Cocaine body packing in pregnancy

Ingesting multiple packets of drugs ("body packing") is a well-described method of smuggling. Although older reports suggested that body packers were mostly young men, the demographics of this group may be changing because children, older patients, and pregnant women may be involved. Pregnant patients represent a challenge in management, particularly in the event of package rupture. Modification of standard management protocols, which were developed for nonpregnant body packers, may be necessary to address the anatomic and physiologic changes of pregnancy. We report the case of a pregnant cocaine body packer who required a perimortem cesarean section after the rupture of a cocaine packet. The care of the pregnant body packer is discussed.     

Reorganized small intestine from fetal mouse as an in vitro wound healing model

BACKGROUND: We developed a method for reorganizing the mouse small intestine. In the present study, we investigated whether the reorganized small intestine was morphologically and histochemically differentiated. We also evaluated the reorganized small intestine as an in vitro wound healing model. METHODS: Fetal mouse small intestines were dispersed into single cells, which were then cultured to a high density. Newly formed small intestine-like organs on a membrane filter were observed by light and electron microscopy. Alkaline phosphatase (ALPase) activity of the epithelium was analyzed. To evaluate the reorganized small intestine as an in vitro wound healing model, a scalpel was used to cut the reorganized intestine on a membrane, and the healing process was morphologically and immunohistochemically examined. RESULTS: After 6 days in culture, the surface was almost completely coveed with epithelial cells, and villus-like structures were observed. These epithelial cells formed microvilli, and in parallel with this development, ALPase activity of the microvilli increased (from day 4). Twenty-four hours after the cutting, the wound surface was almost completely covered with undifferentiated epithelial cells. The number of acetylated low-density lipoprotein labeled with 1,1,dioctadecyll,3,3,3,3, tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL)-positive macrophages increased after cutting. Platelet-derived growth factor (PDGF)-, basic fibroblast growth factor (bFGF)-, matrix metalloproteinase-1 (MMP-1)-positive cells were detected by immunohistochemical staining. CONCLUSIONS: The reorganized small intestine had a morphologically and histochemically differentiated organoid structure, and was useful as an in vitro model for investigating the process of wound healing.     

Transepithelial migration of human neutrophils: an in vitro model system.

An in vitro model system for studying transepithelial migration of human neutrophils has been developed. Canine kidney epithelial cells grown on micropore filters form a confluent, polarized monolayer with an average transepithelial electrical resistance of 181 ohms.cm2. Neutrophils in a chemotactic chamber are stimulated to undergo random migration, chemokinesis, or chemotaxis through the epithelium. When stimulated by a gradient of the synthetic chemoattractant fMet-Leu-Phe, significantly more neutrophils traverse the low-resistance epithelium than do under conditions of random migration or chemokinesis. Transmission and scanning electron microscopy of this process reveal that neutrophils traverse the epithelium through the intercellular space. After leukocyte emigration, lateral epithelial cell membranes reapproximate. Neutrophils undergoing chemotaxis can also traverse the polarized epithelium from the basal epithelial surface, which suggests that the chemotactic gradient and not the apical-basal polarity of the epithelial cells determines the direction of transepithelial migration. The data further suggest that (i) the in vitro model of leukocyte transepithelial migration morphologically simulates the in vivo process, (ii) neutrophils more readily penetrate the epithelium when attracted by a chemotactic factor, and (iii) neutrophils can traverse a low-resistance epithelium in the absence of serum and connective tissue factors.     

Kinetics of in vitro bronchoconstriction in an elastolytic mouse model of emphysema.

