Comparison between adult and foetal adnexa derived equine post-natal mesenchymal stem cells.

BACKGROUND: Little is known about the differences among adult and foetal equine mesenchymal stem cells (MSCs), and no data exist about their comparative ultrastructural morphology. The aim of this study was to describe and compare characteristics, immune properties, and ultrastructural morphology of equine adult (bone marrow: BM, and adipose tissue: AT) and foetal adnexa derived (umbilical cord blood: UCB, and Wharton's jelly: WJ) MSCs. RESULTS: No differences were observed in proliferation during the first 3 passages. While migration ability was similar among cells, foetal MSCs showed a higher adhesion ability, forming smaller spheroids after hanging drop culture (P < 0.05). All MSCs differentiated toward adipogenic, chondrogenic and osteogenic lineages, only tenogenic differentiation was less evident for WJ-MSCs. Data obtained by PCR confirmed MHC1 expression and lack of MHC2 expression in all four cell types. Foetal adnexa MSCs were positive for genes specific for anti-inflammatory and angiogenic factors (IL6, IL8, ILß1) and WJ-MSCs were the only positive for OCT4 pluripotency gene. At immunofluorescence all cells expressed typical mesenchymal markers (α-SMA, N-cadherin), except for BM-MSCs, which did not express N-cadherin. By transmission electron microscopy, it was observed that WJ-MSCs had a higher (P < 0.05) number of microvesicles compared to adult MSCs, and UCB-MSCs showed more microvesicles than BM-MSCs (P < 0.05). AT-MSCs had a lower number of mitochondria than WJ-MSCs (P < 0.05), and mitochondrial area was higher for WJ-MSCs compared to UCB and AT-MSCs (P < 0.05). CONCLUSIONS: Results demonstrate that MSCs from adult and foetal tissues have different characteristics, and foetal MSCs, particularly WJ derived ones, seem to have some charactestics that warrant further investigation into potential advantages for clinical application.

Differences between female suicidal patients with family history of suicide attempt and family history of completed suicide.

BACKGROUND: Family history of suicidal behavior and suicide are both risk factors for suicide. However, the effects of family history of suicide versus suicide attempts on patient suicidal behavior remain unclear. The aim of the present study was to understand if family history of suicide as compared to family history of suicide attempts or no family history of suicidal behavior evidences different associations with suicidal behavior among psychiatric patients. METHOD: Participants included 157 female patients between the ages of 18 and 65years admitted at the Dr. Braulio A. Moyano Neuropsychiatric Women's Hospital. RESULTS: Seventy-nine patients (50.3%) reported no family history of suicidal behavior (NFHSB), while 78 patients (49.7%) reported a family history of suicidal behavior. Specifically, 41 patients (26.1%) reported a family history of suicide attempt (FHSA) and 37 patients (23.6%) reported a family history of suicide (FHS). These groups showed significant differences between family history of psychopathology and number of previous suicide attempts. Patients with an FHSA were more likely to present with a greater number of previous suicide attempts as compared to patients with NFHSB and FHS. CONCLUSION: There is an association between the number of suicide attempts and family history of suicide attempts in female patients hospitalized for suicidal behavior.

The interleukin-1ß/CXCL1/2/neutrophil axis mediates host protection against group B streptococcal infection.

Previous studies have indicated that group B streptococcus (GBS), a frequent human pathogen, potently induces the release of interleukin-1ß (IL-1ß), an important mediator of inflammatory responses. Since little is known about the role of this cytokine in GBS disease, we analyzed the outcome of infection in IL-1ß-deficient mice. These animals were markedly sensitive to GBS infection, with most of them dying under challenge conditions that caused no deaths in wild-type control mice. Lethality was due to the inability of the IL-1ß-deficient mice to control local GBS replication and dissemination to target organs, such as the brain and the kidneys. Moreover, in a model of inflammation induced by the intraperitoneal injection of killed GBS, a lack of IL-1ß was associated with selective impairment in the production of the neutrophil chemokines CXCL1 and CXCL2 and in neutrophil recruitment to the peritoneal cavity. Decreased blood neutrophil counts and impaired neutrophil recruitment to the brain and kidneys were also observed during GBS infection in IL-1ß-deficient mice concomitantly with a reduction in CXCL1 and CXCL2 tissue levels. Notably, the hypersusceptibility to GBS infection observed in the immune-deficient animals was recapitulated by neutrophil depletion with anti-Gr1 antibodies. Collectively, our data identify a cytokine circuit that involves IL-1ß-induced production of CXCL1 and CXCL2 and leads the recruitment of neutrophils to GBS infection sites. Moreover, our data point to an essential role of these cells in controlling the progression and outcome of GBS disease.

