Does tumor necrosis factor-alpha (TNF-α) play a role in post-chemotherapy cerebral dysfunction?

Post-chemotherapy treated cancer patients frequently report cognitive difficulties. The biology of this phenomenon is poorly understood, with uncertainty about possible direct toxic effects on the brain, secondary effects from systemic inflammation, host factors/genetic predisposition to cognitive complaints, or hormonal changes influencing cognitive function. To elucidate possible mechanisms associated with post-treatment cognitive dysfunction among breast cancer survivors, in 2007 we established a prospective, longitudinal, observational cohort study of early stage breast cancer patients, recruited at the end of initial treatments (primary treatment exposure included surgery, ± radiation, ± chemotherapy), and prior to the initiation of adjuvant endocrine therapy. We assessed cognitive complaints, neuropsychological (NP) test performance, markers of inflammation, and brain imaging at baseline, 6 months and 12 months after enrollment. In this analysis of data from the first 93 patients enrolled in the cohort study, we focus on the relationship of circulating levels of proinflammatory cytokines to cerebral functioning and chemotherapy exposure. Among the proinflammatory cytokines tested (IL-1 ra, sTNF-RII, CRP, and IL-6) at baseline, only sTNF-RII was increased among chemotherapy exposed patients, with a significant decline in the year after treatment (p=0.003). Higher baseline sTNF-RII in chemotherapy patients was significantly associated with increased memory complaints. In chemotherapy exposed patients, the longitudinal decline in sTNF-RII was significantly correlated with fewer memory complaints over 12 months (r=-0.34, p=0.04). Higher baseline sTNF-RII was also associated with relatively diminished brain metabolism in the inferior frontal cortex (r=-0.55, p=0.02), as well as relatively increased inferior frontal metabolism after 1 year, in chemotherapy-exposed subjects. These preliminary findings suggest that post-chemotherapy increases in TNF-α may be playing an important role in the manifestations of cognitive complaints in breast cancer survivors.

Preexisting mild sleep disturbance as a vulnerability factor for inflammation-induced depressed mood: a human experimental study.

Sleep disturbance and depression are common, particularly in females, and sleep disturbance is a well-known risk factor for depression. Systemic inflammation has been suggested as a potential mechanism of this association. This study examined whether preexisting sleep disturbance acted as a vulnerability factor for depressed mood induced by an inflammatory challenge in healthy females vs males. In a randomized double-blind placebo-controlled design, volunteers aged 18-50 (N = 111; 67 females) were assigned to placebo or low-dose endotoxin. Before substance administration, sleep disturbance was assessed using the Pittsburgh Sleep Quality Index and dichotomized using median split (⩾ 3 vs < 3). Self-reported depressed mood (profile of mood states) and circulating proinflammatory cytokines (interleukin-6, tumor necrosis factor-α) were repeatedly assessed over 6 h. Among females, moderation of depressed mood by sleep disturbance was significant even after adjustment for covariates (X(2) = 12.73, df = 6, P < 0.05). There was a robust time-by-condition interaction in females with sleep disturbance (X(2) = 26.22, df = 6, P < 0.001), but not in females without sleep disturbance (X(2) = 8.65, df = 6, P = 0.19). Although cytokines increased equally in all females, the correlations between cytokines and depressed mood were significantly stronger in females with sleep disturbance. Among males, no moderating effect of sleep disturbance was observed. Inflammation-induced depressed mood was considerably more severe among females reporting mild sleep disturbance compared with those reporting no sleep disturbance, suggesting that even mild sleep disturbance may increase vulnerability for inflammation-induced depression in females. Furthermore, sleep disturbance appears to increase the vulnerability to depression by augmenting affective sensitivity to cytokines rather than by enhancing cytokine responses to inflammatory challenge in females.

Human herpesvirus 8-encoded interleukin 6 activates HIV-1 in the U1 monocytic cell line.

