Expression of eNOS in the lungs of neonates with pulmonary hypertension.

BACKGROUND: Neonatal hypoxemic respiratory failure (NHRF) is usually associated with reversible persistent pulmonary hypertension (PPHN). Congenital diaphragmatic hernia (CDH), a cause of refractory NHRF, is associated with irreversible pulmonary hypertension. Nitric oxide (NO) generated in the pulmonary vascular endothelium by endothelial nitric oxide synthase (eNOS) plays a pivotal role in perinatal circulatory adaptation. OBJECTIVE: To compare the expression of eNOS using IHC in postmortem lung tissue from newborns diagnosed clinically with PPHN and CDH. DESIGN/METHODS: Formalin-fixed lung tissue from infants who died following treatment for PPHN (n=12) or CDH (n=8) and age and gender matched controls who died from non-respiratory causes (Control, n=14) was evaluated for expression and staining intensity (1-4 scale) of eNOS using IHC. RESULTS: Mean gestational and postnatal age was comparable across groups. Histological evidence of chronic lung disease, pulmonary hypoplasia and pulmonary hypertension were seen more frequently in CDH compared to PPHN and control infants. eNOS expression was increased in arteriolar media of PPHN infants compared to Controls (p=0.027). CDH infants had increased intensity of staining for eNOS in the arteriolar endothelium (p=0.022) compared to control and PPHN infants and in the alveolar lining (p=0.002) compared to Controls. CONCLUSIONS: Upregulation of eNOS was seen both in infants with CDH and PPHN but was more marked in infants with CDH. These findings may have implications for understanding disease pathophysiology in cases with fatal outcome and development of novel therapies for neonatal pulmonary hypertension.

PAMAM dendrimer-azithromycin conjugate nanodevices for the treatment of Chlamydia trachomatis infections.

Chlamydia trachomatis is an important bacterial pathogen known to be etiological in genital infections, as well as several serious disease sequelae, including inflammatory arthritis. Chlamydiae can persist in infection, making treatment with antibiotics such as azithromycin (AZ) a challenge. The authors explore the use of neutral generation-4 polyamidoamine (PAMAM) dendrimers as intracellular drug-delivery vehicles into chlamydial inclusions. Azithromycin was successfully conjugated with the dendrimers, and the conjugate (D-AZ) released ≈ 90% of the drug over 16 hours. The conjugate readily entered both the Chlamydia-infected HEp-2 cells and the chlamydial inclusions. The conjugate was significantly better than free drug in preventing productive infections in the cells when added at the time of infection, and better in reducing the size and number of inclusions when added either 24 hours or 48 hours post infection. These studies show that dendrimers can deliver drugs efficiently to growing intracellular C. trachomatis, even if the organism is in the persistent form. FROM THE CLINICAL EDITOR: In this report, the use of polyamidoamine dendrimers as intracellular drug-delivery vehicles into chlamydial inclusions is investigated. This method results in efficient intracellular delivery of azithromycin to address chlamydia infection.

Effect of aerosolized PGE(1) on the ductus arteriosus of neonatal swine.

BACKGROUND: Inhaled PGE(1) (IPGE(1)) is a potential pulmonary vasodilator in neonatal respiratory failure. However, its effect on the patency of the ductus arteriosus (DA) has not been described. OBJECTIVE: To investigate the effect of IPGE(1) on the DA in healthy piglets. DESIGN/METHODS: IPGE(1) (1200ng/kg/min) [Study] or nebulized saline [Control] was administered using a jet nebulizer. Transthoracic echocardiography (TTE) was performed prior to (T0) and after 24h of aerosol therapy (T24). The DA was also evaluated histomorphologically at autopsy. RESULTS: Fifteen piglets, 1-9 days old (study=9; control=6), were evaluated for DA patency. Study piglets received IPGE(1) for 12-24h. TTE was performed on 12 piglets at T0. Nine animals showed no ductal flow and 3 (1 study, 2 control) had a small DA. TTE at T24 in 5 animals showed no change in DA. At autopsy, the ductal diameter and histologic maturity stage were comparable in study and control animals. CONCLUSIONS: High dose IPGE(1) given for 12-24h does not exert significant effect on the DA of healthy term piglets as evaluated by echocardiography and histomorphology. We conclude that ductal patency in neonates is influenced not only by prostaglandins but also by factors like hypoxemia, prematurity, and heart disease.

Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation.

During the final stage of spermatogenesis (i.e., spermiogenesis), round spermatids differentiate into mature spermatozoa. This transformation is mediated by a suite of nuclear packaging proteins. These include the transition proteins and the protamines. The two human protamines PRM1 and PRM2, and transition protein TNP2, are encoded by a single chromatin domain bounded by two regions of matrix attachment. Previous transgenic studies in our laboratory have shown that mice harboring a 40-kb segment of human chromosome 16p13.13 containing the PRM1--> PRM2-->TNP2 domain express the transgene in a haploid-specific, copy number-dependent, and position-independent manner. While these results indicate that this segment of the genome is a complete structural and functional regulatory unit, the elements governing the haploid expression of this suite of genes remain to be clearly defined. The preparation of spermatogenic cells is required to begin to address this mechanism. The CELSEP (Wescor/Dupont Inc. Wilmington, DE) unit-gravity sedimentation apparatus provides a simple, efficient, and reproducible means to separate testicular germ cells at all stages along this differentiative pathway. The high quality and integrity of germ cells obtained by this means provides a valuable resource for characterizing the molecular mechanisms governing the regulation of the PRM1-->PRM2-->TNP2 domain during spermatogenesis. A discussion of the CELSEP apparatus and the application of this methodology in our laboratory are presented.

Conservation of the PRM1 --> PRM2 --> TNP2 domain.

In mouse and human, the genes encoding protamines PRM1, PRM2 and transition protein TNP2 are found clustered together on chromosome 16. In addition, these three genes lie in the same orientation to one another and are coordinately expressed in a haploid-specific manner during spermatogenesis. Previously, we have shown that the human PRM1 --> PRM2 --> TNP2 locus exists as a single chromatin domain bounded by two male germ cell-specific MARs, i.e. Matrix Attachment Regions. A third, somatic-specific MAR element lies immediately 3' of the PRM1 --> PRM2 --> TNP2 domain. This MAR maps to a conserved CpG island 5' of the human SOCS-1 gene. Similarly, two candidate MARs flank the mouse Prm1 --> Prm2 --> Tnp2 domain. Comparative analysis of the mouse and human promoter regions identified several conserved regulatory motifs for each of the genes of this cluster. This further establishes the synteny of this region. Global structural similarities and the functional relevance of the associated candidate regulatory elements are discussed.

Folate-functionalized dendrimers for targeting Chlamydia-infected tissues in a mouse model of reactive arthritis.

Chlamydia trachomatis is an intracellular human pathogen that causes a sexually transmitted disease which may result in an inflammatory arthritis designated Chlamydia-induced reactive arthritis (ReA). The arthritis develops after dissemination of infected cells from the initial site of chlamydial infection. During Chlamydia-associated ReA, the organism may enter into a persistent infection state making treatment with antibiotics a challenge. We hypothesize that folate receptors (FR), which are overexpressed in Chlamydia-infected cells, and the associated inflammation would allow folate-targeted nanodevices to better treat infections. To investigate this, we developed a folate-PAMAM dendrimer-Cy5.5 conjugate (D-FA-Cy5.5), where Cy5.5 is used as the near-IR imaging agent. Uptake of D-FA-Cy5.5 upon systemic administration was assessed and compared to non-folate conjugated controls (D-Cy5.5), using a mouse model of Chlamydia-induced ReA, and near-IR imaging. Our results suggested that there was a higher concentration of folate-based nanodevice in sites of infection and inflammation compared to that of the control nanodevice. The folate-conjugated nanodevices localized to infected paws and genital tracts (major sites of inflammation and infection) at 3-4 fold higher concentrations than were dendrimer alone, suggesting that the overexpression of folate receptors in infected and inflamed tissues enables higher dendrimer uptake. There was an increase in uptake into thymus, spleen, and lung, but no significant differences in the uptake of the folate nanodevices in other organs including kidney and heart, indicating the 'relative specificity' of the D-FA-Cy5.5 conjugate nanodevices. These results suggest that folate targeting dendrimers are able to deliver drugs to attenuate infection and associated inflammation in Chlamydia-induced ReA.

