Modulation of plasma complement by the initial dose of epirubicin/docetaxel therapy in breast cancer and its predictive value.

BACKGROUND: Despite the widespread use of neoadjuvant chemotherapy in breast cancer patients, prediction of individual response to treatment remains an unsolved clinical problem. Particularly, administration of an inefficient chemotherapeutic regimen should be avoided. Therefore, a better understanding of the molecular mechanisms underlying response to neoadjuvant chemotherapy is of particular clinical interest. Aim of the present study was to test whether neoadjuvant chemotherapy with epirubicin/docetaxel induces early changes in the plasma proteome of breast cancer patients and whether such changes correlate with response to therapy. METHODS: Plasma samples of 25 breast cancer patients obtained before and 24 h after initiation of epirubicin/docetaxel-based neoadjuvant chemotherapy were analysed using two-dimensional differential gel electrophoresis (2D-DIGE). Protein spots found to be differentially expressed were identified using mass spectrometry and then correlated with the pathological response after six cycles of therapy. Markers identified in a discovery set of patients (n=12) were confirmed in an independent validation set (n=13). RESULTS: 2D-DIGE revealed 33 protein spots to be differentially expressed in response to chemotherapy, including the complement factors C1, C3 and C4, inter-α-trypsin inhibitor, α-1-antichymotrypsin and α-2-Heremans-Schmid glycoprotein (AHSG). With respect to cytokines, only interleukin (IL)-6, IL-10 and soluble intracellular adgesion molecule 3 (sICAM3) were minimally modulated. Moreover, two protein spots within the complement component C3 significantly correlated with response to therapy. CONCLUSION: We have identified acute phase proteins and the complement system as part of the early host response to epirubicin/docetaxel chemotherapy. As complement C3 cleavage correlates with the efficacy of docetaxel/epirubicin-based chemotherapy, it has the potential as an easily accessible predictive biomarker.

Retinal ganglion cells that project to the dorsal lateral geniculate nucleus in the macaque monkey.

Horseradish peroxidase was deposited in the optic nerve to retrogradely label and reveal the dendritic form of all classes of ganglion cell, or it was injected into the dorsal lateral geniculate nucleus to reveal only those classes projecting to the thalamus. The results were compared with those of the accompanying paper in which the ganglion cells projecting to the midbrain are selectively revealed. Two major classes of ganglion cells are described, the P alpha and P beta cells. For both classes dendritic field size increases with eccentricity from the fovea and there is no overlap in the two classes at any given eccentricity. Cell body size shows a similar mean difference but with a slight overlap. Both cell bodies and dendritic fields are larger along the temporal horizontal meridian than the nasal horizontal meridian, for P alpha and for P beta cells, but these differences are reduced when naso-temporal differences in ganglion cell density are taken into account, that is, size correlates closely with density. Injections restricted to the parvocellular layers of the lateral geniculate nucleus labelled almost exclusively P beta cells, whereas injections confined to the magnocellular layers labelled almost exclusively P alpha cells. As midbrain injections label no P beta cells and few P alpha cells it can be shown that about 80% of ganglion cells are P beta cells projecting to parvocellular lateral geniculate nucleus, and that about 10% are P alpha cells projecting to magnocellular layers. The coverage factor, that is the number of cells covering each point on the retina, varied from 1.9-2.3 for P beta cells, and from 2-7 for P alpha cells. Comparing the results with those of comparable investigations on cats and rabbits shows a much clearer segregation of the terminal targets of different classes of ganglion cell in monkeys, the greatest difference being the absence in the monkey of a projection to the geniculate from gamma- and epsilon-like cells. Further, axons which branch and innervate both thalamus and midbrain are rare in monkeys but common in other mammals. Comparing the results with those from physiological investigations suggests that the P beta cells correspond to colour-opponent cells, whereas P alpha cells correspond to the achromatic broad-band magnocellular cells.

Cell type-specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia.

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.

The detection of airborne Mycobacterium tuberculosis using micropore membrane air sampling and polymerase chain reaction.

