Preparation of a proteolytic enzyme mixture containing trypsin,elastase, and chymotrypsin for use in tissue disaggregation

Summary We describe a purification method for tissue culture-grade trypsin that yields an enzyme mixture with reproducible activities of trypsin, elastase, and chymotrypsin and eliminates amylase and lipase.     

Genetic dissection of a rat model for rheumatoid arthritis: significant gender influences on autosomal modifier loci

Rheumatoid arthritis (RA) is a common, chronic, autoimmune, inflammatory disease that is influenced by genetic factors including gender. Many studies suggest that the genetic risk for RA is determined by the MHC, in particular class II alleles with a 'shared epitope' (SE), and multiple non-MHC loci. Other studies indicate that RA and other autoimmune diseases, in particular insulin-dependent diabetes mellitus (IDDM) and autoimmune thyroid disease (ATD), share genetic risk factors. Rat collagen-induced arthritis (CIA) is an experimental model with many features that resemble RA. The spontaneous diabetes-resistant bio-breeding rat, BB(DR), is of interest because it is susceptible to experimentally induced CIA, IDDM and ATD, and it has an SE in its MHC class II allele. To explore the genetics of CIA, including potential gender influences and the genetic relationships between CIA and other autoimmune diseases, we conducted a genome-wide scan for CIA regulatory loci in the F(2) progeny of BB(DR) and CIA-resistant BN rats. We identified 10 quantitative trait loci (QTLs), including 5 new ones (Cia15, Cia16*, Cia17, Cia18* and Cia19 on chromosomes 9, 10, 18 and two on the X chromosome, respectively), that regulated CIA severity. We also identified four QTLs, including two new ones (Ciaa4* and Ciaa5* on chromosomes 4 and 5, respectively), that regulated autoantibody titer to rat type II collagen. Many of these loci appeared to be gender influenced, and most co-localized with several other autoimmune trait loci. Our data support the view that multiple autoimmune diseases may share genetic risk factors, and suggest that many of these loci are gender influenced.     

The final demise of Rodriguez lethal acrofacial dysostosis: A case report and review of the literature

We evaluated a newborn with acrofacial dysostosis in whom a clinical diagnosis of Nager syndrome was entertained. Radiographs revealed hypoplasia of the scapulae and bilateral humeroradial synostosis, with absent ulna on the left and hypoplastic ulna on the right. The finding of bilateral humeroradial synostosis had not been seen in cases of Nager syndrome before and we considered other diagnoses. Humeroradial synostosis has been found in three cases of acrofacial dysostosis Rodriguez type, a syndrome characterized by mandibular hypoplasia, upper and lower extremity phocomelia, and oligodactyly of the upper limbs. More recently, haploinsufficiency of the SF3B4 gene has been identified as the cause of both Nager and Rodriguez syndrome, leading many to believe that Rodriguez syndrome represents a more severe end of a Nager syndrome spectrum. An SF3B4 mutation was found in our patient, prompting a review of the previous known cases of Rodriguez syndrome, which revealed no clustering of SF3B4 mutations, and four cases of Rodriguez syndrome with mutations identical to those in cases of Nager syndrome. Rodriguez syndrome was previously thought of as a lethal acrofacial dysostosis distinct from Nager syndrome. A number of more mild cases, as well as our case, intermediate between the two phenotypes, illustrate that Rodriguez syndrome is a severe manifestation of Nager syndrome, and is not lethal with aggressive medical care.     

Stimulation of human monocytes by anti-CD3 monoclonal antibody: Induction of inflammatory mediator release via immobilization of Fc receptor by adsorbed immunoglobulin and T-lymphocytes

Human monocytes released superoxide anion, prostaglandin E₂, leukotriene B₄, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating (cross-linking) second antibody failed to induce mediator release. Monocytes armed with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 and TNF- when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG_2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.     

Reduced Left Ventricular Compliance and Mechanical Efficiency after Prolonged Inhibition of NO Synthesis in Conscious Dogs

