High-resolution 3D scaffold model for engineered tissue fabrication using a rapid prototyping technique.

Rapid prototyping, automatic image processing (computer-aided design (CAD)) and computer-aided manufacturing techniques are opening new and interesting prospects for medical devices and tissue engineering, especially for hard tissues such as bone. The development of a bone high-resolution scaffold prototype using these techniques is described. The results testify to the fidelity existing between microtomographic reconstruction and CAD. Furthermore, stereolithographic manufacturing of this scaffold, which possesses a high degree of similarity to the starting model as monitored by morphological evaluations (mean diameter 569 +/- 147 microm), represents a promising result for regenerative medicine applications.

Increased CD34+ cell peripheral traffic appears to be an unique feature of the allergic inflammation.

Evidence has been cumulated during the last years concerning the immaturity of the cells involved in the local and systemic aspects of allergic inflammation. Hematopoietic precursors (HPC) are mobilized from the bone matrix as multipotent cells or, more often, as progenitors that, after the initial white-lineage commitment reach through the peripheral blood (PB) their final destinations constituted by the target organs of allergy. Although several studies have investigated the CD34+ cells traffic and location at the level of the inflamed peripheral mucosae in allergic populations, limited information is available on their behaviour on the time-course of infectious diseases. The current study thus was designed to asses the peripheral traffic of CD34+ HPC during the infectious inflammation. To this end CD34+ HPCs have been enumerated, by flow-cytometric techniques, in PB of 24 adult healthy beings (Group A), 24 adult subjects with symptomatic extrinsic allergy (Group B) and in PB of 24 adult patients hospitalised for febrile infectious pathology (Group C). CD34+ cell values ranged 0.01-0.08% with a median of 0.03 in Group A. In Group B values ranged 0.17-0.75% with a median of 0.28 and in Group C values ranged 0.00-0.12% with a median of 0.07. Variance analysis test among the three groups was statistically significant (p<0.001) supporting the conclusion that CD34+ HPC mobilizing and increased peripheral traffic is an unique feature of the allergic inflammation.

P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion.

We have recently reported that hepatic ischemia/reperfusion (I/R) is associated with a biphasic increase in the expression of P-selectin in the liver microvasculature, with peak expression levels observed at 20 min and 5 h after reperfusion. This I/R-induced upregulation of P-selectin expression is accompanied by leukocyte-endothelial cell adhesion in terminal hepatic venules (THV). The objective of this study was to determine whether the early expression of P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in THV after liver I/R. Left hepatic lobe ischemia was induced for 30 min in anesthetized C57B1/6 and P-selectin knockout (KO) mice. The number of rolling, saltating, and adherent leukocytes in THV was measured at 0, 15, 30, 60, and 120 min after reperfusion using intravital video microscopy. Hepatic I/R elicited significant increases in the number of rolling, saltating, and adherent leukocytes, with peak values observed at 30 min after reperfusion. All of these responses were absent in P-selectin KO mice and in C57B1/6 mice treated with a blocking antibody to P-selectin. Our findings suggest that P-selectin is the primary determinant of leukocyte-endothelial cell adhesion observed in hepatic venules in the initial period after I/R. Hence, this adhesion molecule may represent a target for therapeutic intervention in liver transplantation and other conditions associated with hepatic I/R.

Small-bowel tumors.

