this residue is additionally phosphorylated by protein kinase C. In vivo, HSF1 is repressed through phosphorylation of this residue by protein kinase Calpha or -zeta but not MAPK. Regulation at 37 degrees C, therefore, involves the action of three protein kinase cascades that repress HSF1 through phosphorylation of serine residues 303, 307, and 363 and may promote growth by suppressing the heat shock response.

Objectives: To identify risk factors for culling of dairy cows from eight New South Wales dairy herds., Design: A longitudinal population study of dairy cow culling in eight non-seasonally calving dairy herds in the Camden district of New South Wales. Cox's proportional hazards model was used to evaluate various risk factors for culling for a specific reason (sales, deaths, reproductive failure, disorders of the udder and low milk production)., Results: Age at first calving was not a significant risk factor for culling. Milk production in the first lactation greater than the population mean did not influence length of productive life overall, but was associated with a greater hazard of removal for disorders of the udder. Risk of culling for reproductive failure differed significantly between farms, and was not related to events in the previous lactation such as calving-to-first service interval or calving-to-conception interval. Shorter calving intervals were associated with increased risk of removal for low milk production and disorders of the udder., Conclusion: Longitudinal surveys to accurately identify reasons for removal from a wide range of herds, identification of herds with low culling rates (especially for reproductive failure and udder disorders), and the identification of practices associated with these culling rates would be worthwhile to the Australian dairy industry.

Objectives: To describe the production and reproduction characteristics of the population under investigation and to compare them with the target population of non-seasonally calving dairy herds, to describe the pattern of culling with respect to length of productive life and length of time after calving, and to identify stages of the production cycle that are associated with increased risk of culling from the dairy herd., Design: A longitudinal population study of eight non-seasonally calving dairy herds in the Camden district of New South Wales., Results: The production and reproduction characteristics of this population were broadly similar to herds participating in the New South Wales Agriculture Dairy Herd Improvement scheme. In agreement with recent surveys, risk of culling increased with increased parity. This was accounted for specifically by increased risk of culling for calving disorders, reproductive tract lesions, and disorders of the udder. Risk of culling for failure to conceive appeared to remain constant throughout productive life. The overall risks for culling were greatest during the first 9 days after calving and 300 days after calving., Conclusion: Reducing the incidence of calving-associated disorders, attention to reproductive management and control of herd mastitis offer opportunities to reduce involuntary culling rates in this population.

Heat shock factor 1, the critical molecular regulator of the stress response is conserved throughout eukaryotic organisms and activates the transcription of heat shock genes. We now show that heat shock factor 1 inhibits the expression of c-fos, an immediate early gene that controls responses to extracellular stimuli for growth and differentiation. Heat shock factor 1 inhibits the transcription of the c-fos gene and antagonizes the activating effects of the signal transducing protein Ras on the c-fos promoter and on the promoter of another Ras responsive gene uPA. This property was specific for heat shock factor 1; c-fos repression was not seen with the structurally related protein heat shock factor 2. Repression involved different molecular mechanisms compared with those involved in transcriptional activation by heat shock factor 1 and specifically did not require binding to the c-fos promoter. Thus, in addition to its known role as a transcriptional activator of the cellular heat shock response, heat shock factor 1 also antagonizes the expression of Fos, a key component of the ubiquitous AP-1 transcription factor complex and as such could influence multiple aspects of cell regulation.

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.

CD97 is a receptor that spans the membrane seven times, a defining feature of G protein-coupled receptors. CD97 is predominantly expressed in leukocytes, but the function and accurate protein structure of this receptor have not been described. We show here that CD97 has the novel property among G protein-coupled receptors characterized to date of being processed intracellularly in either the endoplasmic reticulum or early Golgi from a proprotein into a noncovalently associated two-subunit structure that becomes expressed on the cell surface and is composed of a large extracellular protein (CD97alpha) and a seven-membrane spanning protein (CD97beta). CD97beta is part of an evolutionarily conserved subfamily of four proteins, including two Caenorhabditis elegans proteins of as yet unknown function, which is distinct from but most closely related to the glucagon receptor family. CD97alpha exists in three alternatively spliced isoforms that contain between three and five epidermal growth factor (EGF)-like repeats that are related to the calcium binding EGF-like repeats in the microfibril protein fibrillin. Leukocytes strongly positive for CD97 are concentrated at sites of inflammation relative to CD97 expression in normal lymphoid tissues. Soluble CD97alpha was found in body fluids from inflamed tissues, suggesting that a functional consequence of the CD97 heterodimeric structure is the stable existence of CD97alpha in a cellfree form. CD97 appears to be a multifunctional protein that may play a signal transduction role associated with the establishment or development of an inflammatory process.

Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.

The major dose-limiting adverse effect of ricin A chain-containing immunotoxin (IT) therapy is vascular leak syndrome (VLS). Since plasma fibronectin (Fn) plays a role in maintaining microcirculatory integrity and since the gradient between plasma and tissue Fn can be altered in various pathological situations, we determined whether the administration of IT-ricin A chain to patients resulted in changes in the levels of serum Fn and, if so, whether these changes correlated with the severity of VLS. We also measured the serum levels of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine which has been implicated in tissue damage and in interleukin 2-mediated VLS. Our results indicate that the most severe manifestations of VLS were associated with the highest pretreatment levels of Fn, the largest decreases in Fn immediately after starting IT therapy, increases in the levels of serum TNFalpha, higher concentrations of circulating IT, and the lowest numbers of circulating tumor cells. These parameters should, therefore, be useful for predicting which patients will have severe VLS.

Peripheral neuropathy is the major dose-limiting toxicity of the hypoxic cell sensitiser, etanidazole. Previous work from this laboratory using culture neuronal cell lines suggested that nitroimidazole-induced degradation of neurofilament proteins might be the critical biological event mediating this neurotoxicity. The purpose of the present study was to develop the neurofilament degradation assay in an organotypic spinal cord culture system with the goal of developing strategies for optimising sensitiser efficacy as well as ameliorating nitroimidazole-induced neurotoxicity. Spinal cord cultures were treated with etanidazole and neurofilament protein degradation was analysed by immunoblot analysis. Spinal cord cultures exposed to etanidazole exhibited a dose-dependent loss of parent neurofilament proteins, with concomitant appearance of low molecular weight degradation products. The potential neurotoxic effect of L, S-buthionine sulphoximine (BSO), a compound that enhances the radiosensitising effectiveness of 2-nitroimidazoles, was also screened in this assay system. BSO alone, at concentrations up to 100 microM, did not promote neurofilament degradation. BSO (20 microM) enhanced the effect of etanidazole on neurofilament degradation by a dose-modifying factor of 1.6 +/- 0.5. Since 20 microM BSO is expected to enhance etanidazole radiosensitisation of hypoxic cells by a larger factor, this suggests that a therapeutic gain could be achieved using BSO in combination with etanidazole in radiation therapy.

Improved understanding of the underlying biologic mechanisms that pertain to radiation oncology is providing an explanation for the cellular and tissue responses to ionizing radiation and is leading to the potential for novel therapeutic strategies. Among the areas of intensive investigation are: DNA recombination and repair, signal transduction, gene regulation, apoptosis, the cellular stress response, and the effect of the tumor microenvironment. These new biologic concepts, coupled with the superior technical capabilities now available for treatment delivery, are paving the way for new clinical approaches to improving both the quality and quantity of life for the cancer patient.

Radiation therapy remains a mainstay for the treatment of urologic malignancies, offering the opportunity for organ preservation as the primary treatment and serving in combination with surgery and systemic therapy for the more advanced tumors. Using prostate cancer as a model, classic radiation biology will be linked to the newer aspects of cellular and molecular biology of radiation oncology. Knowledge of classic radiation biology has provided new therapeutic approaches in dose fractionation, radiation sensitization, and normal tissue protection. Radiobiologic parameters derived from measuring radiation survival curves, tumor cell kinetics, and tumor oxygenation may provide the means to predict response to treatment. The basic mechanisms behind tumor and radiation biology that are under investigation include DNA recombination and repair, signal transduction, gene regulation, apoptosis, the cellular stress response, and the effect of the tumor microenvironment. The knowledge derived from improved understanding of the new biologic concepts, coupled with the superior technical capabilities now available for treatment delivery, is producing novel approaches in the clinic that may improve both the quality and quantity of life for the urologic cancer patient.

The authors report the flow cytometric detection of neoplastic T cells in the peripheral blood of four out of five (80%) patients with peripheral blood involvement with mycosis fungoides (Sezary syndrome) based on the levels of T-cell receptor expression as measured by CD3 and TCR-alpha beta staining. Antigen receptor expression was abnormal in terms of increased density of surface CD3 or TCR-alpha beta per cell. Other immunophenotypic abnormalities were present in three of these patients. However, in one patient abnormal T-cell receptor expression was the only immunophenotypic evidence of neoplasia, although morphologically abnormal lymphocytes were present and a T-cell clone was detected by polymerase chain reaction (PCR). In another patient, the authors were able to detect development of a new, more aggressive neoplastic T-cell population based on levels of T-cell receptor expression. Levels of T-cell receptor expression may be of diagnostic utility in the evaluation of peripheral blood for the presence of neoplastic T-cell populations.