OBJECTIVE: Human herpesvirus 8 (HHV-8) encodes a viral interleukin 6 (vIL-6) which is structurally and functionally similar to human interleukin 6 (hIL-6). Since hIL-6 has been shown to upregulate the expression of HIV-1, the objectives of this study were to examine the ability of vIL-6 to upregulate HIV-1, and to determine the interactions of this virokine (viral cytokine) with the components of the interleukin 6 (IL-6) receptor complex. DESIGN AND METHODS: Recombinant HHV-8 vIL-6 (rvIL-6) was assayed for bioactivity in the IL-6-dependent cell line MH60.BSF2. HIV-1 p24 production by the U1 monocytic and ACH-2 T-cell lines, which are chronically infected with HIV-1, was used to assess the ability of vIL-6 to affect HIV-1 expression. hIL-6 and vIL-6 receptor utilization was determined by quantifying HIV-1 p24 production after neutralization of components of the IL-6 receptor complex, CD126'IL-6R' and CD130'gp130', on U1 cells with blocking antibodies. RESULTS: HHV-8 rvIL-6 was seen to have IL-6-like bioactivity in MH60.BSF2 cells, and readily upregulated HIV-1 p24 production in U1 monocytic cells, but not in ACH-2 T cells. The vIL-6 appeared to utilize the IL-6-specific component of the IL-6 signaling complex, CD126'IL-6R', in U1 cells. CONCLUSIONS: HHV-8 vIL-6 clearly has the potential to upregulate HIV-1 expression in monocytic cells, and therefore may play a role in AIDS pathogenesis in individuals infected with both viruses.

Serum soluble CD23 level correlates with subsequent development of AIDS-related non-Hodgkin's lymphoma.

The cytokine soluble CD23 (sCD23) has been shown to act as a B cell growth factor and to be elevated in serum prior to development of AIDS-related non-Hodgkin's lymphoma (AIDS NHL). To further characterize the elevation of serum sCD23 in AIDS NHL patients and investigate its potential as a diagnostic test, a matched case-control study of AIDS NHL (n = 101) was nested within the Multicenter AIDS Cohort Study. Serum sCD23 was measured in cases' and controls' serum specimens at three different time periods (0-6, 6-12, and 12-18 months) and CD4+ thresholds (0-99, 100-199, and 200-299 cells/microl) prior to the case's NHL diagnosis. Changes in serum sCD23 over time were examined in AIDS NHL cases relative to controls, and t tests were performed to determine whether cases' serum sCD23 exceeded that of controls at each time period and CD4+ threshold. Overall, cases' median serum sCD23 levels were approximately double those of controls. Serum sCD23 concentration was positively correlated with lymphocyte counts for both cases and controls. The difference in cases' and controls' serum sCD23 levels became greater as AIDS NHL diagnosis date approached: in the 18 months preceding the case's NHL diagnosis, serum sCD23 was stable in cases but dropped in controls. Although this difference was statistically significant (P < 0.05), it was not clinically significant. It is unlikely that serum sCD23 would make a useful test for AIDS NHL because the magnitude of the difference between cases and controls was small and there was no change in serum sCD23 in cases that would indicate disease.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro.

Tissue remodeling is an adaptive response to mechanical tension in the lung. However, the role of pulmonary fibroblasts in this response has not been well characterized. This study investigates the influence of extracellular matrix on the response of fibroblasts to mechanical strain. Cells were cultured on flexible-bottom surfaces coated with fibronectin, laminin, or elastin and exposed to strain. Under these conditions, fibroblasts align perpendicular to the force vector. This stimulus results in an increase in alpha(1)(I) procollagen mRNA in cells cultured on laminin or elastin but not fibronectin. Increased alpha(1)(I) procollagen mRNA was detected 6 h after exposure to strain and reached control levels by 72 h. [(3)H]proline incorporation into newly synthesized procollagen reflects changes in mRNA levels. Strained fibroblasts cultured on laminin or elastin incorporated 190 and 114%, respectively, more [(3)H]proline into procollagen than did unstrained cells. No difference was detected in strained fibroblasts cultured on fibronectin. These results suggest that fibroblasts respond to mechanical strain in vitro, and this response is signaled by cell-extracellular matrix interactions.

High vascular and airway pressures increase interstitial protein mRNA expression in isolated rat lungs.

We hypothesized that wall stresses produced by high peak airway (Paw) and venous (Ppv) pressures would increase mRNA levels for structural proteins of the interstitial matrix in isolated rat lungs. Groups of lungs (n = 6) were perfused for 4 h at a peak Paw of 35 cmH2O (HiPaw), cyclical peak Ppv of 28 cmH2O (HiPv), or baseline vascular and airway pressures (LoPress). In two separate groups, comparable peak pressures increased capillary filtration coefficient fourfold in each group. Northern blots were probed for mRNA of alpha 1(I), alpha 1(III), and alpha 2(IV) procollagen chains, laminin B chain, fibronectin, and transforming growth factor-beta 1, and densities were normalized to 18S rRNA. mRNA was significantly higher in the HiPv group for type I (4.3-fold) and type III (3.8-fold) procollagen and laminin B chain (4.8-fold) and in the HiPaw group for type I (2.4-fold) and type IV (4.5-fold) procollagen and laminin B chain (2.3-fold) than in the LoPress group. Only fibronectin mRNA was significantly increased (3.9-fold) in the LoPress group relative to unperfused lungs. Estimated wall stresses were highest for alveolar septa in the HiPaw group and for capillaries in the HiPv group. The different patterns of mRNA expression are attributed to different regional stresses or extent of injury.