Acute inhibition of NO synthesis decreases left ventricular (LV) work and external efficiency, but it is unknown whether compensatory mechanisms can limit the alterations in LV mechanoenergetics after prolonged NO deficiency. Eight chronically instrumented male mongrel dogs received 35 mg kg⁻¹ day⁻¹ of N ^ω-nitro-L-arginine methyl ester orally for 10 days to inhibit NO synthesis. At spontaneous beating frequency, heart rate, coronary blood flow, peak LV pressure, end-diastolic LV pressure and the maximum derivative of LV pressure (d P /d t _max) were not significantly different vs. baseline, whereas LV end-diastolic diameter (32.5 ± 1.0 vs. 37.6 ± 1.4 mm) and LV stroke work (515 ± 38 vs. 650 ± 44 mmHg mm), were reduced (all P < 0.05). The slope of the LV end-systolic pressure-diameter relationship was increased at 10 days vs. baseline (13.9 ± 1.0 vs. 9.6 ± 0.9 mmHg mm⁻¹, P < 0.05), while the end-diastolic LV diameter was smaller at matched LV end-diastolic pressures. At fixed heart rate (130 beats min⁻¹), cardiac oxygen consumption was increased (12.2 ± 1.5 vs. 9.9 ± 1.0 ml min⁻¹), and the ratio between stroke work and oxygen consumption was decreased by 33 ±7 % (all P < 0.05) after NO inhibition. We conclude that sustained inhibition of NO synthesis in dogs causes a decrease in LV work despite an increased contractility, which is most probably due to reduced diastolic compliance and a decrease in external efficiency. Thus, prolonged NO deficiency is not compensated for on the level of LV mechanoenergetics in vivo .     

Long-term maintenance of hematopoietic stem cells does not require contact with embryo-derived stromal cells in cocultures

We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.     

Hyperplastic synoviocytes from rats with streptococcal cell wall-induced arthritis exhibit a transformed phenotype that is thymic-dependent and retinoid inhibitable.

It has been suggested that streptococcal cell wall-induced arthritis in LEW/N rats resembles a localized neoplasm consisting of, in part, a proliferative and invasive population of fibroblast-like synoviocytes. To further pursue this concept, the synoviocytes from diseased rats were characterized in situ and in vitro for various parameters of "transformation." The spindle-shaped synoviocytes were found throughout the synovium and were the predominant cell type at sites of invasion of bone and cartilage by synovium. They stained intensely for vimentin, a microfilament prominently expressed in immature and transformed mesenchymal cells. They stained variably for Ia antigens and did not exhibit T cell surface antigens nor did they stain with histochemical stains characteristic of monocytes or granulocytes. Electron microscopy confirmed their fibroblastlike morphology and suggested high grade metabolic activity. In primary culture, the abnormal synoviocytes were adherent, grew rapidly and did not contact inhibit. Moreover, they grew under anchorage-independent conditions. These abnormal growth characteristics were inhibited by all-trans retinoic acid. Finally, explants of the arthritic synovium formed short-lived tumorlike nodules in athymic nude mice. These observations, considered in the context of other data, support the concept that the pathologic process represents a thymic-dependent, nonmalignant, locally invasive inflammatory neoplasm.     

Loss of p16 (INK4A) expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected malignant peripheral nerve sheath tumors.

The p16 is a tumor suppressor gene on the short arm of chromosome 9p21. The product of the p16 acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases that phosphorylate the retinoblastoma protein. This study was designed to assess the frequency of genetic loss of 9p21 and to determine the role of p16 the pathogenesis of sporadic and neurofibromatosis 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs). The authors examined 15 cases for p16 protein expression and 10 cases for allelic imbalance (AI)/loss of heterozygosity (LOH) of chromosome 9p. DNA was microdissected from normal and neoplastic tissues. AI/LOH analysis was performed using six microsatellite markers on the 9p region. On immunohistochemical analysis 80% of cases showed abnormal expression of p16. Similarly, 8 of 10 cases revealed genetic loss with at least one microsatellite marker. The most frequent deletion was that within the coding sequence. Of p16 at me D9S974 locus. These findings emphasize the role of loss of p16 in the development of both sporadic and NF1-associated MPNSTs.     

Inflammatory Markers in Acute Myocardial Infarction Patients: Preliminary Evidence of a Prospective Association With Depressive Symptoms

Inflammatory activity has been associated with both coronary disease and depressive symptoms. We sought to determine whether inflammatory markers in myocardial infarction (MI) patients are prospectively associated with depressive symptomatology. Participants were a convenience sample of MI patients. Depressive symptoms were assessed soon after the MI and again 7 months postdischarge. Inflammatory markers examined were interleukin-6 (IL-6) and interleukin-1β. Results suggest no significant cross-sectional association between inflammatory markers and depressive symptoms at baseline. However, bivariate and multiple regression analyses revealed a significant positive prospective association between baseline IL-6 and depressive symptoms 7 months later ( β  = .57, p  < .01). The results suggest that temporal considerations are important in understanding relationships between inflammation and depressive symptoms following MI.