DAB486IL-2 is a novel fusion toxin in which the ADP-ribosyltransferase and membrane-translocating domains of diphtheria toxin have been combined with the interleukin-2 (IL-2) gene, creating a recombinant protein capable of selectively intoxicating cells bearing the high-affinity IL-2 receptor. Clinical activity has been documented in Hodgkin disease and the non-Hodgkin lymphomas; toxicities have been minimal and include mild hepatic transaminitis, proteinuria, and hypersensitivity reactions. In this report, a patient with tumor-stage cutaneous T-cell lymphoma developed clinical adrenal failure with bilateral adrenal hemorrhage and necrosis 7 weeks after completing a 5-day course of treatment with DAB486IL-2. The relationship of fusion toxin therapy to the development of this unusual toxicity is discussed.

Purpose: To examine interleukin-7 (IL7)- and interleukin-2 (IL2)-induced proliferation of Sézary lymphoma cells and to consider if an autocrine or paracrine growth-stimulatory circuit involving IL7 exists in the Sézary syndrome (SS)., Materials and Methods: Fresh Sézary lymphoma cells were maintained in short-term culture in the presence of cytokines, and growth was measured by incorporation of (3H)-thymidine (TdR). Expression of IL7 and IL7 and IL2 receptors (IL7-R and IL2-R, respectively) was assessed by polymerase chain amplification of first-strand complementary DNA (RT-PCR), by affinity cross-linking of radioactive iodine-125-IL7, and by dual-color fluorescence-activated cell analysis. IL-7 production was measured by immunoassay., Results: Sézary lymphoma cells from seven patients showed synergistic (five of seven) or additive (two of seven) proliferation when cultured in the presence of IL2 and IL7, as compared with culture with either cytokine alone. Two patients with evidence of synergistic stimulation of [3H]-TdR incorporation showed IL7-R gene expression by RT-PCR and IL7 affinity cross-linking. Incubation of all seven patients' cells with IL7 induced coexpression to varying degrees of IL7-R and IL2-R. Sézary lymphoma cells from at least three of five patients studied expressed IL7 mRNA, and skin from three of five patients studied, as well as normal skin, expressed IL7 mRNA by RT-PCR., Conclusion: Sézary lymphoma cells respond by proliferation to IL7 plus IL2, and in some instances produce IL7. Therapeutic maneuvers should be pursued to take advantage of this potential autocrine or paracrine growth-stimulatory mechanism.

Extracellular signal-regulated kinases (ERKs), also known as mitogen-activated protein (MAP) kinases, are rapidly phosphorylated and activated in response to a number of external factors which promote growth and differentiation (T. G. Boulton, S. H. Nye, D. J. Robbins, N. Y. Ip, E. Radziejewska, S. D. Morgenbesser, R. A. DePinho, N. Panayotatos, M. H. Cobb, and G. D. Yancopoulos, Cell, 65: 663-675, 1991; S. L. Pelech and S. S. Jasbinder, Science (Washington DC), 257: 1355-1356, 1992; G. Thomas, Cell, 68: 3-6, 1992). We have identified two novel stimulators of MAP kinase activity, ionizing radiation and H2O2. Both radiation and H2O2, as well as the known agonist 12-O-tetradecanoylphorbol 13-acetate activate MAP kinase through the production of reactive oxygen intermediates. Our results demonstrate a direct link between the MAP kinase signal transduction pathway and reactive oxygen species and provide a unifying mechanism for activation of early- and late-response genes by inducers of oxidative stress.

The heat shock response in mammals consists of a complex array of intracellular reactions initiated by stress, although its regulation is poorly understood. We have investigated the role of transmembrane signal transduction in the response, examining mechanisms involved in the activation of phospholipase C (PLC) by heat shock. In rodent fibroblasts permeabilized with digitonin, heat shock and receptor-mediated PLC activity exhibited a strict GTP analog dependency. This indicates that heat shock-mediated phospholipase activation, in common with receptor mediated stimulation, does not involve direct effects on the phospholipases and suggests the participation of GTP binding (G) proteins in the activation process. When cells were treated with the inhibitor pertussis toxin (PTX), the phospholipases retained their inducibility by heat shock, but became refractory to thrombin treatment, indicating that heat shock may influence PLC activity through a distinct population of G proteins compared to thrombin. The data seem to exclude a role for PTX sensitive G proteins in the production of IP3 after heating and suggest a pathway involving the direct thermal activation of the Gq class of G proteins, which are coupled to the PLC beta 1 isoform.

Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat-shocked cells of phosphoinositide-specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shock, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response.