Physician cooperation in outpatient cancer care. An amplified secondary analysis of qualitative interview data.

The importance of outpatient cancer care services is increasing due to the growing number of patients having or having had cancer. However, little is known about cooperation among physicians in outpatient settings. To understand what inter- and multidisciplinary care means in community settings, we conducted an amplified secondary analysis that combined qualitative interview data with 42 general practitioners (GPs), 21 oncologists and 21 urologists that mainly worked in medical practices in Germany. We compared their perspectives on cooperation relationships in cancer care. Our results indicate that all participants regarded cooperation as a prerequisite for good cancer care. Oncologists and urologists mainly reported cooperating for tumour-specific treatment tasks, while GPs' reasoning for cooperation was more patient-centred. While oncologists and urologists reported experiencing reciprocal communication with other physicians, GPs had to gather the information they needed. GPs seldom reported engaging in formal cooperation structures, while for specialists, participation in formal spaces of cooperation, such as tumour boards, facilitated a more frequent and informal discussion of patients, for instance on the phone. Further research should focus on ways to foster GPs' integration in cancer care and evaluate if this can be reached by incorporating GPs in formal cooperation structures such as tumour boards.

Stimulation of megakaryocytopoiesis in mice by human modified C-reactive protein (mCRP)

The prototypic human acute phase reactant, C-reactive protein (CRP), and a structurally modified form of CRP (mCRP) were studied as agents which could stimulate thrombopoiesis in both in vitro and in vivo mouse models. mCRP, but not the widely studied (native) pentameric form of CRP, demonstrated significant megakaryocyte colony-stimulating activity. This activity was measured in plasma clot cultures incubated with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). mCRP increased the number of mouse megakaryocyte colonies in a dose-dependent manner. While significantly more colonies were observed in mCRP-treated cultures compared to controls, the kinetics of megakaryocyte growth and maturation were similar to those measured in cultures stimulated with PWM-SCM lacking mCRP. A low level of megakaryocyte growth-promoting activity was noted when mCRP was added to plasma clot cultures not incubated with spleen cell conditioned medium. However, the most striking activity of mCRP was in potentiating stimulated megakaryocyte colony formation (i.e., as a Meg-POT factor). In in vivo experiments, mCRP injected subcutaneously into normal mice resulted in significant increases in blood platelet numbers compared to control mice receiving sham injections. These results suggest that a modified form of CRP has thrombopoietic activity in both in vitro and in vivo mouse models, Therefore, one important biological role for CRP during an acute-phase response might be to contribute, after a structural modification, to the hematopoietic regulation of blood platelets.

Monitoring the activity of antiviral therapy for HIV infection using a polymerase chain reaction method coupled with reverse transcription.

AIM: To monitor the anti-HIV-1 activity of antiretroviral agents in patients with HIV-1 infection. METHOD: Quantification of viral RNA copy in plasma or serum using a polymerase chain reaction method coupled with reverse transcription. CONCLUSIONS: The HIV-1 RNA copy number represents the HIV-1 viremia status in patients with HIV-1 infection. This copy number is likely to be useful in monitoring the effectiveness of antiviral therapy and the method is likely to be built into every clinical trial of anti-HIV-1 therapy in the near future.

Analogues of natural phloroglucinols as antagonists against both thromboxane A2 and leukotriene D4.

Antagonists against both thromboxane A2 and leukotriene D4 were prepared from phloroglucinol. These compounds showed almost the same activity as the chinesins which were isolated from Hypericum chinese L. The correlation between the structures and activity was studied in the synthesized and naturally occurring phloroglucinol derivatives.

Escherichia coli mediated biosynthesis and in vitro anti-HIV activity of lipophilic 6-halo-2',3'-dideoxypurine nucleosides.