STUDY OBJECTIVES: Mycobacterium tuberculosis (MTb) bacilli are carried on airborne droplet nuclei produced by aerosolization that can occur from coughing, talking, or even singing. Because of their prolonged period of suspension, they can be filtered from the air onto a porous medium and readily detected using polymerase chain reaction (PCR). DESIGN: Prospective cohort analysis. SETTING: Samples of circulating air were collected over a 12-month period from within the rooms of 10 hospitalized patients who were under respiratory isolation to rule out MTb infection. A small laboratory pump was used to draw ambient air at a rate of 2 L/min over a 6-h period through a 0.2-microm polycarbonate membrane filter placed near the patient's bed. Analysis of the membrane filters was conducted using PCR. Sputum cultures for MTb were performed simultaneously, and the results of smears stained for acid-fast bacilli (AFB) were noted. MEASUREMENTS AND RESULTS: MTb complex was successfully detected by PCR in six of seven patients in whom sputum MTb cultures were subsequently positive, and in zero of three with subsequently negative sputum cultures. Sampling in one patient with a positive culture, in whom PCR results were negative, was only carried out for 2 h due to pump malfunction. One of the six PCR-positive patients was AFB-smear negative at the time of air sampling. CONCLUSIONS: Our preliminary findings indicate that the technique of Micropore membrane air sampling with PCR analysis has important applications in the epidemiology and diagnosis of MTb.

BK virus.

BK virus is a human polyoma virus that infects the renal epithelium and remains latent until immunosuppression triggers reactivation. After reactivation, BK virus can be detected in the urine by methods currently available in the clinical laboratory. Correlations can be made between BK viruria and the occurrence of both renal and hepatic pathologies. BK virus is emerging as a significant pathogen in transplant patients. Additionally, the presence of BK virus DNA in primary brain and pancreatic tumors suggests that it may have oncogenic potential. Thus far, attempts to treat BK virus infection have been ineffective, though research has opened new avenues for treatment possibilities. Prevention of BK virus and other latent viral reactivation remains a challenge to viral research.

Postoperative glycyl-glutamine infusion reduces immunosuppression: partial prevention of the surgery induced decrease in HLA-DR expression on monocytes.

BACKGROUND AND AIMS: Surgery, trauma and inflammation reduce HLA-DR expression on monocytes, which is associated with an increased susceptibility to infection and sepsis. Furthermore, surgery decreases plasma glutamine (GLN) levels. The expression of HLA-DR on human monocytes in vitro is dependent on the concentration of GLN in the culture medium. We therefore hypothesized that postoperative infusions of glutamine-dipeptides would prevent the decreased HLA-DR expression on monocytes. METHODS: Thirty patients undergoing major abdominal surgery were randomly allocated to receive either 1500 ml Vamin (control) or an isonitrogenic formulation containing Vamin and 500 ml glycyl-glutamine (35 g GLN; 0.5g/kg BW) (GLY-GLN), or Vamin and 500 ml alanyl-glutamine (35 g GLN; 0.5 g/kg BW) (ALA-GLN) as a continuous infusion over 48 h post-operatively. Immediately and 48 h after surgery blood samples were collected to determine HLA-DR expression on monocytes by flow cytometry. RESULTS: The groups were comparable with respect to age, gender distribution and operation time. In patients receiving GLY-GLN mean HLA-DR expression on monocytes at 48 h was significantly better preserved than in controls (65.0 %+/-7 % vs 42.5 %+/-4 %;P<0.05), whereas HLA-DR expression on monocytes in patients receiving ALA-GLN was not significantly different. CONCLUSION: This is the first study comparing the dipeptides GLY-GLN and ALA-GLN in the postoperative setting. The GLY-GLN induced preservation of HLA-DR on monocytes following surgery may prevent infectious complications in these patients.

HSP70 expression in granulocytes and lymphocytes of patients with polytrauma: comparison with plasma glutamine.

Heat shock proteins (HSP's) are a set of conserved proteins which confer tolerance to stress. These proteins play a major role in the pathophysiology of infection and inflammation. Induction of HSP's before onset of sepsis is able to reduce or prevent organ damage and death. GLN is known to influence the expression of HSP70 in different cell types. In this work we tried to find out if there is an association between plasma GLN levels and HSP70 expression in immune cells. We investigated six polytraumatized patients and a control group of six healthy donors. HSP70 expression was investigated by western blot analysis and immune-histochemistry. We demonstrated that granulocytes and lymphocytes behave differently in the expression of HSP70 in polytraumatized patients. In healthy donors both lymphocytes and granulocytes showed a pronounced expression of HSP70. In contrast, most of the polytraumatized patients showed no HSP70 expression in granulocytes. In lymphocytes of these patients, however, a pronounced expression similar to that of healthy volunteers was observed. Plasma glutamine levels were reduced in all patients and at normal range in healthy donors. These results suggest that lymphocytes and granulocytes behave different when confronted with a reduction of plasma GLN levels.