"A lactose-modified chitosan accelerates chondrogenic differentiation in mesenchymal stem cells spheroids".

Spheroids derived from human mesenchymal stem cells (hMSCs) are of limited use for cartilage regeneration, as the viability of the cells progressively decreases during the period required for chondrogenic differentiation (21 days). In this work, spheroids based on hMSCs and a lactose-modified chitosan (CTL) were formed by seeding cells onto an air-dried coating of CTL. The polymer coating can inhibit cell adhesion and it is simultaneously incorporated into spheroid structure. CTL-spheroids were characterized from a morphological and biological perspective, and their properties were compared with those of spheroids obtained by seeding the cells onto a non-adherent surface (agar gel). Compared to the latter, smaller and more viable spheroids form in the presence of CTL as early as 4 days of culture. At this time point, analysis of stem cells differentiation in spheroids showed a remarkable increase in collagen type-2 (COL2A1) gene expression (~700-fold compared to day 0), whereas only a 2-fold increase was observed in the control spheroids at day 21. These results were confirmed by histological and transmission electron microscopy (TEM) analyses, which showed that in CTL-spheroids an early deposition of collagen with a banding structure already occurred at day 7. Overall, these results support the use of CTL-spheroids as a novel system for cartilage regeneration, characterized by increased cell viability and differentiation capacity within a short time-frame. This will pave the way for approaches aimed at increasing the success rate of procedures and reducing the time required for tissue regeneration.

Cutaneous foetal injuries related to amniocentesis.

Amniocentesis is one of the most important prenatal diagnostic procedures available to assess congenital abnormalities. It is performed worldwide due to its simplicity of execution and lack of risk. The most frequent known accidents in amniocentesis are abortion, oligohydramnios, amniositis and placental abruption, while direct fetal injuries produced by contact with the needle are rarely seen. The injuries produced are extremely variable in severity, but the most frequent is skin wounds, which usually heal as small, round depressed scars. The cases we describe concern the occurrence of iatrogenic cutaneous wound lesions to a fetus during amniocentesis. The medical-legal analysis of the cases required dermatological expertise in order to exclude a different pathogenesis for the skin injuries to the child and were assigned by the court, in order to assess the administrative compensation due to the parents of the child as a result of medical malpractice.

The role of endosomal toll-like receptors in bacterial recognition.

BACKGROUND: Infections caused by extracellular Gram positive bacteria are still a major health problems. Better understanding of the mechanisms underlying immune responses to these organisms is key to develop pharmacological agents, including vaccines, to control these infections. OBJECTIVE AND PERSPECTIVES: The objective of this review is to highlight the importance of nucleic acid-sensing, intracellular Toll-like receptors in innate immune recognition and in host defenses against extracellular bacteria. CONCLUSIONS: Toll-like receptors 7 and 9 have a major role in inducing host-protective type I interferon responses in conventional dendritic cells in response to streptococci and other extracellular gram positive bacteria. Moreover an as yet unidentified MyD88-dependent receptor is likely responsible for proinflammatory cytokine induction in response to these pathogens.

Neonatal mouse immunity against group B streptococcal infection by maternal vaccination with recombinant anti-idiotypes.

We investigated whether immunization with recombinant anti-idiotypic antibody fragments mimicking the conformation of the capsular antigen can protect against infection by group B streptococcus, an important neonatal pathogen. Single-chain fragment-variable anti-idiotypes competed with the type III carbohydrate for binding to type-specific antibodies and elicited, in mice, the production of protective immunoglobulins reacting against the type III polysaccharide. Moreover, maternal immunization with soluble or phage-displayed fragments protected neonatal mice against streptococcal infection. These data indicate that recombinant anti-idiotypic antibodies may be useful in developing protein images of relevant carbohydrate epitopes and, ultimately, in preventing infections by encapsulated bacteria.