A series of 6-substituted 2',3'-dideoxypurine ribofuranosides (ddP) was enzymatically synthesized with live E. coli in an effort to enhance the lipophilicity of this class of anti-human immunodeficiency virus (HIV) compounds and thereby facilitate drug delivery into the central nervous system. All 6-halo-substituted ddPs were substantially more lipophilic, as defined by their octanol-water partition coefficient (P), than their nonhalogenated congeners 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG). For this class of compounds, log P's ranged from +0.5 to -1.2 in the following order: 6-iodo, 2-amino-6-iodo greater than 6-bromo, 2-amino-6-bromo greater than 6-chloro, 2-amino-6-chloro greater than 6-fluoro, 2-amino-6-fluoro much greater than ddG greater than ddI. These compounds were evaluated in vitro for ability to suppress the infectivity, replication, and cytopathic effect of HIV. 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddP exhibited a potent activity against HIV comparable to that of ddI or ddG and completely blocked the infectivity of HIV without affecting the growth of target cells. The comparative order of in vitro anti-HIV activity was 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro greater than 2-amino-6-bromo greater than 2-amino-6-iodo, 6-chloro greater than 6-bromo greater than 6-iodo. These compounds also exhibited potent in vitro activity against HIV-2 and 3'-azido-3'-deoxythymidine-resistant HIV-1 variants. All 2-amino-6-halo-ddPs and 6-halo-ddPs were substrates for adenosine deaminase (ADA) and were converted to ddG or ddI, respectively. In the presence of the potent ADA inhibitor 2'-deoxycoformycin, 6-halo-substituted ddPs failed to exert an in vitro antiretroviral effect. These dideoxypurine nucleoside analogues represent a new class of lipophilic prodrugs of ddG and ddI that possess the potential for more effective therapy of HIV-induced neurologic disorders.

HTLV type 1 envelope glycoprotein gp46 evokes necrosis by binding to receptor complex.

In syncytium formation induced by HTLV-1-bearing cells, 71-kDa heat shock cognate protein (HSC70) functions as a receptor molecule and the receptor complex with beta-actin and palmitoyl(16:0)-oleoyl(18:1)-phosphatidylglycerol (PG) is thus formed. We now have evidence that the molecular association between HTLV-1 gp46 envelope protein and HSC70 led to pore formation on the surface of target cell membrane and cell death followed. The peptide segment corresponding to the region from Asp-197 to Leu-216 (gp46-197), and which serves as a binding site to both HSC70 and PG for syncytium formation, also had cytotoxic effects on target cell MOLT-4. This cytotoxicity was due to necrosis, not apoptosis. On the other hand, two other receptor-binding sites, Lys-111 to Asp-138 on gp46 (gp46-111) and Cys-400 to Leu-429 on gp21 (gp21-400), and which bound only with PG, had no cytotoxic effects on MOLT-4 cells. The HTLV-2 peptide (gp46-194; Glu194 to Leu-213) corresponding to the region of HTLV-1 gp46-197 showed no cytotoxicity, and interacted only with PG, not with either HSC70 or beta-actin. Amino acid alterations between HTLV-1 gp46-197 and HTLV-2 gp46-194 were significant on the hydrophilic face of the amphipathic structure. Taken together, the interaction between HSC70 and gp46 of HTLV-1 through the hydrophilic face of gp46-197 may lead to pore formation in lipid bilayers to be followed by membrane fusion or cell death.

Phosphatidylglycerol participates in syncytium formation induced by HTLV type 1-bearing cells.