beta-Lactamase recovery from E. coli cell lysate via two-phase electrophoresis.

beta-Lactamase was recovered from Escherichia coli cell lysate by a novel cell debris removal method using two-phase electrophoresis. The cells were harvested by centrifugation after fermentation, resuspended in a low ionic strength electrophoresis buffer, lysed, and combined with a poly(ethylene glycol)/dextran aqueous two-phase system in the same buffer. The cell lysate was subjected to a 40 V/cm electric field oriented perpendicular to the phase interface for 90 min. Experiments were conducted both with and without a nucleic acid precipitation step using poly(ethylene imine) (PEI). For PEI-treated lysate at pH 5, the positively charged beta-lactamase was directed to the upper phase, while negatively charged contaminants (including cell debris, nucleic acid/PEI precipitates, and negatively charged proteins) were directed to the lower phase with the applied field. beta-Lactamase yield in the upper phase was 81%, while cell debris and nucleic acids partitioned almost exclusively to the lower phase. For untreated lysate, beta-lactamase did not move in the electric field due to strong interaction with nucleic acids in solution.

MHC-class-I expression in human breast cancer correlates with nuclear localization of the 90 kDa heat-shock-protein.

Breast cancer cells frequently exhibit a reduction in expression of major-histocompatibility-complex (MHC) class I proteins which blocks cytotoxic T-lymphocyte (CTL) mediated apoptosis. Recent studies indicate that the 90 kD heat-shock-protein (HSP90) plays a major role in the transfer of antigenic peptides to the MHC class I complex. HSP90 is a molecular chaperone which is involved in signal transduction and regulation of apoptosis. Since HSP90 is described to be elevated in breast cancer, its relationship with MHC class I expression was investigated. Using immunohistochemistry we analyzed the expression and localization of HSP90 and MHC class I in 17 human breast tumors. Positive correlation (p < 0.025) between strong nuclear staining for HSP90 and high MHC class I expression was observed. In tumors with reduced MHC class I expression, no nuclear localization of HSP90 was detectable. These findings lead to the hypothesis that tumor cells with high MHC class I expression and susceptibility to CTL action may escape apoptosis by a mechanism which involves increased nuclear HSP90.

Non-micelle forming bile acids increase biliary gentamicin excretion in the rat.

To investigate whether non-micelle forming bile acids are able to increase biliary gentamicin excretion, male Sprague-Dawley rats were anesthetized with pentobarbital and fitted with a biliary fistula. After a control period of 30 min, dehydrocholate, taurodehydrocholate, or norursodeoxycholate were administered iv at doses of 2 or 10 mumol.min-1.kg-1. Taurodehydrocholate increased bile flow and biliary gentamicin clearance similarly in a dose-dependent fashion. Its unconjugated analogue, in contrast, increased gentamicin clearance fourfold, while increasing bile flow only 1.6-fold. This suggests that other than purely osmotic phenomena were involved. This effect was even more marked for the short-chain bile acid, norursodeoxycholate. At a dose of 2 mumol.min-1.kg-1 it increased bile flow by 30%, but gentamicin clearance by 210%; a similar discrepancy between choleresis and gentamicin clearance was observed at the higher dose tested. It may be concluded that conjugated triketo bile acids increase biliary gentamicin clearance by osmotic choleresis. Unconjugated triketo bile acids and nor-bile acids, to an even greater extent, increase gentamicin clearance much more markedly than bile flow; other effects, such as the putative cholahepatic shunt pathway, are responsible for this phenomenon. This novel therapeutic principle might be useful in achieving therapeutic biliary antibiotic concentrations or in treating gentamicin intoxication in patients with renal insufficiency.

Characterisation of genotoxic properties of 2',2'-difluorodeoxycytidine.

The genotoxic properties of 2',2'-difluorodeoxycytidine (dFdC) were characterised using diploid, mortal low-passage fibroblasts (LPF cells) and the spontaneously transformed fibroblast cell line V79. In both cell types, incorporation of dFdC into the DNA led to an increase of DNA single-strand breaks evaluated by an in situ nick translation assay and to an accumulation of cells in the S-phase of the cell cycle. At concentrations below those leading to cell cycle arrest, dFdC neither induced sister chromatid exchange (SCE) nor structural chromosome aberrations in LPF cells, whereas V79 cells accumulated SCEs as well as chromosome breaks over a broad dose range. In LPF cells treated with dFdC, chromosomal alterations were detected by the micronucleus assay within a narrow concentration range, whereas in V79 cells, a dose-dependent increase in the appearance of micronuclei was seen up to cytotoxic concentrations. In addition, V79 cells went into apoptosis, as evaluated by nuclear fragmentation and condensation, whereas this phenomenon was not detectable in LPF cells.