Targeted delivery of antibiotics to intracellular chlamydial infections using PLGA nanoparticles.

Chlamydia trachomatis and Chlamydia pneumoniae are intracellular bacterial pathogens that have been shown to cause, or are strongly associated with, diverse chronic diseases. Persistent infections by both organisms are refractory to antibiotic therapy. The lack of therapeutic efficacy results from the attenuated metabolic rate of persistently infecting chlamydiae in combination with the modest intracellular drug concentrations achievable by normal delivery of antibiotics to the inclusions within which chlamydiae reside in the host cell cytoplasm. In this research, we evaluated whether nanoparticles formulated using the biodegradable poly(d-L-lactide-co-glycolide) (PLGA) polymer can enhance the delivery of antibiotics to the chlamydial inclusion complexes. We initially studied the trafficking of PLGA nanoparticles in Chlamydia-infected cells. We then evaluated nanoparticles for the delivery of antibiotics to the inclusions. Intracellular trafficking studies show that PLGA nanoparticles efficiently concentrate in inclusions in both acutely and persistently infected cells. Further, encapsulation of rifampin and azithromycin antibiotics in PLGA nanoparticles enhanced the effectiveness of the antibiotics in reducing microbial burden. Combination of rifampin and azithromycin was more effective than the individual drugs. Overall, our studies show that PLGA nanoparticles can be effective carriers for targeted delivery of antibiotics to intracellular chlamydial infections.

The structural organization of sperm chromatin.

The packaging of the male haploid genome within the differentiating spermatid nucleus is facilitated by small basic nuclear proteins called protamines. Although the majority of the DNA in human sperm chromatin is bound by these proteins, a small percentage retains a nucleosomal-like component. These histone-enriched regions may possess enhanced nuclease sensitivity and have been postulated to designate certain genes involved in early embryogenesis. We have shown previously that the chromatin domain containing the two human protamines PRM1 and PRM2 and the transition protein TNP2 forms a DNase I-sensitive conformation in pachytene spermatocytes, a requisite event prior to the haploid expression of its members in round spermatids (Kramer, J. A, McCarrey, J., Djakiew, D., and Krawetz, S. A. (1998) Development 125, 4749-4755). Interestingly, this configuration persists in mature spermatozoa subsequent to the transcriptional silencing of the locus. It was therefore postulated that the retained, enhanced DNase I-sensitive conformation of the PRM1-->PRM2-->TNP2 domain in human sperm may be preferentially histone-enriched. To address this tenet, we examined the chromatin structure of the human PRM1--> PRM2--> TNP2 domain using a PCR-based assay. The results show that this retained, enhanced DNase I sensitive domain reflects an enrichment of histones at discrete regions across the locus. In addition, a similar examination of other genes and repetitive sequences suggests the non-random distribution of histones and protamines within the sperm nucleus. A discussion of these results and their functional significance is presented.

Chromatin loops are selectively anchored using scaffold/matrix-attachment regions.

The biological significance of nuclear scaffold/matrix-attachment regions (S/MARs) remains a topic of long-standing interest. The key to understanding S/MAR behavior relies on determining the physical attributes of in vivo S/MARs and whether they serve as rigid or flexible chromatin loop anchors. To analyze S/MAR behavior, single and multiple copies of the S/MAR-containing constructs were introduced into various host genomes of transgenic mice and transfected cell lines. These in vivo integration events provided a system to study the association and integration patterns of each introduced S/MAR. By utilizing FISH to visualize directly the localization of S/MARs on the nuclear matrix or chromatin loop, we were able to assign specific attributes to the S/MAR. Surprisingly, when multiple-copy S/MARs were introduced they were selected and used as nuclear matrix anchors in a discriminatory manner, even though they all contained identical primary sequences. This selection process was probably mediated by S/MAR availability including binding strength and copy number, as reflected by the expression profiles and association of multi-copy tandem inserted constructs. Whereas S/MARs functioned as the mediators of loop attachment, they were used in a selective and dynamic fashion. Consequently, S/MAR anchors were necessary but not sufficient for chromatin loops to form. These observations reconcile many seemingly contradictory attributes previously associated with S/MARs.