We previously reported that 71-kDa heat shock cognate protein (HSC70) was expressed on the cell surface of human T cell lymphotropic virus type 1 (HTLV-1)-susceptible cells and that HSC70, beta-actin, and a lipid-like component on the target cell membrane participated in syncytium formation by HTLV-1. We have now identified this lipid-like component to be palmitoyl (16:0)-oleoyl (18:1)-phosphatidylglycerol (POPG), using preparative thin-layer chromatographic fractionation and tandem mass spectrometric analysis. In the syncytium formation assay, exogenously added PG inhibited cell-to-cell transmission of HTLV-1 in a dose-dependent manner. Other phospholipids showed less (PE) or no effect (PC, PS, PI, PA, lysoPC, lysoPE, and CL). Binding experiments showed that PG interacted with three synthetic peptides, gp46--111, gp46--197, and gp21--400, which correspond to regions Lys111--Asp138 and Asp197--Leu216 on the gp46 surface glycoprotein, and to region Cys400--Leu429 on the gp21 transmembrane glycoprotein, respectively, as well as with intact gp46 and gp21 proteins of HTLV-1. On the other hand, HSC70 and beta-actin interacted with gp46--197 and gp46, not with gp46--111. However, the eluate from an affinity column coupled with gp46--111 contained not only PG but also HSC70 and beta-actin, despite the lack of direct interaction between gp46--111 and these proteins. In the in vitro binding assay, HSC70 showed interaction with both PG and beta-actin, while there was no evidence of any interaction between PG and beta-actin. These results suggest that HSC70 molecules on target cell surface interact with both PG in lipid bilayers and intracellular beta-actin and that these three cellular components form a receptor complex that plays a critical role in syncytium formation induced by HTLV-1-bearing cells.

A C2 symmetry-based HIV protease inhibitor, A77003, irreversibly inhibits infectivity of HIV-1 in vitro.

A C2 symmetry-based HIV protease inhibitor, A77003, exerts potent antiviral activity against a wide spectrum of HIV isolates in vitro. In this study, we asked whether A77003 could cause irreversible conformational changes to HIV-1, whether the amounts of viral RNA and p24 capsid protein per virion were altered, and how the infectivity of the virus produced in the presence of the drug was affected. We found that the number of viral particles and per-virion viral RNA content of the virus produced in the presence of A77003 did not significantly differ from those of the virus produced in the absence of the drug, whereas significant morphological changes were observed as assessed by transmission electron microscopy. However, the virus produced in the presence of A77003 contained substantially less p24gag protein per virion particle as compared to those produced in the absence of the drug or in the presence of AZT. Virions produced in the presence of A77003 showed up to 50-fold less infectious capability in subsequent tissue culture than control virions produced in the absence of drug or in the presence of AZT. This reduction in infectivity was maintained for at least 10 days in culture. The present data suggest that A77003 impairs HIV-1 protease-mediated Gag processing, interferes with the assembly and maturation of the virus, and leads to an irreversible loss of the infectivity of the virus, although a low but positive level of reversion to infectivity during the 10-day assay occurs. These features of A77003 (and perhaps similar HIV protease inhibitors as well) anti-HIV activity should represent desirable properties for antiviral therapy of AIDS and related diseases.

Identification of drug-related genotypic changes in HIV-1 from serum using the selective polymerase chain reaction.

We attempted to detect drug-related HIV-1 pol gene mutations by selective polymerase chain reaction (PCR) using both proviral DNA and viral RNA isolated from patients (pts) with AIDS or ARC receiving antiretroviral therapy. Peripheral blood mononuclear cell (PBM)-associated proviral DNA and serum-derived viral RNA were obtained from eight patients before and after receiving an alternating regimen of AZT and ddC for 15-41 months or ddI monotherapy for 12-26 months. These specimens were examined for the presence of mutations at positions 70, 74, 215 and 219. We noted that selective PCR results can be ambiguous depending on the quantity of DNA template employed. We, therefore, used the minimal quantity of DNA templates that yielded evaluable PCR products in this study. For all the eight pairs of pre- and post-therapy proviral DNA samples, selective PCR results agreed with independently determined nucleotide sequences. Results of reverse transcription of serum-derived viral RNA followed by selective PCR differed in some cases from those using the proviral DNA. In particular, the use of serum viral RNA appeared to allow earlier detection of changes in drug-related mutations than the use of PBM-associated proviral DNA. We conclude that (i) selective PCR using the minimum and sufficient number of PBM-associated proviral DNA and serum viral RNA copies successfully detects the presence of known pol gene mutations; (ii) drug-related mutations may be distinguished earlier in virions in serum (or plasma) than in proviral DNA in PBM; and (iii) quantification of HIV-1 prior to selective PCR may be an important component in monitoring the therapy of HIV-1 infection.