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity.

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

Determining the time of death by morphological and immunohistochemical evaluation of collagen fibers in postmortem gingival tissues.

The estimation of the post mortem interval (PMI) is still one of the most challenging variables to determine and the different approaches currently used in its estimation generally yield to large post mortem windows. In the present study we combined morphological and immunohistochemical analysis in order to reach a more detailed knowledge on tissue organization and degradation after death. Ultrastructural cellular changes and the extracellular matrix of gingival tissues, collected at different post mortem intervals, were observed by a Transmission Electron Microscopy (TEM), in combination with the immunohistochemical detection of extracellular matrix proteins (i.e. collagen type I and collagen type III) as potential post mortem biochemical markers. The final goal was to find a correlation between morphological modifications, biomarkers expression and the time of death. Samples of gingival tissues obtained from 10 cadavers at different post mortem intervals (short post mortem interval, 1-3 days; mid post mortem interval, 4-6 days; long post mortem interval, 7-9 days) were processed for light microscopy and TEM and they were also immunostained with anti-collagen type I and type III antibodies. Results showed gradual degradation of extracellular matrix in the suboral connective tissue in relation to the different time of death. Moreover PMI was related to an increase of nuclear chromatin condensation and cytoplasmic vacuolization both in epithelial and connective tissues. In conclusion, in addition to traditional forensic approaches to estimate PMI, the combined analyses of cellular morphology, ultrastructure and immunohistochemical expression of collagen proteins allow to better infer the PMI.

Analysis of the pattern of hypersensitivity reactions in patients receiving carboplatin retreatment for recurrent ovarian cancer.

The aim of this paper was to assess hypersensitivity reactions in 69 patients who received carboplatin (CBDCA) retreatment for recurrent ovarian cancer. Hypersensitivity reactions developed in 15 (21.7%) patients and occurred during the second cycle of retreatment in 13 (86.7%) of them. Reactions consisted of skin rash, flushing, itching, or abdominal cramping in eight (53.3%) and severe respiratory or cardiovascular events in seven patients (46.7%). One patient had a chest pain, without any other symptoms suggestive of hypersensitivity, followed by cardiac arrest unresponsive to standard resuscitative measures. All the other cases promptly recovered from symptoms. Logistic regression analysis showed that allergy history and CBDCA retreatment interval (interval time between the last cycle of first-line chemotherapy and CBDCA retreatment) were independent predictive variables for the risk of hypersensitivity, whereas patient age, first-line chemotherapy, total CBDCA dose given during first-line treatment, recurrence treated with CBDCA (first versus other), and CBDCA regimen at recurrence had no predictive value. Hypersensitivity reaction rate was higher in patients with CBDCA retreatment interval longer than 23.4 months compared to those with a shorter interval (36.3% versus 8.3%, P = 0.0132). Nine patients were subsequently treated with cisplatin, and two (22.2%) still developed allergic reactions. In conclusion, hypersensitivity reactions to CBDCA retreatment can occur in approximately one fifth of the cases, and a CBDCA retreatment interval longer than 2 years appears to be the strongest predictive variable for the development of allergic reactions.

Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria.

Two recombinant strains of Streptococcus gordonii, secreting or displaying a microbicidal single-chain antibody (H6), and stably colonizing rat vagina, were used to treat an experimental vaginitis caused by Candida albicans. A post-challenge intravaginal delivery of the H6-secreting strain was as efficacious as fluconazole in rapidly abating the fungal burden. Three weeks after challenge, 75% and 37.5% of the rats treated with the H6-secreting or displaying bacteria, respectively, were cured of the infection, which persisted in 100% of the animals treated with a S. gordonii strain expressing an irrelevant single-chain antibody. Thus, a human commensal bacterium can be suitably engineered to locally release a therapeutic antibody fragment.

High resolution morphological analysis of in situ human chromosomes.