Chronic hypoxia attenuates resting and exercise-induced VEGF, flt-1, and flk-1 mRNA levels in skeletal muscle.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic mitogen. However, chronic hypoxia is generally not found to increase mammalian skeletal muscle capillarity. We sought to determine the effect of chronic hypoxia (8 wk, inspired O2 fraction = 0.12) on skeletal muscle gene expression of VEGF, its receptors (flt-1 and flk-1), basic fibroblast growth factor, and transforming growth factor-beta1. Wistar rats were exposed to chronic hypoxia (n = 12) or room air (n = 12). After the exposure period, six animals from each group were subjected to a single 1-h treadmill exercise bout (18 m/min on a 10 degrees incline) in room air while the remaining six animals served as rest controls. Morphological analysis revealed that chronic hypoxia did not increase skeletal muscle capillarity. Northern blot analyses showed that chronic hypoxia decreased resting VEGF, flt-1, and flk-1 mRNA by 23, 68, and 42%, respectively (P < 0.05). The VEGF mRNA response to exercise was also decreased (4.1- and 2.7-fold increase in room air and chronic hypoxia, respectively, P < 0.05). In contrast, neither transforming growth factor-beta1 nor basic fibroblast growth factor mRNA was significantly altered by chronic hypoxia. In conclusion, prolonged exposure to hypoxia attenuated gene expression of VEGF and its receptors flt-1 and flk-1 in rat gastrocnemius muscle. These findings may provide an explanation for the lack of mammalian skeletal muscle angiogenesis that is observed after chronic hypoxia.

Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia.

Gene expression of vascular endothelial growth factor (VEGF), and to a lesser extent of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF), has been found to increase in rat skeletal muscle after a single exercise bout. In addition, acute hypoxia augments the VEGF mRNA response to exercise, which suggests that, if VEGF is important in muscle angiogenesis, hypoxic training might produce greater capillary growth than normoxic training. Therefore, we examined the effects of exercise training (treadmill running at the same absolute intensity) in normoxia and hypoxia (inspired O(2) fraction = 0.12) on rat skeletal muscle capillarity and on resting and postexercise gene expression of VEGF, its major receptors (flt-1 and flk-1), TGF-beta(1), and bFGF. Normoxic training did not alter basal or exercise-induced VEGF mRNA levels but produced a modest twofold increase in bFGF mRNA (P < 0.05). Rats trained in hypoxia exhibited an attenuated VEGF mRNA response to exercise (1.8-fold compared 3.4-fold with normoxic training; P < 0.05), absent TGF-beta(1) and flt-1 mRNA responses to exercise, and an approximately threefold (P < 0.05) decrease in bFGF mRNA levels. flk-1 mRNA levels were not significantly altered by either normoxic or hypoxic training. An increase in skeletal muscle capillarity was observed only in hypoxically trained rats. These data show that, whereas training in hypoxia potentiates the adaptive angiogenic response of skeletal muscle to a given absolute intensity of exercise, this was not evident in the gene expression of VEGF or its receptors when assessed at the end of training.

Nitric oxide synthase inhibition attenuates the skeletal muscle VEGF mRNA response to exercise.

Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta(1) (TGF-beta(1)) mRNA increase in rat skeletal muscle in response to a single acute exercise bout. Nitric oxide (NO) is released locally by muscle vascular endothelium and muscle fibers during exercise, contributes to the blood flow response to exercise, and regulates mitochondrial respiration. We hypothesized that a reduction in NO production, via NO synthase inhibition, would demonstrate a link between NO and the VEGF, bFGF, and TGF-beta(1) gene responses to exercise. To investigate this hypothesis, 9-wk-old female Wistar rats were divided into eight treatment groups (n = 6 each): 1) saline + rest, 2) saline + exercise, 3) 30 mg/kg N(omega)-nitro-L-arginine methyl ester (L-NAME, a known NOS inhibitor) + rest, 4) 30 mg/kg L-NAME + exercise, 5) 300 mg/kg L-NAME + rest, 6) 300 mg/kg L-NAME + exercise, 7) 300 mg/kg N(omega)-nitro-D-arginine methyl ester (D-NAME, inactive enantiomer of L-NAME) + rest, and 8) 300 mg/kg D-NAME + exercise. Exercise consisted of 1 h of running at 20 m/min on a 10 degrees incline. VEGF, TGF-beta(1), and bFGF mRNA from left gastrocnemius were analyzed by quantitative Northern blot. Submaximal exercise for 1 h increased VEGF mRNA 4.2-fold and TGF-beta(1) mRNA 1.5-fold in untreated rats but did not increase bFGF mRNA. The exercise-induced increase in VEGF mRNA was attenuated approximately 50% by 30 and 300 mg/kg L-NAME; the TGF-beta(1) mRNA increase was unaffected by 300 mg/kg L-NAME. In addition, 300 mg/kg D-NAME had no effect on the exercise-induced increase in VEGF mRNA. Administration of 300 mg/kg L-NAME had no effect on bFGF mRNA. These findings suggest that NO is important in the regulation of the VEGF gene response to exercise through increases in VEGF transcription or by increases in the VEGF mRNA half-life.

Effect of captopril on skeletal muscle angiogenic growth factor responses to exercise.

Acute exercise increases vascular endothelial growth factor (VEGF), transforming growth factor-beta(1) (TGF-beta(1)), and basic fibroblast growth factor (bFGF) mRNA levels in skeletal muscle, with the greatest increase in VEGF mRNA. VEGF functions via binding to the VEGF receptors Flk-1 and Flt-1. Captopril, an angiotensin-converting enzyme inhibitor, has been suggested to reduce the microvasculature in resting and exercising skeletal muscle. However, the molecular mechanisms responsible for this reduction have not been investigated. We hypothesized that this might occur via reduced VEGF, TGF-beta(1), bFGF, Flk-1, and Flt-1 gene expression at rest and after exercise. To investigate this, 10-wk-old female Wistar rats were placed into four groups (n = 6 each): 1) saline + rest; 2) saline + exercise; 3) 100 mg/kg ip captopril + rest; and 4) 100 mg/kg ip captopril + exercise. Exercise consisted of 1 h of running at 20 m/min on a 10 degrees incline. VEGF, TGF-beta(1), bFGF, Flk-1, and Flt-1 mRNA were analyzed from the left gastrocnemius by quantitative Northern blot. Exercise increased VEGF mRNA 4.8-fold, TGF-beta(1) mRNA 1.6-fold, and Flt-1 mRNA 1.7-fold but did not alter bFGF or Flk-1 mRNA measured 1 h after exercise. Captopril did not affect the rest or exercise levels of VEGF, TGF-beta(1), bFGF, and Flt-1 mRNA. Captopril did reduce Flk-1 mRNA 30-40%, independently of exercise. This is partially consistent with the suggestion that captopril may inhibit capillary growth.

High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma.

Remodeling of pulmonary capillaries occurs after chronic increases in capillary pressure (e.g., mitral stenosis). Also, remodeling of pulmonary arteries begins within 4 h of increased wall stress and is endothelium dependent. We have previously shown that high lung inflation increases wall stress in pulmonary capillaries. This study was designed to determine whether high lung inflation induces remodeling of the extracellular matrix (ECM) in lung parenchyma. Open-chest rabbits were ventilated for 4 h with 9-cmH2O positive end-expiratory pressure (PEEP) on one lung and 1-cmH2O PEEP on the other (High-PEEP group), or with 2-cmH2O PEEP on both lungs (Low-PEEP group). An additional untreated control group was also included. We found increased levels of mRNA in both lungs of High-PEEP rabbits (compared with both the Low-PEEP and untreated groups) for alpha1(III) and alpha2(IV) procollagen, fibronectin, basic fibroblast growth factor, and transforming growth factor-beta1. In contrast, alpha2(I) procollagen and vascular endothelial growth factor mRNA levels were not changed. We conclude that high lung inflation for 4 h increases mRNA levels of ECM components and growth factors in lung parenchyma.

Angiogenic growth factor mRNA responses in muscle to a single bout of exercise.