Genotypic and phenotypic characterization of HIV-1 isolated from patients receiving (--)-2',3'-dideoxy-3'-thiacytidine.

We attempted to determine whether HIV-1 developed resistance to (--)-2',3'-dideoxy-3'-thiacytidine ((--)-3TC or 3TC, lamivudine) in patients with advanced human immunodeficiency virus type 1 (HIV-1) infection during therapy with 3TC. Genotypic analysis of HIV-1 strains isolated from 6 patients receiving 3TC revealed that as early as 2 months of therapy, HIV-1 developed a Met to Val amino acid substitution at codon 184 (Met184-->Val) in the reverse transcriptase-coding region of the pol gene. A detailed study of a series of HIV-1 strains isolated from a patient demonstrated that Met at codon 184 was first substituted with Ile by 2 weeks of 3TC therapy, followed by the substitution with Val by 8 weeks. All HIV-1 strains with the Met184-->Val substitution were profoundly less susceptible to 3TC (1800- to 5500-fold decreased sensitivity) as compared to pretherapy virus strains. These strains were also moderately less sensitive to 2',3'-dideoxycytidine (4.5- to 9-fold), but more sensitive to 3'-azido-2',3'-dideoxythymidine (2- to 14-fold). A decrease in viremia levels and an increase in CD4 counts were observed early in therapy; however, these changes were only transient. Our data suggest that reversal of such beneficial changes is associated with the Met184-->Val substitution of the pol gene of HIV-1. The data also suggest that 3TC, as a single agent, may induce virologic and immunologic improvement in patients with advanced HIV-1 infection, but only transiently.

Adaptive response in embryogenesis: I. Dose and timing of radiation for reduction of prenatal death and congenital malformation during the late period of organogenesis.

An adaptive response was demonstrated during embryogenesis in mice. Whole-body irradiation at a dose of 0-50 cGy was given to condition pregnant ICR mice on day 9 to day 11 of gestation. Then their whole bodies were exposed to a challenging dose of 5 Gy on the next day. The numbers of living fetuses, prenatal deaths and living fetuses with external gross malformations were determined on day 19. A conditioning dose of 30 cGy on day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations induced by a 5-Gy dose on day 12. This indicates the existence of a critical dose and timing for administering a conditioning dose for radioadaptation during the late period of organogenesis in mice. The possible mechanisms involved are discussed.

The treatment of unruptured tubal pregnancy with intratubal methotrexate injection under laparoscopic control.

Nine patients with unruptured tubal pregnancies of 6-9 weeks' duration were treated with methotrexate intratubal injection under laparoscopic control. Urinary hCG levels decreased immediately after completion of the procedure, with a median time of 11 days (range 1-29) to resolution. Tubal patency on the side of the ectopic gestation was confirmed by hysterosalpingography 1-3 months after the procedure in all cases. This method requires a reduced methotrexate dosage compared with intramural or intravenous therapy. The indications are unruptured tubal pregnancy of 4 cm or less in diameter and urinary hCG levels of 8000 mIU/mL or lower.

Antiviral effects of 6-chloro-2',3'-dideoxyguanosine in rhesus monkeys acutely infected with simian immunodeficiency virus.