BACKGROUND: The rarity, delayed presentation, and diagnostic difficulty of small-bowel tumors prompted this study. STUDY DESIGN: Charts were reviewed retrospectively for 85 patients with 89 small-bowel tumors (22 primary malignant, 23 primary benign, and 44 metastatic) over a 10-year period (1986-1996) at Louisiana State University Medical Center-Shreveport and two affiliated hospitals in Shreveport. RESULTS: Of the primary malignant tumors, 10 carcinoids and 11 duodenal adenocarcinomas were identified. Most primary benign tumors were adenomatous or hyperplastic polyps, diagnosed by esophagogastroduodenoscopy. Metastatic tumors accounted for nearly 50% of all small-bowel tumors. Across all three tumor types, the most common presenting signs and symptoms were abdominal pain and nausea and vomiting. In addition, patients with benign tumors were more commonly presented with gastrointestinal hemorrhage, and those with metastatic tumors were more likely to present with obstruction. The mean interval from the onset of signs and symptoms to operation was 54 days for primary malignant tumors and 330 days for primary benign tumors. Esophagogastroduodenoscopy and computed tomography of the abdomen were occasionally helpful in diagnosis. Among the 22 primary malignant tumors, curative resections were performed in 11 patients (for 9 carcinoids and 2 adenocarcinomas) and palliative resections were performed in 10 patients (for 9 adenocarcinomas and 1 myxoliposarcoma). One patient had carcinomatosis from colon cancer and an incidentally discovered ileal carcinoid; this carcinoid was not included in this group of resections for primary malignant small-bowel tumors. All operations for 39 (of 44) patients with metastatic tumors were palliative. The remaining 5 (of 44) patients had metastatic duodenal cancer (confirmed by esophagogastroduodenoscopy or endoscopic retrograde cholangiopancreatography with biopsy) and did not undergo laparotomy. Surgical complications occurred more commonly with metastatic than with primary malignant tumors. Patients with primary malignant tumors had a 5-year survival rate of 36%. CONCLUSIONS: These findings demonstrate that small-bowel tumors are difficult to diagnose because of delayed presentation, nonspecific signs and symptoms, and lack of accurate diagnostic studies. If the overall survival of patients with small-bowel tumors is to be improved, clinicians must have a high index of suspicion and be willing to perform exploratory celiotomy early.

Colorectal cancer in the young patient.

Although predominantly a disease of older adults, colorectal cancer affects the younger population with an incidence of two to six per cent. It is thought to carry a less favorable prognosis in the young than in the general population. This may be due to advanced stage of the tumor at diagnosis. This study is composed of 37 patients, aged 40 and younger, treated over a 20-year period for colorectal cancer at Louisiana State University Medical Center-Shreveport and E. A. Conway Hospital. It was performed to investigate the incidence, stage at diagnosis, and prognosis of colorectal cancer in these young patients. The location of the primary tumor was fairly evenly distributed throughout the colon and rectum in this population. Pain, weight loss, rectal bleeding, and nausea and vomiting were the most common presenting symptoms. A family history of colon cancer or premalignant lesions were not risk factors in this study. Seventy per cent of all patients were treated with curative intent, and 42 per cent of these patients developed recurrent disease. The patients in this review presented with a higher incidence of advanced disease. Thirty-seven per cent of the lesions were Duke's C and 22 per cent were Duke's D, with poor 5-year survival (11% and 0%, respectively) when compared with national studies. The absolute 5-year survival for all young patients with colorectal cancer was 26 per cent (5 of 19 patients). It is important for the surgeon to be aware of the potential for colorectal cancer in young patients and to take an aggressive approach to the diagnosis and early treatment of the disease.

Diverticulitis in the young patient.

Although predominantly a disease in older adults diverticulitis does affect younger patients. The disease has been described as not only rare but virulent by some authors, and a young patient age is considered to be a relative indication for early sugery. The goal of this study was to evaluate the experience of the Louisiana State University Health Sciences Center-Shreveport and affiliated hospitals with diverticulitis in young patients. This study was a retrospective chart review of 22 patients with diverticulitis age 40 years and younger over the past 20 years. Inclusion criteria were either a diagnosis of diverticulitis confirmed at surgery or positive CT findings and/or a positive contrast enema. The mean age in this study was 32.1 years (range 16-40). All 22 patients presented with abdominal pain. The next most common symptom was nausea and/or vomiting in 45 per cent followed by fever and chills in 36 per cent. Twelve patients had abdominal CTs on admission, and 87 per cent had positive findings. Eighteen patients underwent an operation. Four patients were treated nonoperatively. Nineteen patients had diverticulitis of the sigmoid colon. The remaining three had right-sided diverticulitis. Two patients underwent right hemicolectomy, and one underwent cecectomy. Of the 15 patients with sigmoid diverticulitis 12 (80%) underwent a two-stage procedure of sigmoid colectomy, end colostomy, and Hartmann's pouch. Three patients (20%) underwent a one-stage procedure of sigmoid colectomy and primary anastomosis. Two of three patients undergoing a one-stage procedure required reoperation. Postoperative complications occurred in 10 of 18 patients for an overall incidence of 56 per cent. Two of these patients had septic complications. Both of these patients had a delay in time from admission until operation: one for 7 days and the other for 10 days. There was one death in the series. Colostomy closure was performed successfully in nine of 12 (75%) patients. The mean time interval before closure was 7.7 months, (range 3-14). Patients with two-stage procedures on initial admission fared better than those with one-stage procedures. The overall mortality was 4.5 per cent. There was a high overall complication rate of 56 per cent in patients undergoing an operation. Two patients who had a delay in time from admission to operation had septic complications. Early surgical intervention should be considered in this clinical setting. In summary, although rare, diverticulitis in the young patient is often a fulminant illness requiring operation early in the disease process.