A major adaptation to exercise is new capillary formation in skeletal muscle. On the basis of angiogenesis in tumors and during development, several angiogenic growth factors may be involved, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1 (TGF-beta 1). In 9-wk-old female Wistar rats, mRNA expression for these three growth factors in gastrocnemius muscle was examined by quantitative Northern analysis after a single 1-h run at 15 or 20 m/min at 10 degrees incline in room air. A third group ran at 15 m/min in 12% O2, and resting control groups were included at inspired O2 fractions of 0.21 and 0.12. Exercise significantly increased mRNA levels two- to fourfold, which was evident over the first 4 h postexercise; by 8 and 24 h, mRNA levels returned to baseline. For all three factors, mRNA levels were significantly higher after exercise at 20 than at 15 m/min. Hypoxia at rest doubled VEGF and TGF-beta 1 message but had no effect on bFGF. Hypoxic exercise further raised VEGF mRNA levels but had no effect on the other factors. We suggest that VEGF, bFGF, and TGF-beta 1 may be involved in the angiogenic response to exercise and that reduced intracellular PO2 (as occurs during normoxic exercise) may be part of the stimulus to such growth factor production.

The association between pro-inflammatory cytokines, regional cerebral metabolism, and cognitive complaints following adjuvant chemotherapy for breast cancer.

To examine relationships following adjuvant chemotherapy between circulating pro-inflammatory cytokines, regional cerebral metabolism, and cognitive complaints in early stage breast cancer patients. 33 breast cancer patients who had completed initial treatment (surgery, ± radiation, 23 chemotherapy, 10 no chemotherapy) obtained resting (18)F-FDG PET/CT brain imaging at baseline and 1 year later. Pro-inflammatory cytokine markers (IL-1ra, sTNF-RII, CRP, and IL-6) and cognitive complaints were also assessed at both time points. At baseline, consistent correlations were seen between the left medial frontal and right inferior lateral anterior temporal cortices and inflammatory markers within the chemotherapy group, and not in the no chemotherapy group. After 1 year, correlations persisted in the medial frontal cortex and the temporal cortex, the latter shifting superiorly. Both of these regional correlations demonstrated the highest levels of significance when looking across the 1 year time frame (IL-1ra: peak voxel p < 0.0005; cluster size p < 0.0005, p = 0.001 after correction (medial prefrontal), p < 0.0005; cluster size p = 0.001, p = 0.029 corr. (anterior temporal), sTNF-RII: p < 0.0005; cluster size p = 0.001, p = 0.040 corr. (medial prefrontal)). Positive correlations were also seen within the chemotherapy group between baseline memory complaints and the medial frontal (p < 0.0005; cluster size p < 0.0005, p < 0.0005 corr.) and anterior temporal (p < 0.0005; cluster size p < 0.0005, p = 0.002 corr.) cortices at baseline and 1 year later. Metabolism in the medial prefrontal cortex and anterior temporal cortex was found to correlate with both memory complaints and cytokine marker levels in chemotherapy patients.

Tubulin-targeting agents in hybrid drugs.

The targeting of tubulin is an important mechanism for cancer chemotherapy. However, limitations such as resistance, toxicity and incomplete tumour elimination associated with individual anti-cancer drugs have led to a need for combination therapy in cancer. It is therefore relevant to ask whether two or more drugs might be combined in a single hybrid molecule to advantageous effect. This review provides an overview of the hybrid drugs thus far investigated, in which at least one component targets tubulin. The rationale behind this approach is that the hybrid drug may have activity enhanced above and beyond that of the equivalent drug combination, or have an otherwise improved clinical outcome. Particular emphasis is placed on the investigation of activity in multidrug-resistant cancer cell lines. Attention is drawn to the difficulties encountered when developing hybrid drugs, with respect to in vivo metabolism-tracking, increased molecular bulk, and optimisation of the drug dosage ratio. The actual and potential advantages and disadvantages of such hybrid drugs when compared to single drugs or drug combinations are discussed critically and promising directions for future research is highlighted.

Calcyclin gene expression is increased by mechanical strain in fibroblasts and lung.