A lipophilic dideoxynucleoside analogue, 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG), was expected to be effective against AIDS-related dementia. In this study, we tested the effect of 6-Cl-ddG on simian immunodeficiency virus (SIVmac239) replication in vitro and on acute infection of six rhesus monkeys (Macaca mulatta) with SIVmac239. This compound inhibited SIV-induced cytopathic effect in CEM x 174 cells and SIV replication in vitro with an ED50 value of 2.5 microM. A dose of 25 mg/kg 6-Cl-ddG was administered to three monkeys every 8 h for 10 days and an untreated group of three monkeys was injected with the solvent without drug. Although 6-Cl-ddG was not detected in the plasma, the metabolite ddG was maintained at a concentration of more than 3 microM for 8 h after administration. In the cerebrospinal fluid, the ddG concentration was 2 microM at 2 h after administration. SIV antigen (p27) and antibody appearance in the plasma were delayed for 5-8 days compared with the mock-treated group. The occurrence of lymphadenopathy in treated monkeys was delayed for 6 days compared with the mock-treated group. Signs of 6-Cl-ddG toxicity were minimal after the treatment. The results of this study provide further evidence that 6-Cl-ddG may act as a potent anti-human immunodeficiency virus agent in vivo.

Primary motor cortex isolation: complete paralysis with preserved primary motor cortex.

We present a left-sided hemiplegic patient with a cerebrovascular lesion involving the medial part of the right frontal and parietal lobes and the corpus callosum, but sparing the hand area of right primary motor cortex (M1). Several studies using transcranial magnetic stimulation demonstrated functional integrity of the efferent pathways from the right M1, intact sensory afferents to M1, an impairment of transcallosal connection between the bilateral motor cortices, and reduced ipsilateral cortico-cortical inhibition within the right M1. Based on these results, we conclude that the paralysis of this patient was caused by disconnection of the intact M1 from any structures requisite for initiation of movements. The present patient also suggests the importance of various afferents to M1 in voluntary movement. We propose a term of 'primary motor cortex isolation' to designate the paralysis reported here.

Mechanism of short-term memory and repetition in conduction aphasia and related cognitive disorders: a neuropsychological, audiological and neuroimaging study.

To evaluate the role of the sub-cortical white matter and cortical areas of the supramarginal gyrus in short-term memory impairment (shortened digit or letter span) and repetition difficulty, four patients with conduction aphasia and impaired short-term memory and two patients with only short-term memory impairment were given digit span, letter span, speech audiometry and dichotic listening tests. The results showed that in most of the patients letter span was inferior to digit span and that bilateral ear suppression in the dichotic listening test was observed in two patients with a lesion in the inferior part of the supramarginal gyrus, suggesting that what was affected was phonological information and that the supramarginal gyrus was the storage site. The overlapped lesion of conduction aphasia patients with short-term memory impairment was the periventricular white matter at the upper to middle part of the trigone, while patients with only short-term memory impairment had a lesion in the inferior supramarginal gyrus in common. Thus, damage to the periventricular white matter at the trigone may yield the phonemic paraphasia characteristic of conduction aphasia, while damage to the inferior part of the supramarginal gyrus may result in the impairment of short-term memory. We believe that as a part of the mechanisms of short-term memory and repetition, phonological information is processed in the primary auditory cortex and goes through the periventricular white matter to the inferior part of the supramarginal gyrus and is temporarily stored there.

An immunoglobulin G inhibiting lactate dehydrogenase activity.

We found extremely low (6 U/l) serum lactate dehydrogenase (LDH, EC 1.1.1.27) activity in a patient with uterine myoma. The patient's serum inhibited purified LDH isoenzymes. One milliliter serum neutralized 73 U purified LDH-1 isoenzyme, equivalent to 300-500-fold the amount of LDH in 1 ml of normal serum. The serum inhibitor was purified by ordinary procedures using chromatographies and identified as IgG which contains both kappa- and lambda-chains. The IgG is very unique in showing higher affinity to the H- than M-subunit of the LDH tetramer, in contrast with the IgGs already reported. After removal of the myoma, the anti-LDH activity gradually decreased with a half-life of 20 days corresponding to that of IgG and finally almost disappeared. This indicates a possibility that the myoma cells produce some factors such as B-cell growth and differentiation factors.

Trans-Tenon's retrobulbar triamcinolone infusion for the treatment of uveitis.