Thin-slice videomicroscopy was used to examine the kinetics of constriction in small airways in situ. Balb/C mice inhaled elastase (0-20 IU), and were then left to recover for 14 days before euthanisation and lung removal. Cholinergic responsiveness was assessed in thin lung slices. Magnitude and velocity of narrowing in response to 10(-5) M acetylcholine (ACh), as well as the full concentration-response relationship for ACh (10(-8)-10(-5) M) were assessed. In vivo exposure to elastase was accompanied by statistically significantly decreased magnitudes and velocities of contraction, but no change in the ACh concentration-response relationship. Conversely, overnight, in vitro exposure of slices from control animals to elastase (2.5 microg.mL(-1)) resulted in increased magnitudes and velocities of airway narrowing, with impaired relaxation, as well as marked tearing of the airways from the surrounding parenchyma. These changes are characteristic of decreased tethering forces on the airway wall. Thus, the lung slice technique coupled with videomicroscopic analysis of airway contraction velocities provides a powerful tool to study airway-parenchymal interactions. The elastolytic model of emphysema, which manifests with airspace enlargement and loss of parenchymal attachments, is accompanied by decreased airway contraction kinetics. The mechanism(s) underlying this loss of function remain to be elucidated.     

Cocaine produces coronary artery vasoconstriction independent of an intact endothelium.

Studies have demonstrated that cocaine causes coronary vasoconstriction, but this has been unassociated with myocardial ischemia. Therefore, cocaine seems unlikely to precipitate myocardial infarction in the absence of potentiating factors. We hypothesized that injury to coronary endothelium could potentiate cocaine-induced coronary vasoconstriction by decreasing EDRF. The effect of cocaine on LAD diameter was measured in dogs subjected to coronary endothelial denudation and compared with that in a non-denuded control group. Endothelial denudation was accomplished by abrasion with an inflated angioplasty balloon and confirmed in vivo by demonstrating a vasoconstrictive response to infused acetylcholine and by postmortem scanning electron microscopy. Cocaine produced a similar maximal reduction in LAD diameter in both groups. Thus, cocaine induces endothelium-independent coronary artery vasoconstriction. Failure of endothelial injury to potentiate the coronary vasoconstrictive effect by cocaine suggests that factors other than endothelial dysfunction may be important in pathogenesis of cocaine-associated myocardial infarction.     

KBM-3, an in vitro model of human acute myelomonocytic leukemia.

A human acute myelomonocytic leukemia cell line, KBM-3, was developed to study the pathophysiology of human acute myeloid leukemia. This cell line was characterized by morphology, immunophenotype, Giemsa-banding pattern, in vitro proliferation capacity, and tumorigenicity in nude mice. The KBM-3 cell line was established in the presence of exogenous lymphokines (human placenta-conditioned medium, HPCM), but medium for later passages did not contain HPCM. We found high cellular expression of the mRNA message for granulocyte-macrophage colony-stimulating factor (GM-CSF), which we suggest may be important for the immortalization of the cell line. KBM-3 cells have an immature myelomonocytic phenotype. Cytogenetic analysis revealed a pseudodiploid karyotype with five characteristic marker chromosomes and ranging in total number from 45 to 49. In suspension cultures, the cells had a doubling time of 23 h and a cloning efficiency of about 30% in soft agar independent of exogenous lymphokines. Two-thirds of nude mice injected with 1 x 10(4) KBM-3 cells and all animals injected with 1 x 10(5) cells developed S.C. granulocytic sarcomas within 6-8 weeks. These tumors were locally invasive but did not give rise to distant metastases. When transplanted to a new set of nude mice, all tumors formed secondary sarcomas at the site of implant. We conclude that the KBM-3 cell line may have value for studying the molecular events that underlie the neoplastic transformation in human myeloid leukemia.     

Prevention of CMV-induced myelosuppression by anti-CMV antibodies: an in vitro model.

Pathogenesis of cytomegalovirus (CMV)-induced myelosuppression is not clearly understood and could be related to a direct toxic effect on the marrow progenitors and/or an alteration of the marrow environment. Myeloid progenitors (granulocyte-macrophage colony-forming units, CFU-GM) were not affected by incubation with increasing titers of CMV (10-10(5) plaque-forming units [pfu]/ml) during 2-6 h. By contrast, using the blast colony-forming cell (Bl-CFC) assay, we confirmed that CMV induced myelosuppression through an alteration of the marrow-derived stromal layer. Using this experimental model, we compared the capacity of nonspecific human immunoglobulins (IgG), specific polyclonal anti-CMV IgG, and a human monoclonal anti-CMV IgG to prevent the myelosuppressive effect of 10(4) pfu/ml of CMV. Specific anti-CMV IgG (polyclonal or monoclonal) at the concentration of 10 micrograms/ml were able to prevent the CMV-induced myelosuppressive effect, whereas nonspecific human IgG was not effective in this model. Our results suggest that 1) CMV-induced myelosuppression is related to an alteration of the marrow microenvironment, 2) specific monoclonal and polyclonal anti-CMV IgG prevent this myelosuppressive effect in vitro, and 3) human monoclonal anti-CMV IgG could be useful in vivo in the immunoprophylaxis of CMV infections.     