T-cell receptor Vbeta repertoire in mite-allergic subjects after sublingual immunotherapy.

Sublingual immunotherapy has been recognized as an alternative to injected immunotherapy for the treatment of allergic diseases. Even if compelling clinical evidence supports such a view, few studies are available on its mechanisms of action. This study was carried out to investigate the peripheral lymphocyte Vbeta repertoire of subjects with mite-allergic respiratory allergy who were either not treated or treated for 2 years with mite-specific sublingual immunotherapy. The T-cell receptor Vbeta distribution was studied by flow-cytometric techniques in three subject groups. Group A (untreated) included 19 subjects with symptomatic, mite-allergic, low to moderate asthma and/or rhinitis. Group B (treated) was made up of 10 asymptomatic subjects treated for 2 years with mite-specific sublingual-swallow immunotherapy for low to moderate asthma and/or rhinitis. Group C (controls) included 10 healthy subjects. The Vbeta usage was investigated with monoclonal antibodies specific to the diverse beta segments V3, V5a, V5b, V5c, V6a, V8a, V8b and V12a. The comparison between the group A and group C repertoires showed a lower expression (p < 0.05) of the beta V8b+ T-cell subset. The group B repertoire, when compared with group A, showed a significantly greater usage of the beta V5a (p <0.05), 8a (p <0.05) and 12a (p <0.01) segments. The significantly lower expression of beta V8b observed in the symptomatic untreated group was not present in the group that was asymptomatic after treatment. The oligoclonal expansion observed in the treated group was consistent with the development of suppressor T-cell and/or of Th1 clones but not with deletion mechanisms of induced tolerance.

Effects of calmodulin and protein kinase C antagonists on muscle in the filariid, Acanthocheilonema viteae.

Drugs that act on calmodulin and protein kinase C (PKC) were investigated in the filariid Acanthocheilonema viteae. The filariid was slit open longitudinally and attached to an isotonic muscle transducer in a warmed (37 C) chamber containing physiologic solution bubbled with 95% N2-5% CO2. The calmodulin inhibitors, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), increased the spontaneous contractions of the parasite at low concentrations and induced a contraction followed by a flaccid paralysis at high concentrations. Trifluoperazine and W-7 also reduced the contractions from acetylcholine (ACh) and KCl in a concentration-dependent manner. The phorbol esters, phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate, which activate PKC, were either inactive or only weakly active at inducing contractions. Staurosporine (10(-6) M), a PKC inhibitor, enhanced and then blocked the spontaneous contractions of the filariid. Two other PKC inhibitors, H-7 (10(-4) M) and sphingosine (3 x 10(-5) M), induced much smaller increases in the spontaneous contractions and did not inhibit them. Staurosporine and sphingosine inhibited the ACh contractions; however, staurosporine only slightly reduced the maximal KCl contraction. These results support a role for calmodulin, but not for PKC, in filarial muscle contraction.

Probiotics reduce the CD34+ hemopoietic precursor cell increased traffic in allergic subjects.