[Glutamine: effects on the immune system, protein balance and intestinal functions]. / Glutamin: Wirkungen auf das Immunsystem, auf Eiweisshaushalt und Darmfunktionen.

Glutamine is the most abundant free amino acid of the human body. In catabolic stress situations such as after operations, trauma and during sepsis the enhanced transport of glutamine to splanchnic organs and to blood cells results in an intracellular depletion of glutamine in skeletal muscle. Glutamine is an important metabolic substrate for cells cultivated under in vitro conditions and is a precursor for purines, pyrimidines and phospholipids. Increasing evidence suggests that glutamine is a crucial substrate for immunocompetent cells. Glutamine depletion in the cultivation medium decreases the mitogen-inducible proliferation of lymphocytes, possibly by arresting the cells in the G0-G1 phase of the cell cycle. Glutamine depletion in lymphocytes prevents the formation of signals necessary for late activation. In monocytes glutamine deprivation downregulates surface antigens responsible for antigen preservation and phagocytosis. Glutamine is a precursor for the synthesis of glutathionine and stimulates the formation of heat-shock proteins. Moreover, there are suggestions that glutamine plays a crucial role in osmotic regulation of cell volume and causes phosphorylation of proteins, both of which may stimulate intracellular protein synthesis. Experimental studies revealed that glutamine deficiency causes a necrotising enterocolitis and increases the mortality of animals subjected to bacterial stress. First clinical studies have demonstrated a decrease in the incidence of infections and a shortening of the hospital stay in patients after bone marrow transplantation by supplementation with glutamine. In critically ill patients parenteral glutamine reduced nitrogen loss and caused a reduction of the mortality rate. In surgical patients glutamine evoked an improvement of several immunological parameters. Moreover, glutamine exerted a trophic effect on the intestinal mucosa, decreased the intestinal permeability and thus may prevent the translocation of bacteria. In conclusion, glutamine is an important metabolic substrate of rapidly proliferating cells, influences the cellular hydration state and has multiple effects on the immune system, on intestinal function and on protein metabolism. In several disease states glutamine may consequently, become an indispensable nutrient, which should be provided exogenously during artificial nutrition.

Kaposi's sarcoma mimicking cellulitis.

Although Kaposi's sarcoma is not the most common cause of death in AIDS patients, it is often one of the initial opportunistic illnesses associated with human immunodeficiency virus infection. Extensive plaque formation and edema in the lower extremities may take on the appearance of cellulitis, and in dark-skinned persons, the lesions of the neoplasm may not be noticeable. Treatment is palliative; therapy for local effect is appropriate unless lesions are extensive or systemic involvement is present.

Serum-dependent processing of late apoptotic cells for enhanced efferocytosis.

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.

Spatial interactions in the rhesus monkey retina: a behavioural study using the Westheimer paradigm.

For two trained rhesus monkeys, increment thresholds for a small test-spot of 100 ms duration were determined as a function of background size, at 10 retinal eccentricities along the horizontal meridian. Typical 'Westheimer-functions' were obtained, i.e. threshold first increases with increasing background size, reaches a maximum, then decreases with further increasing backgrounds and finally reaches a plateau. With increasing retinal eccentricities, the position of the peak of the functions is shifted towards larger background sizes, indicating an increase of perceptive field centre size from 0.25 degrees at 5 degrees eccentricity to 1.5 degrees at 40 degrees eccentricity. The perceptive field centres tend to be slightly smaller in the nasal retina. Total perceptive field sizes, as indicated by the beginnings of the plateaus, increase from about 1 degree near the fovea to about 3 degrees at 40 degrees eccentricity. The perceptive field centre sizes of two human observers, tested under the same experimental conditions, closely resemble those of the monkeys. The total perceptive fields are larger in the human subjects. The retinal ganglion cells determining threshold in this experiment are most likely the broad-band cells. The agreement between the behaviourally determined perceptive field centre sizes and the receptive field centre sizes of broadband cells (measured by DeMonasterio and Gouras 1975) is excellent. The dendritic fields of P-alpha-ganglion cells, most likely the morphological substrates of the broad-band cells (Perry, Oehler and Cowey 1984) are somewhat smaller at all eccentricities.(ABSTRACT TRUNCATED AT 250 WORDS)