Cell-type specificity of preconditioning in an in vitro model

We investigated whether preconditioning could protect several cultured cell lines, to determine whether the protection is specific for cells derived from different myogenic and non-myogenic sources. Ischemia was simulated by centrifugation of cells into a pellet, and cell viability was determined by hypotonic trypan blue solution. Preconditioning was produced by brief exposures to either glucose-free solution or metabolic inhibition. Freshly isolated rabbit ventricular myocytes were studied to confirm that preconditioning occurs in this model. We then compared these results to those in several cultured cell lines, including HEK 293 cells derived from human embryonic kidney, HIT-T15 cells from Syrian hamster pancreatic islets, and C₂C₁₂ cells from mouse skeletal muscle. In the latter cell line, we also determined whether differentiation alters preconditioning. Preconditioning protected rabbit ventricular myocytes: the percentage of dead cells was decreased from 36.8±4.7% in the control group to 23.0±5.2% in the preconditioned group after 60 min and from 50.7±2.1% in the control group to 25.5±4.5% in the preconditioned group after 120 min ischemia (p<0.02). In contrast, there was no protection from preconditioning in HEK 293 cells or HIT-T15 cells. Preconditioning did not protect C₂C₁₂ myoblasts either. Interestingly, after C₂C₁₂ myoblasts had differentiated into myotubes (induced by exposing the cells to low-serum medium), they could then be protected by preconditioning (46.3±3.6% in the control group vs 26.0±2.7% in the preconditioned group after 60 min and 67.4±3.6% in the control group vs 46.0±4.6% in the preconditioned group after 120 min ischemia; p<0.05). In conclusion, protection from preconditioning is cell-type specific. The presence of endogenous K_ATP channels (which are plentiful in HIT-T15 cells) is insufficient to enable preconditioning of the cell. Among the various cell types studied, only differentiated muscle cells (rabbit ventricular myocytes and C₂C₁₂ myotubes) exhibited preconditioning.     

Osteoclast-like cells in an in vitro model of bone destruction by rheumatoid synovium.

OBJECTIVE: Osteoclasts may be involved in the process of rheumatoid bone destruction. To test this hypothesis, we developed an in vitro model of bone destruction by osteoclast-like cells derived from cultured rheumatoid synovial tissue without using any inducers. METHODS: Synovial tissues were obtained from rheumatoid arthritis and osteoarthritis patients and tissue pieces of about 2 mm(3) that contained synovial lining were cultured. Multinucleated cells derived from cultured synovial tissues were studied cytochemically and morphologically for osteoclast-specific markers. RESULTS: Fibroblast-like and macrophage-like cells from the tissue pieces proliferated in the coexistence of lymphocytes. After 14 days of culture, multinucleated cells with tartrate-resistant acid phosphatase activity appeared. These cells expressed vacuolar H(+)-ATPase, the vitronectin receptor and cathepsin K. Although binding of (125)I-labelled salmon calcitonin was very low, the cells contained ringed structures of F-actin and showed strong bone-resorbing activity on ivory slices. Proliferation of macrophage-like cells and formation of multinucleated cells continued during 6 months of culture in the presence of fibroblast-like cells. The bone-resorbing activity of multinucleated cells derived from rheumatoid synovial tissue was much higher than that of cells from osteoarthritis synovial tissue, and was related to the disease activity of rheumatoid arthritis. CONCLUSION: Our culture system reproduced in vitro the process of bone destruction by rheumatoid synovium, including the proliferation and fusion of precursor cells, polarization, activation and bone tissue resorption. This system may provide a tool for understanding the mechanisms of bone destruction in rheumatoid arthritis and for the development of new therapies to prevent bone destruction.     