An increased traffic of circulating CD34+ Hemopoietic Precursors Cells (HPC) is an important feature of systemic allergic inflammation. Bacteria and bacterial products are capable of stimulating the transcription of the maturational cytokines IL12 and IFNs through the activation of Toll-Like-Receptor and the subsequent nuclear translocation of the NF-kappaB factor. In this study the probiotics differentiation/maturational effect potential on CD34+ HPC has been investigated. Fourteen consecutive subjects, 9M and 5F, aged 6-48, with clinical symptoms of asthma and /or conjunctivitis, rhinitis, urticaria, atopic dermatitis, food allergy and irritable bowel syndrome were enrolled. Allergen-specific serum IgE were found in twelve patients. Flow-cytometric measurement of peripheral blood CD34dim/bright HPC values were assessed before and after 30 days of therapy, consisting in the oral administration of one sachet a day of ENDOLAC (UCB Pharma, Turin, Italy). Each sachet contained a mixture of Lactobacillus acidophilus, L. delbrueckii and Streptococcus thermophilus for a total of 1 x 10(9) live bacteria. Circulating CD34+ cell values significantly (p < 0.001) reduced after the treatment. ENDOLAC, thus, may improve the efficacy of the standard treatments of allergic diseases.

Quantitative expression of the major homing integrins alphaL and alpha4 on human cord blood hematopoietic progenitor and stem cells.

An increased traffic of hematopoietic progenitor cells (HPC) between bone marrow and peripheral organs is a peculiar feature of the allergic inflammation. It has been recently reported that the sublingual form of specific immunotherapy (SLIT) is capable of reducing such an increased HPC traffic. The House Dust Mite major antigen Der p1 has been proved to up-regulate the expression of the ICAM-1 and VCAM-1 endothelial addressins, supporting the view of an inflammatory cell recruiting at the site of allergen extract administration. In the present work we have investigated, by flow-cytometric techniques, the expression of the two major integrins CD11a (LFA-1) and CD49d (VLA-4) that are the homing receptor cognate for ICAM-1 and VCAM-1 on human cord blood CD34 hematopoietic progenitor and stem cells. Even if both the investigated molecules resulted detectable on CD34+ HPC surfaces, being the system redundant, the density of the cellular expression was significantly higher for CD49d (median value: 158) than CD11a (median value: 20.5), suggesting a preferential usage of the homing axis VLA-4/VCAM-1. Results consistency with outcomes of clinical trials that relate SLIT efficacy to allergen dosage is discussed.

Specific sublingual immunotherapy in atopic dermatitis. Results of a 6-year follow-up of 35 consecutive patients.

BACKGROUND: allergen-specific immunotherapy has proved to be effective in selected patients with IgE-mediated respiratory allergic diseases, and alternative routes of administration are being studied. Atopic Dermatitis (AD) is currently regarded as an allergic inflammatory disease. METHODS: we conducted a cohort study to evaluate the safety and effectiveness of sublingual-swallow immunotherapy (SLIT) in selected patients with allergic (extrinsic) AD. Thirty-five patients, 16 suffering from AD without respiratory allergic symptoms (Group A) and 19 with AD associated to mild asthma and/or rhinitis (Group B), were enrolled in the study. The severity of the skin lesions (eczema) was scored on a 0 to 4 scale (and subsequently related to the more recent SCORAD Index), where 0 indicated complete healing of the eczema and 4 indicated maximal spread of the lesions. Only patients with an eczema score of 1 to 3 were started on allergen-specific SLIT for 36 months. Eczema scores, symptoms and side effects were recorded every two months during the first 2 years and then after 36 months. After SLIT was completed, all patients attended 3 yearly follow-up visits to evaluate the long-term effects of the treatment. All patients followed a set of rules designed to control for identified confounding variables. All patients received ketotifen during the first 3 months of SLIT. RESULTS: only the complete disappearance of skin lesions (score 0) was considered to indicate effectiveness. In Group A this was observed in 12.6% of the patients after 6 months of SLIT, in 31,2% after 12 months and 68.8% after 24 months. In Group B, eczema disappeared in 0% after 6 months, in 36.8% after 12 months and 73.7% after 24 months. No patients in Group A developed asthma during SLIT, and 1 patient developed asthma 3 years after immunotherapy had ended. Three focal reactions consisting of 2 cases of mild eczema and one case of diarrhoea were recorded. One case of urticaria, due to violation of the administration schedule was the only systemic reaction observed. No life-threatening reactions appeared at any time of the study. CONCLUSIONS: the outcomes obtained, taken into account the limitations of the study design, suggest that sublingual allergen-specific immunotherapy for the treatment of the extrinsic form of Atopic Dermatitis is safe and well tolerated by patients, and may favourably affect the natural course of the disease.