Mechanical tension extending throughout the structural elements of the lung is a potential stimulus for cell proliferation and gene expression. Pulmonary fibroblasts located in the interstitial space of the capillary wall throughout the lung parenchyma and within the large vessels and airways are uniquely situated to sense changes in mechanical force. Therefore, we used the polymerase chain reaction-based method of differential display analysis to screen for altered gene expression in fetal human lung fibroblasts exposed to increased cyclic stretch. IMR-90 cells were seeded at 3 x 10(4) cells/cm(2) on laminin-coated plates. Cells were subsequently exposed to mechanical strain on a Flexercell apparatus, resulting in a maximal elongation of 20% at a rate of 60 cycles/min over a period of 48 h. A complementary DNA corresponding to the cell cycle-regulated gene calcyclin was identified in mechanically strained fibroblasts. Increased calcyclin messenger RNA levels were confirmed by Northern blot analysis. Further, calcyclin gene expression was upregulated in isolated-perfused rat lungs exposed to increased mechanical strain by ventilation at high states of lung inflation for 4 h. These data suggest that calcyclin gene expression plays a role in the response of pulmonary fibroblasts to increased mechanical tension and may alter the regulation of the fibroblast cell cycle.

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons.

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.

Cytokine gene expression in normal human lymphocytes in response to stimulation.

Sequential gene expression of two type 1 cytokines (interleukin 2 [IL-2] and gamma interferon), one type 2 cytokine (IL-10), two monokines (IL-6 and tumor necrosis factor alpha), and one cytokine receptor (IL-2 receptor [IL-2R]) in normal human peripheral blood mononuclear cells (PBMC) following in vitro stimulation was investigated by reverse transcription-PCR methods. Two stimuli were utilized: phytohemagglutinin (PHA), which acts on the CD2 molecule and T-cell receptors, and anti-CD3 monoclonal antibody, which acts on the CD3 molecule and on T-cell receptors. Increased expression of all studied genes occurred between 1 and 4 hours after stimulation, except for that of the gene encoding IL-10, which was delayed. Expression of all but one of the genes was transient, with a maximal mRNA accumulation at about 8 h on average. IL-2R mRNA expression was an exception, showing a prolonged increase (72 h). The general profiles of expression of the five cytokine genes were similar but not identical, suggesting some shared regulatory mechanisms. When responses to four additional stimuli (pokeweed mitogen, Candida albicans, and IL-2 at high and low doses) were compared, similar profiles of cytokine gene expression were found. Thus, the various stimuli caused induction of all cytokines with quantitative, not qualitative, differences. Altogether, the present data are useful for defining the kinetics of gene expression for key cytokines in response to standard immune-cell stimuli.

Alveolar hypoxia increases gene expression of extracellular matrix proteins and platelet-derived growth factor-B in lung parenchyma.

The walls of pulmonary capillaries are extremely thin, and wall stress increases greatly when capillary pressure rises. Alveolar hypoxia causes pulmonary vasoconstriction and hypertension, and if this is uneven, some capillaries may be exposed to high transmural pressure and develop stress failure. There is evidence that increased wall stress causes capillary remodeling. In this study we exposed Madison strain Sprague-Dawley rats to normobaric hypoxia (10% oxygen) for 6 h or 3 d (short-term group), and for 3 d or 10 d (long-term group). Peripheral lung tissue was then collected and messenger RNA (mRNA) levels were determined for extracellular matrix (ECM) proteins and growth factors. Collagen content (hydroxyproline) was also measured. Levels of mRNA for alpha2(IV) procollagen increased sixfold after 6 h of hypoxia and sevenfold after 3 d of hypoxia, and then decreased after 10 d exposure. Levels of mRNA for platelet-derived growth factor-B (PDGF-B) doubled after 6 h of hypoxia but returned to control values after 3 d. mRNA levels for alpha1(I) and alpha1(III) procollagens and fibronectin were increased after 3 d of hypoxia (by seven- to 12-fold, 1.6- to eightfold, and 12-fold, respectively), then decreased toward control values after 10 d. In contrast, neither levels of mRNA for vascular endothelial growth factor (VEGF) nor collagen content changed. These results suggest that alveolar hypoxia causes vascular remodeling in lung parenchyma, and are consistent with capillary wall remodeling in response to increased wall stress.

In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.

The steroid hormone vitamin D is a principal mediator of skeletal homeostasis. 1,25-Dihydroxyvitamin D3 treatment of ROS 17/2.8 osteoblast-like cells results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. This transcriptional upregulation requires the positive cis-acting vitamin D responsive element (VDRE). We have used the ligation-mediated polymerase chain reaction to demonstrate that protein occupancy of the VDRE within the intact cell correlates with increased synthesis of osteocalcin transcripts. These protein-DNA contacts were not present in the absence of vitamin D or in osteosarcoma cells (ROS 24.1) lacking the vitamin D receptor. Our results establish in intact cells the requirement for both ligand- and receptor-dependent occupancy of the VDRE for vitamin D responsive enhancement of osteocalcin gene transcription.