Prototype of an in vitro model of the microcirculation

We have used microfabrication technology to construct a network of microchannels, patterned after the dimensions and architecture of the mammalian microcirculation. The network is cast in transparent silicone elastomer and the channels are coated with silanated mPEG to provide lubrication. Flow of red and white blood cells through the network is readily visualized by the use of high-speed digital image acquisition. The acquired sequences of high-quality images are used to calculate hematocrits and rates of red cell movement in the microchannels. Our prototype system has significant advantages over scaled-up room-size experimental systems in that it permits experimentation with actual human blood cells. Experiments can be carried out under well-controlled conditions in a network of microchannels with precisely known dimensions using cell suspensions of defined composition. Moreover, there is no need to counteract or anticipate the host's adaptive responses that may confound live animal experiments. Notwithstanding its limitations, the current prototype demonstrates certain features characteristic of the microcirculation, such as parachute and bullet shapes of red cells deformed in capillary channels, rouleaux formation, plasma skimming, and the utilization of collateral flow pathways due to flow obstruction caused by a white cell blocking a microchannel. We present this device as a prototype scale-to-scale model of the mammalian microcirculation. Limitations of the system as well as a variety of possible applications are described.     

Cocaine: an independent risk factor for aortic sudanophilia. A preliminary report.

Several recent autopsy reports indicate an increased prevalence of coronary atherosclerosis in ischemic heart disease temporally associated with cocaine abuse. The objective of this study was to conduct a retrospective analysis of sudanophilic lesions in young asymptomatic individuals who abused cocaine. Twenty-six cases (15-34-year-old black males) were examined from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Sixteen subjects (mean age 25 +/- 1 years) had a positive toxicologic screen for cocaine and/or its major metabolites at autopsy and were confirmed habitual cocaine abusers. The remaining 10 cases (mean age 24 +/- 2 years) were subjects with a negative toxicologic screen at autopsy and no history of illicit drug abuse. Post-mortem blood was collected for lipoprotein analysis and determination of smoking status. The aorta and right coronary arteries were stained with Sudan IV and the degree and extent of sudanophilia was quantitated by image analysis. Multiple linear regression analysis of cocaine, age, smoking status, VLDL+LDL-C/HDL-C ratio and HDL-C as predictor variables of percentage intimal surface involvement, revealed an association between cocaine abuse and the extent of sudanophilia in both the thoracic and abdominal aorta (P = 0.002 and 0.049, respectively). Analysis of risk factors or of cocaine abuse as predictors of sudanophilia did not achieve statistical significance in the right coronary artery. These preliminary results suggest that habitual use of cocaine, through unknown mechanism(s), increases aortic sudanophilia independent of traditional risk factors.     

Ventricular reentry around a fixed barrier. Resetting with advancement in an in vitro model

We studied an in vitro model of reentrant tachycardia in a ring of ventricular endocardial tissue surrounding the canine mitral and aortic valves to understand how the response of a reentrant tachycardia to premature impulses can provide insight into the underlying tachycardia mechanism, circuit characteristics, and nature of the central barrier. Reproducible regular reentrant tachycardias (cycle length range, 177-450 msec) were induced with programmed stimulation in 19 intact preparations studied at 34-38 degrees C. Tachycardias were sustained and stable until terminated by programmed stimulation in 95% of preparations. Reentry was reliably reinitiated during experiments lasting 2-15 hours. Data supporting reentry as the mechanism of these tachycardias included sequential activation around the ring that spanned the cycle length of the tachycardia, unidirectional block during initiation of the reentrant rhythm, and termination of the tachycardia after interruption of the circuit. Tachycardias in 13 preparations were systematically reset by premature stimuli. During reentry, each of these preparations had full recovery of excitability by the end of their excitable gap as evidenced by a flat portion along their resetting response curve (eight of 13) or by lack of faster conduction velocity during the second poststimulus beat after premature impulses that produced a long return cycle (13 of 13). From analysis of the conduction of premature impulses and their return cycles, we reached several conclusions useful for interpreting resetting response curves when the reentrant circuit is not fully accessible for study. The duration of a flat portion of the resetting response curve indicated the duration of the shortest fully recovered excitable gap in the reentrant circuit. The window of reset of the tachycardia reflected only the local excitable gap at the site of stimulation and did not define the shortest excitable gap within the circuit. The extent of advancement of the tachycardia provided a lower-limit estimate of the shortest excitable gap in the reentrant circuit. Advancement of a tachycardia in time by premature stimuli indicated advancement at each point in the circuit. Finally, for tachycardias advanced by premature impulses, the length of the reentrant path cannot be determined by the recovery of a refractory barrier.     