The purpose of this study was to analyze the inner structure of chromosomes in cells arrested, fixed and cryosectioned in metaphase. The chromosomes in metaphase maps prepared using standard cytogenetic protocols, are usually covered by cellular debris, which obscures the structural details on the surface and limits analysis by techniques when using nanometric resolution. By using cryosectioning, the debris is removed and it is possible to analyze the internal structure of the chromosomes. We described the ultrastructure of chromosome sections fixed with either acetic acid, methanol or glutaraldehyde, evaluating the effect and the influence of the fixative on the morphology. Furthermore, we subjected those cells previously fixed with glutaraldehyde to osmic maceration in order to better visualize the intracellular structure. All samples were examined with a Field Emission In Lens Scanning Electron Microscope (FEISEM), which allows high-resolution analysis of biological samples without any metal coating. The results showed a package morphology in samples fixed with glutaraldehyde, mainly due to the high capacity of the fixative to strongly crosslink the proteins. In contrast, the fibrillar structure seen in cryosections fixed with acetic acid/methanol is due to the propensity of the fixatives to extract and remove proteins. We propose that in situ chromosomes fixed with glutaraldehyde and then osmicated are a good model for studying the inner structure of chromosomes by using high resolution scanning electron microscopy.

Liver Graft Allocation by Means of a New, Regionally Shared "Mixed" Model: The Experience in Lazio.

INTRODUCTION: Since 2013, the regional network of transplantation centers "LAZIO TRANSPLANT" have adopted a new, mixed system for the allocation of liver grafts. METHODS: The organs from donors aged <65 are assigned to patients with higher Model for End-stage Liver Disease (MELD) scores on a common regional waiting list, whereas those from donors aged >65 are allocated to patients with higher MELD scores on a specific local waiting list (LWL) at each center, on a rotational basis. RESULTS: The new mixed allocation model grants a more rational allocation of the "standard" organs to the patients with the actual worst MELD score in the entire region, avoiding the possibility that a patient in relatively better clinical condition might be transplanted before a more severely ill patient on another center's waiting list. Nonstandard organs, presenting slightly increased transplant risks, are still allocated on a rotational basis among the different transplant centers, ensuring them the possibility to select, on the basis of a global clinical risk evaluation, those patients in their LWL whose MELD score would not grant any possibility to compete for the "standard" organ allocation. CONCLUSIONS: The application of the new model had no negative impact on the overall number of transplants performed or on the global list-satisfaction percentages, but has slightly improved the cumulative mortality of the patients in the waiting list, granting to the clinically worst patients a prompt graft allocation, independent of the local center belonging.

RANK/RANKL/OPG signaling pathways in necrotic jaw bone from bisphosphonate-treated subjects.

Osteonecrosis of the jaw (ONJ) is a chronic complication affecting long-term bisphosphonate-treated subjects, recognized by non-healing exposed bone in the maxillofacial region. The pathophysiological mechanism underlying ONJ has not been fully elucidated. The aim of the present study was to investigate the role of RANK/RANKL/OPG signaling pathway and, in parallel, to evaluate angiogenic and matrix mineralization processes in jaw bone necrotic samples obtained from bisphosphonate-treated subjects with established ONJ. Necrotic bone samples and native bone samples were processed for Light and Field Emission in Lens Scanning Electron Microscope (FEISEM) analyses, for Real-Time RT-PCR to evaluate the gene expression of TNFRSF11A (RANK), TNFSF11 (RANKL), and TNFSF11B (OPG) and for immunohistochemical analyses of VEGF and BSP expression. Morphological analyses performed by Light microscope and FEISEM show empty osteocytic lacunae and alteration of lamellar organization with degradation of the mineralized bone matrix in necrotic bone samples. A significant increase in TNFRSF11A, TNFSF11, TRAF6 and NFAT2 gene expression, and a reduction of TNFSF11B gene transcription level compared is also showed in necrotic bone compared to control samples. No significant difference of VEGF expression is evidenced, while lower BSP expression in necrotic bone compared to healthy samples is found. Even if the pathogenesis of bisphosphonate-associated ONJ remains unknown, a link between oral pathogens and its development seems to exist. We suppose lipopolysaccharide produced by bacteria colonizing and infecting necrotic bone and the surrounding viable area could trigger RANK/RANKL/OPG signaling pathway and, in this context, osteoclasts activation could be considered as a protective strategy carried out by the host bone tissue to delimitate the necrotic area and to counteract infection.