AIM: To assess efficacy and complications of trans-Tenon's retrobulbar infusion of triamcinolone acetonide for posterior uveitic inflammation. METHODS: Non-randomised, uncontrolled, retrospective study of 51 eyes of 37 patients who underwent triamcinolone infusion for vitritis, cystoid macular oedema (CMO), or posterior retinal vasculitis using a long blunt cannula via an incision made through conjunctiva and Tenon's capsule. RESULTS: Overall clinical efficacy was 86%; 96% for vitritis, 82% for CMO, and 33% for posterior retinal vasculitis. Mean visual acuity improved within 1 month after triamcinolone infusion (p <0.05). Cataract progression and intraocular pressure elevation were observed in 31% and 27% of eyes, respectively. CONCLUSION: Trans-Tenon's retrobulbar triamcinolone infusion may be a safe and effective treatment for posterior uveitic inflammation.

High-performance liquid chromatography of N-terminal tryptophan-containing peptides with precolumn fluorescence derivatization with glyoxal.

A precolumn fluorescence derivatization method combined with high-performance liquid chromatography is described for the sensitive and selective determination of N-terminal tryptophan-containing peptides. The peptides and tryptophan were converted into fluorescent derivatives with glyoxal in a moderately acidic medium (pH 4.5). The derivatives were separated on a reversed-phase column with isocratic elution with an aqueous mobile phase composed of acetonitrile, methanol and phosphate buffer (pH 6.0), and subsequently detected by fluorimetry. The derivatization technique provided the respective N-terminal tryptophan-containing oligopeptides with single fluorescent peaks in chromatography. The detection limits for the peptides were 55-382 fmol per 100-microliters injection volume at a signal-to-noise ratio of 3. The method also allowed the facile detection of an N-terminal tryptophyl fragment in the enzyme reaction mixture of dynorphin A with trypsin.

Effects of 5-fluorouracil on hematopoietic stem cells in normal and irradiated mice.

The effects of 5-fluorouracil (5-FU) on hematopoietic stem cells (CFU-S) and nucleated cells in mouse femur and spleen were studied in normal and X-irradiated mice (ddY-SLC male, 8-10 weeks old). Changes in the number of circulating blood cells also were investigated in mice treated with 5-FU. A single dose of 5-FU (150 mg/kg) was injected i.p. The femoral CFU-S decreased after 5-FU treatment up to day 3 when it reached 2% of the control value. The cells then increased, reaching a maximum about twice that of control by day 12. A very similar profile was found for splenic CFU-S. Post-irradiation recovery for femoral and splenic CFU-S in mice treated with 5-FU 3 days before X-irradiation (1.9 Gy) was faster than in mice given irradiation alone. Regrowth rates of CFU-S were almost the same as in mice treated with 5-FU alone. The radiosensitivity of the CFU-S population in mice treated first with 5-FU at different times before X-irradiation (1.9 Gy) changed day-by-day after treatment. The maximal survival for femoral CFU-S was found in mice treated with 5-FU 5 days before irradiation, and for splenic CFU-S in mice treated 12 days before irradiation.

Growth and hydrocarbon production of microalga Botryococcus braunii in bubble column photobioreactors.

Microalga Botryococcus braunii, which produces high levels of liquid hydrocarbons called botryococcenes, was cultivated in bubble column photobioreactors. Algal cells, adapted to low irradiance (3 klx) in the preculture, showed lower biomass and hydrocarbons productivity in the photobioreactor illuminated at high irradiance (10 klx) due to the effects of photoinhibition. The degree of photoinhibition was reduced by partial shading, the lighted volume ratio being varied from 25 to 100%. The algal cells adapted to high irradiance in the preculture showed a high biomass productivity at 10 klx: the cell concentration reached higher than 7 kg/m3 and the hydrocarbon content was 50% based on cell dry weight. Hydrocarbon production kinetics of this alga in the linear growth phase was found to be growth-associated irrespective of the experimental conditions of illumination and preculture.