Biological pacemakers: characterization in an in vitro coculture model

Background

Biological pacemakers could be an alternative or complement to electronic pacemakers. Embryonic stem cells (ESCs) can be differentiated in vitro to spontaneously active cells. Although numerous studies show that ESC-derived cardiomyocytes (ESC-CMs) and other cell types are capable to exert pacemaker function in vivo, detailed analyses of pattern and safety of conduction on a tissue level are rare.

Methods

Murine ESCs (mESCs) expressing enhanced green fluorescent protein and puromycin resistance under control of the promoter of α-myosin (heavy chain) were differentiated to cardiomyocytes (mESC-CMs) and purified by negative antibiotic selection. Ventricles of mouse embryonic hearts (embryonic day 16.5) were embedded in agarose and sliced along the short axis. Clusters of mESC-CMs and the murine, vital heart slices were cocultured on multielectrode arrays for 4 days. Field potentials and videos were recorded daily to investigate beating behavior and excitation spreading within the slice.

Results

On the first day of coculture, the mean beating rate of the tissue slices cocultured with mESC-CMs (n = 19) did not differ significantly from the beating rate of control slices (n = 19) (37 ± 10 versus 19 ± 7 bpm, P = .133). After 4 days of coculture, beating rates were significantly higher in cocultures than in control slices (154 ± 22 versus 49 ± 8 bpm, P < .001). On day 4, 1:1 coupling could be found in 1 of 19 preparations; 2:1, 3:1, or 4:1 coupling in another 4 of 19 preparations; 14 of 19 propagation patterns were irregular.

Conclusion

In this in vitro model, the increase of the beating rate suggests that purified mESC-CMs can pace native heart tissue, albeit with low efficiency.     

Cocaine vaccines: antibody protection against relapse in a rat model

The efficacy of active immunization with the cocaine immunogen GNC-keyhole limpet hemocyanin (KLH) in preventing cocaine self-administration reinstatement was assessed in rats. An animal model of relapse was used where rats were trained to self-administer cocaine, subjected to a period of extinction by substituting the drug for saline, vaccinated, and re-exposed to cocaine. Compared with controls, animals immunized with GNC-KLH did not reinstate cocaine self-administration behavior when given a noncontingent cocaine infusion on two consecutive days. Upon double and triple infusions, 38-62% of vaccinated animals failed to reinstate as compared with full reinstatement in all control animals. Exposure to ad libitum cocaine reinstated baseline values in control animals and resulted in double to triple the baseline values of self-infusions in vaccinated animals, suggesting a partial antibody-mediated blockade of cocaine access to the central nervous system. This compensating effect was blocked by passive immunization pretreatment with the monoclonal IgG GNC92H2 in both vaccinated and control groups. To further assess the surmountability potential of GNC-KLH-induced antibody titers by cocaine self-administration, and the capacity of these titers to block the reinforcing effects of the drug, rats were tested at various doses of cocaine (0.015-0.5 mg/infusion). Active immunization with GNC-KLH produced approximately an 8-fold rightward shift of the dose-effect function for cocaine. The results reported suggest that immunopharmacotherapy may offer a promising means to treat cocaine abuse by aiding in the prevention of relapse.     

Pressure recovery in aortic stenosis: an in vitro study in a pulsatile flow model.