In vitro anti-angiogenic effects of Hemidesmus indicus in hypoxic and normoxic conditions.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for many purposes and poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. In the context of anticancer pharmacology, anti-angiogenic therapy has become an effective strategy for inhibiting new vessel formation and contrast tumor growth. These considerations are supported by the evidence that most tumors originate in hypoxic conditions and limitation of oxygen diffusion stimulates the formation of tumor abnormal microvasculature. Aim of this study was to evaluate the in vitro anti-angiogenic potential of Hemidesmus indicus (0.31-0.93 mg/mL) on human umbilical vein endothelial cells and delineate the main molecular mechanisms involved in its anti-angiogenic activity both in normoxia and hypoxia. MATERIALS AND METHODS: The decoction of Hemidesmus indicus was subjected to an extensive HPLC phytochemical characterization. Its in vitro anti-angiogenic potential was investigated in normoxia and hypoxia. Cell proliferation, apoptosis induction, and inhibition of endothelial cell migration and invasion were analyzed by flow cytometry. The endothelial tube formation assay was evaluated in matrix gel. The capillary tube branch points formed were counted using a Motic AE21 microscope and a VisiCam videocamera. The regulation of key factors of the neovascularization process such as VEGF, HIF-1α and VEGFR-2 was explored at mRNA and protein level by real time PCR and flow cytometry, respectively. RESULTS: Treatment with Hemidesmus resulted in a significant inhibition of cell proliferation and tube formation in both normoxia and hypoxia. Hemidesmus differently regulated multiple molecular targets related to angiogenesis according to oxygen availability. In normoxia, the inhibition of VEGF was the main responsible for its anti-angiogenic effect; the angiogenesis inhibition induced in hypoxia was regulated by a more complex mechanism involving firstly HIF-1α inhibition, and then VEGF and VEGFR-2 down-regulation. Additionally, the inhibition of endothelial cell migration and invasion by Hemidesmus was more pronounced in normoxia than in hypoxia, possibly due to the physiological enhanced induction of invasion characteristic of hypoxia. CONCLUSIONS: Our results indicate that Hemidesmus might represent a promising therapeutic strategy for diseases in which the inhibition of angiogenesis could be beneficial, such as cancer. The antiangiogenic activity of Hemidesmus is based on multiple interactions with critical steps in the angiogenic cascade. VEGF expression stimulated by HIF-1α as well as endothelial cell migration and differentiation represent important targets of Hemidesmus action and might contribute to its cancer therapeutic efficacy that is presently emerging and offer a scientific basis for its use in traditional medicine.

Cytochalasin A inhibits B-lymphocyte capping and activation by antigens.

Cytochalasin B (CB) has been shown to be a potent depressant of the antigen-induced clone expansion and terminal differentiation of mouse B-lymphocytes to antibody-forming cells. This effect could be the result of the microfilament-disrupting effect of CB with subsequent inhibition of antigen-sIg complex redistribution, a series of events which seems to be necessary for B-lymphocyte activation. CB is not very active in depressing capping and will inhibit glucose transport. To further investigate the mechanism of action of cytochalasins, the effect of cytochalasin A (CA) on cap formation and plaque-forming cell generation was studied, since CA is less inhibitory of glucose transport and more inhibitory of cap formation. The results presented here indicate that complexes of anti-Ig-sIg will be prevented from capping by as little as 1 microgram of CA, a quantity sufficient to depress markedly the generation of plaque-forming cells to SRBC in culture. These results further confirm our conclusion that the depression of B-lymphocyte activation may be related to the depression of cap formation. It also strongly suggested that inhibition of glucose transport can be regraded as a negligible factor in this depression.

Depression of direct plaque-forming cell response in mouse spleen cell cultures by aggregated IgG2b-induced factors.