Urease activity in gastrointestinal tract of rabbit and electrophoretic behaviour of urease.

Urease activity in the stomach (fundus and antrum), caecal content and soft faeces of rabbit was studied. Significant differences between fundus and antral content (P less than 0.01) and between caecum and soft faeces (P less than 0.05) were observed. The urease zymograms from caecal content and soft faeces of rabbit presented two different bands. The fundus content and the caecal ureolytic Clostridium innocuum bacterium exhibited only one band. Band B of caecal content was not evident at pH 4, whereas band A, also present in the stomach, was observed both at pH 4 and at pH 6. The optimal pH of urease activity of stomach and caecal content was in the range of 4-5 and 5-6, respectively. A comparison of intestinal urease zymograms with those of the single ureolytic bacterial species was suggested in order to clarify their role in urea metabolism.

CD34+ cells in peripheral blood of healthy human beings and allergic subjects: clue to acute and minimal persistent inflammation.

BACKGROUND: There is compelling evidence that hemopoietic precursor cells (HPC) play a crucial role in establishing cellular inflammation in allergic diseases. Increased levels of circulating CD34+ HPC committed to the myeloid lineage have been extensively reported in allergic rhinitis, asthma and eczema, whereas CD34+ cells have been identified within the cellular infiltrates of tissues, at peripheral sites of inflammation. METHOD: We conducted a pilot study to evaluate CD34+ traffic in the peripheral blood of 22 consecutive patients (13 men and nine women; mean age 28.9 years), independently of treatment. The patients presented rhinitis, asthma, eczema, urticaria and adverse food reactions of suspected allergic origin. Allergic reactions were extrinsic in 18 patients and intrinsic in four. In 12 patients who underwent sublingual specific immunotherapy, CD34+ cells were quantified at enrollment (T0), one year later (T1) and two years later (T2). The severity of symptoms was graded on a five-point scale (0 = absence of symptoms and 4 = severe symptoms). Twenty healthy human subjects (10 men and 10 women; mean age 24.5 years) were evaluated as controls. To obtain information about the total amount of circulating HPC, independently of the lineage commitment (Lin+/-) and the degree of differentiation (CD34bright/dim), we used a modification of the Milan protocol of peripheral blood CD34+ cell estimation. The cells were analyzed using a BD FACScan or FACSCalibur and the results were expressed as the percentage of positive cells. RESULTS: CD34+ cell traffic in the control group was very low since all values were < 0.10 (median value: 0.03 %). Values in the patient group were increased in both extrinsic and intrinsic forms with a median value of 0.25 % (interquartile range: 0.13- 0.33 %). The relationship between CD34+ traffic and the severity score was highly significant (Spearman's rho = 0.954; test of Ho: CD34; independent score: Pr > t = 0.000). CONCLUSIONS: The data reported herein suggest that the method employed is effective in assessing acute allergic inflammation, as well as minimal persistent inflammation underlying an asymptomatic clinical condition. Evaluation of CD34bright/dim peripheral traffic, if confirmed by the outcomes of a multicenter study currently being planned together with traditional study of circulating IgE, could be a reliable non-invasive laboratory tool for monitoring allergic inflammation.

Mite antigens enhance ICAM-1 and induce VCAM-1 expression on human umbilical vein endothelium.