OBJECTIVES. This study was designed to study pressure recovery in various models of aortic valve stenosis by performing hemodynamic measurements under physiologic conditions in a pulsatile aortic flow circuit. The results were used to validate calculations of pressure recovery based on theoretic considerations derived from fluid dynamics. BACKGROUND. Pressure recovery in aortic stenosis has not been systematically analyzed. METHODS. Stenoses varying in size, shape (circular, Y-shaped, slitlike) and inlet configuration (sharp-edged, nozzle-shaped inlet, artificially stenosed bioprostheses) were used. Aortic pressures were measured at multiple sites distal to the stenotic orifice to determine pressure gradients and recovery. RESULTS. With decreasing orifice area (2, 1.5, 1 and 0.5 cm2) pressure recovery increased (5, 7, 10 and 16 mm Hg, respectively) and the index pressure recovery to maximal peak to peak gradient decreased (56%, 37%, 24% and 14%, respectively). For a given orifice size of 0.5 cm2, this index ranged between 12% for a Y-shaped orifice and 15% for a circular orifice with a nozzle (cardiac output 4 liters/min). Increasing the cardiac output increased pressure recovery, whereas the ratio of pressure recovery to maximal pressure gradient remained constant. CONCLUSIONS. The index pressure recovery to transvalvular pressure gradient, which expresses the hemodynamic relevance of pressure recovery, decreases with increasing severity of aortic stenosis but is independent of transvalvular flow. Thus, pressure recovery is of minor importance in severe aortic stenosis but may account for discrepancies between Doppler and manometric gradients observed in patients with mild to moderate aortic stenosis or a prosthetic valve in the aortic position.     

Development and application of an in vitro model for screening anti-hepatitis B virus therapeutics.

The development of effective anti-hepatitis B virus agents has been hampered by the lack of reliable in vitro systems for the screening of new therapeutics. In an effort to circumvent this problem, we have developed an in vitro system for screening anti-hepatitis B virus drugs using hepatitis B virus DNA-transfected Hep G2 cells. The cell line designated 2.2.15 produces replicative viral DNA intermediates, mature Dane particles and high levels of viral antigens. Subconfluent 2.2.15 cells were treated with a variety of commonly used anti-hepatitis B virus therapeutics, and their efficacy was determined by analyzing changes in the replicative cellular or extracellular hepatitis B virus DNA content by Southern blotting or slot-blot hybridization. The slot-blot method was sensitive, reproducible and rapid and correlated well with Southern blotting. Analysis of the media for hepatitis B virus DNA was indicative of changes in intracellular, replicative hepatitis B virus DNA, permitting sampling of the media. Therefore 2.2.15 cells may provide a valuable method for identifying and monitoring effective anti-hepatitis B virus therapeutics. Using this system to test various agents, we confirm that 2'-deoxyguanosine strongly inhibited viral replication, whereas others tested were less effective. Correlation with in vivo systems is now needed.     

Application of inflammation-responsive promoter for an in vitro arthritis model

OBJECTIVE: The application of inflammation-regulated therapeutic gene expression in arthritis conditions increases the efficiency of gene therapy by self-limiting the transgene. Incidentally, constitutive overexpression of transgenes typically leads to detrimental effects in disease conditions; therefore, regulation of expression is warranted. We undertook this study to validate a new gene therapy approach using a cell culture-based inflammation model and a novel self-limiting, inflammation-responsive promoter construct. METHODS: We designed a self-limiting promoter construct that expresses an antiinflammatory gene (interleukin-4 [IL-4]) only in the presence of inflammation. Our construct featured a truncated promoter sequence of cyclooxygenase 2 (COX-2) upstream of the IL-4 gene. We triggered inflammation in vitro in articular chondrocytes by applying the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) together exogenously, and we studied the extent of IL-4 expression and its effect on the inflammatory cascade. RESULTS: Using articular chondrocytes, we showed that our COX-2 promoter construct expressed IL-4 only in the presence of IL-1beta and TNFalpha. IL-4 expressed in the presence of IL-1beta and TNFalpha down-regulated a series of inflammation mediators, prostaglandins, and matrix metalloproteinases. CONCLUSION: The use of this construct for the expression of antiinflammatory genes allows production of a therapeutic gene product that is controlled by the severity of the disease. The effectiveness of this promoter construct for combating inflammation makes it a suitable candidate for the development of a new local gene therapy strategy for the treatment of osteoarthritis, in which IL-1beta and TNFalpha trigger a signal cascade that elevates COX-2 levels.