Mouse spleen cells were treated with concanavalin A (Con A) or aggregated mouse IgG2b for 48 h in culture. When cells thus treated were added to fresh mouse spleen cell cultures immunized with SRBC they depressed the response of B lymphocytes as measured by enumerating plaque forming cells (PFC) on the fourth day of culture. When supernatant from cells cultured with IgG2b was added to immunized cultures this resulted in depression of PFC generation similar to that observed by addition of treated cells. The depression observed was essentially in the same range as that observed by addition of Con A treated cells or their supernatant. These observations extend previous work suggesting that IgG2b-induced PFC depression may result from activation of suppressor T cells with elaboration of soluble suppressor factors. This mechanism of immunomodulation may be important in the pathogenesis of immune complex disorders.

Anti-idiotypic vaccination against group B streptococci.

We describe the antigenic properties of an anti-idiotypic single chain fragment variable (scFv) recombinant antibody mimicking the type III capsular polysaccharide of group B streptococci (GBS), an important cause of neonatal sepsis. This scFv could compete with the nominal antigen for binding to specific mouse or rabbit antibodies. Moreover, the scFv elicited, in mice, the production of antibodies which reacted against the type IlI polysaccharide and passively protected neonatal pups from GBS disease. Maternal immunization with the scFv also protected neonatal mice against GBS infection. Next, the scFv was expressed on the surface of the commensal bacterium Streptococcus gordonii. Intravaginal inoculation of mice with these recombinant bacteria induced significant elevations in serum titers of anti-GBS type III antibodies. Therefore, the expression scFv in commensal bacteria may be a convenient and effective way of delivering anti-idiotypic vaccines.

Recombinant Streptococcus gordonii for mucosal delivery of a scFv microbicidal antibody.

The gram-positive bacterium Streptococcus gordonii was engineered to express the microbicidal molecule H6, which is an antiidiotypic single chain antibody mimicking a yeast killer toxin. S. gordonii is a human commensal which we developed as a model system for mucosal delivery of heterologous proteins. The in vivo candidacidal activity of both H6-secreting and H6-surface-displaying streptococcal strains were assayed in a well-established rat model of vaginal candidiasis. At day 21 full clearance of Candida albicans infection was observed in 75% of animals treated with the H6-secreting strain, and in 37.5% of animals treated with the strain expressing H6 on the surface, while all animals treated with the control strain were still infected. The observed candidacidal effect was comparable with that observed with the antimycotic drug fluconazole. These data confirm the potential of H6 as a candidacidal agent and show how promising is the approach of using recombinant bacteria for mucosal delivery of biologically active molecules.

Effect of nitric oxide donors and nitric oxide synthase inhibitors in neonatal rat endotoxic shock.

Previous studies have shown an increased mortality in response to endotoxin in 24-hr-old neonatal rats compared with older neonates and adults. This increased susceptibility may be related to increased nitric oxide (NO) and thromboxane (TxB2) production. Twenty-four-hour-old neonatal rat pups were given either N(G)-nitro-L-arginine methyl ester (L-NAME; a nonspecific NO synthase inhibitor), S-methylthioisourea (SMT; a specific NO synthase inhibitor), or molsidomine (a NO donor) subcutaneously prior to or after an LD50 of intracardiac endotoxin. Mortality was followed for 72 hr. There was no statistically significant difference in mortality between control animals and those pretreated with L-NAME, SMT, or molsidomine. A trend toward increased mortality with nonspecific NO synthase inhibition and decreased mortality with the NO donor was noted. Splenic cells were obtained for in vitro cytokine stimulation studies. In vitro adherent splenic cell stimulation studies confirmed an increase in NO production with NO donor pretreatment and decreased production of NO with NO synthase inhibition pretreatment. There was no difference in TxB2 production with either the NO synthase inhibitor or the NO donor. In conclusion, at the several doses employed, neither nonselective or selective NO synthase inhibitors nor NO donors prevented endotoxin-induced mortality in rat neonatal shock. Although these findings do not preclude possible involvement of NO in neonatal pathophysiology, increased NO production thus does not appear to be the primary determinant of the increased susceptibility of the neonatal rat to endotoxic shock.