Although sublingual allergen-specific immunotherapy has been proved to be effective in the treatment of allergic diseases, controversy surrounds the means by which such a local therapy can induce systemic immunological changes. Adhesion molecules are critical in the regulation of leukocyte traffic. It has been hypothesized that allergenic extract, administered locally, may induce an up-regulation of the mucosal vessel vascular adhesion molecules (CAMs) resulting in local recruitment of circulating inflammatory cells. In the present study we investigated whether the mite antigens, Der p1 and Der p2, can modulate CAM expression of human endothelial cells (HEC). To do this, slices of whole human umbilical cord vein underwent short-term (8 hours) cultures in the presence or absence of mite antigen (baseline, unstimulated controls). Cryostatic sections of the specimens were then evaluated immunohistochemically for expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) molecules. The results revealed that while Der p1 is capable of significantly up-regulating ICAM-1 and VCAM-1 on HEC, Der p2 antigen moderately up-regulates ICAM-1 expression but is ineffective in modulating VCAM-1. Although preliminary, these results clearly support the hypothesis that at least some of the effects of sublingual immunotherapy may derive from inflammatory cell recruitment at the site of allergen release.

IgE responses to Dermatophagoides pteronyssinus native major allergens Der p 1 and Der p 2 during long-term specific immunotherapy.

We investigated by ELISA the IgE response to whole extract of the house-dust mite Dermatophagoides pteronyssinus (Dp) and to the native major allergens, Der p 1 and Der p 2, in sera from 18 adult patients (group A) with Dp-allergic asthma before (t0) and 1, 2, 3, and 4 (t1-t4) years after subcutaneous specific immunotherapy (SIT). A qualitative reduction (P = 0.05) of the IgE responses to Dp and Der p 2 was observed from t1 to t4, but a highly statistical significant decrease appeared at t3 (P < 0.01). With regard to Der p 1 IgE values, the immunotherapy induced a significant decrease (P < 0.01) at t3, but not before. In group A, the IgE responses to Der p 1 and Der p 2 were not correlated at t0 (rs = 0.31; P = 0.21) but were correlated at t3 (rs = 0.78; P = 0.001). We also examined sera from 14 adult patients (group B, same SIT schedule as group A) who were without respiratory symptoms at the end of the third year (t3) of Dp SIT. At this time (t3), there were no significant differences in Der p 1 and Der p 2 IgE levels between group A and group B.

The role of ischemic preconditioning in the recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion.

BACKGROUND: We have recently shown that hepatic ischemia/reperfusion (I/R) results in rolling and adherence of leukocytes in terminal hepatic venules (THV) followed by hepatic enzyme elevation and tissue destruction. The objective of this study was to determine the effect of ischemic preconditioning on the recruitment of leukocytes in THV after liver I/R. METHODS: Left hepatic lobe ischemia was induced for 5 min (preconditioning) in anesthetized C57B1/6 mice followed by reperfusion for 10 min and then prolonged ischemia for 30 min. The number of rolling, saltating, and adherent leukocytes in THV was measured at 0.5, 2, 5, 12, and 24 h after reperfusion using intravital video microscopy. Matching sham groups were evaluated after 30 min of ischemia. RESULTS: Hepatic I/R elicited significant increases in the number of rolling, saltating, and adherent leukocytes, with peak values observed at 30 min and 5 h after reperfusion. All of these responses were significantly attenuated in mice undergoing ischemic preconditioning. Rolling leukocytes in THV following I/R without preconditioning reached peak levels of 25.2 +/- 1.4 leuk/2 min (leukocytes/2 min) at 30 min reperfusion and 31.4 +/- 1.5 leuk/2 min at 5 h reperfusion. With ischemic preconditioning these values fell to 12.3 +/- 0.9 leuk/2 min and 14.4 +/- 1.0 leuk/2 min, respectively (P < 0.001). Similarly, adherent leukocytes in nonpreconditioned mice reached peak values of 4.8 +/- 1.3 leuk/2 min at 30 min reperfusion and 8.3 +/- 1.2 leuk/2 min at 5 h reperfusion compared with 2.0 +/- 1.5 leuk/2 min and 1.6 +/- 1.1 leuk/2 min in preconditioned mice, respectively (P < 0.001). CONCLUSION: Ischemic preconditioning attenuates the initial events leading to leukocyte-mediated hepatic destruction following I/R injury. Delineating these mechanisms may play an important role in hepatic transplantation, resection, shock, and sepsis.