Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the intestinal villus to crypt ratios of Ross 708 broilers during early post-hatch development1,2,3.

Villus to crypt ratio (VCR) is used to quantify the microanatomical response of the intestine to various treatments. In early age chickens, comparative effects of the in ovo (i.o.) and s.c. methods of administration (moa) of the Marek's disease (MD) vaccine on 2 types of measurement of small intestinal VCR at 0 and 4 h post-hatch (poh) were investigated. The effects of moa and 4 and 18 h pre-placement holding times (pht) on the VCR measurements at 168 h (7 d) poh were also investigated. In the jejunum of the small intestine, a standard method for VCR determination, based on 10 villus and crypt length measurements, was utilized for the calculation of villus to crypt length ratio (VCLR). In that same region, a single histomorphometric determination of the crypt and total mucosa areas using image analysis software was also used. Subtraction of the crypt area from the total mucosa area provided the villus area, allowing for calculation of the villus to crypt area ratio (VCAR). Across 0, 4, and 18 h of poh bird age, the VCLR of birds that received an s.c. vaccination was higher in comparison to that of those that received an i.o. vaccination. The highest and lowest VCAR values were observed in the s.c. treatment at 0 h poh and in the i.o. treatment at 4 h poh, respectively. Furthermore, at 168 h poh, VCLR values in the 18 h pht and s.c. vaccination group were higher than those in the 4 h pht and s.c. vaccination or 18 h and i.o. vaccination groups. In conclusion, the effects of pht and MD vaccine moa on VCR were dependent on the use of either the VCLR or VCAR method of measurement. However, regardless of method, s.c. injection overall led to a higher VCR through 4 h poh in Ross 708 broilers, and the effects of moa on VCLR at 168 h were influenced by pht.

Comparison of two methods for determination of intestinal villus to crypt ratios and documentation of early age-associated ratio changes in broiler chickens,.

The determination of intestinal villus to crypt ratios (VCR) is a common method utilized to evaluate effects of various diet regimens on gut microanatomy and for the histologic quantification of intestinal responses to disease processes. Two methods for the determination of small intestinal VCR were compared in early age chickens. A standard method for VCR determination based on 10 villus and crypt length measurements in the jejunal region of the small intestine was employed for the calculation of villus to crypt length ratio (VCLR). That method was compared to a new approach based on a single histomorphometric determination of the crypt and total mucosal areas using image analysis software. Subtraction of the crypt area from the total area provided the villus area and allowed for the subsequent calculation of villus to crypt area ratio (VCAR). At 4 and 18 h posthatch, VCLR was higher than that of VCAR, but there was no significant difference between VCLR and VCAR at 0 h (hatch) and at 168 h (d 7) posthatch. Nevertheless, the pattern of age-associated changes for VCLR and VCAR were comparable throughout the early posthatch period. Furthermore, the new method used in determining VCAR is subject to less human error, allows for an appreciable reduction in the number of measurements required, and facilitates a larger intestinal segment evaluation. Standard linear measurements require the selection of variable numbers of villi and crypts, whereas the area method only requires selection of a single region that incorporates numerous villi and crypts of variable sizes in providing a less subjective approach. This is particularly advantageous in studies on intestinal disease conditions resulting in marked multifocal variation in villus stature. This study further documented age-associated changes occurring in the VCR of the small intestine during the early posthatch period. Across the 2 methods used for VCR determination, a major and highly significant reduction in the VCR was observed to occur between 18 h and 168 h posthatch.

A "whole-blood" technique for the quantitation of canine "T-lymphocyte" progenitors using a semisolid culture system.

A whole blood technique is described for the growth of concanavalin A (Con A) stimulated canine lymphocyte colonies in semisolid medium. By eliminating the routine Ficoll-Paque (F-P) gradient lymphocyte isolation, this method avoids potential problems of growth modulation due to elimination of non-lymphoid accessory cells and the influences on colony formation associated with the selective effects of F-P on lymphocyte subpopulations. Thus, the technique more closely approximates the in vivo milieu. The whole-blood method also produces higher cloning efficiencies than methods using gradient isolation of lymphocytes. Studies over a wide range of blood concentration produced a linear response of in vitro colony formation although extrapolation of the cell-dose colony-response curve did not intersect zero. Mitogen titration data indicates that a relatively large dose of Con A is required for whole blood colony formation compared to the standard F-P method. The colonies ultrastructurally were composed of lymphoblastic and lymphocytic elements which were negative for non-specific esterase activity. Characterization of cells retrieved from the colonies using rosetting techniques indicates a high percentage of the colony cells relative to canine peripheral blood cells form rosettes with human erythrocytes.

The selective growth of human T lymphocyte colonies from whole blood in a semisolid culture system.

Human T lymphocyte colonies may be selectively grown from whole blood in a single phase semisolid culture system following stimulation with PHA-P, Con-A, or PPD. This technique eliminates the requirement for gradient-enriched lymphocyte fractions, and provides a sensitive system for the study of T lymphocyte progenitors that more closely approximates the in vivo milieu. Whole blood colonies were composed of lymphoblasts and mature lymphocytes. Individual colony cells, identified as T lymphocytes, lacked lipase and specific esterase activity, formed E rosettes, did not phagocytize latex beads, and were largely ANAE positive. Whole blood was plated at a final concentration of 3%. Optimal mitogen/antigen concentrations were 125 microgram Con-A, 80 microgram PHA-P and 50 microgram PPD/ml culture media. Peak colony growth occurred between days 7 and 8. Colony formation increased as a power function over a wide range of cell concentrations (5 x 10(3)-5 x 10(4) lymphocytes plated). Maximal whole blood colony formation occurred when 5 x 10(4)-10(5) lymphocytes were plated. There was a significant increase in the cloning efficiency using whole blood as compared to gradient-separated cells. This method has wide application for the study of radiation effects, lymphocyte alterations in various disease states, antigen recognition, and the induction and amplification of T cell function.

The formation of human lymphocyte colonies in semisolid cultures in response to allogeneic mixed-lymphocyte stimulation.

A technique is described for the growth of human lymphocyte colonies in semisolid culture systems in response to allogeneic lymphocyte stimulation. Colonies did not form to any major extent using autologous lymphocyte stimulation. Both one-way and two-way mixed-lymphocyte reactions were investigated. Ultrastructurally, such colonies are composed of cells with lymphoblastic and lymphocytic morphology. The majority of the lymphoid elements composing the colonies were T-cells based on their ability to rosette with sheep red blood cells. Our studies suggest that the colonies are clonogenic in origin and therefore the technique offers the potential for isolation of specific clones, or subpopulations of lymphocytes involved in allogeneic reactions and characterization of their function. Studies directly comparing the stimulation indices achieved with standard mixed lymphocyte cultures utilizing 3HTdr-incorporation to the colony-forming assay indicate that the cloning technique produces higher stimulation indices for allogeneic/autologous reactions and produces less autologous (background) response than the 3HTdr incorporation technique. In addition to lymphocyte colonies, we also observed colonies of surface-adherent populations of macrophages, including multinucleated giant cells. Thus, the technique appears to provide a new and potentially more sensitive method for the study of transplantation immunology and cell-mediated immunity in humans.

Graft-versus-host disease in two immunocompetent dogs.

Graft-versus-host disease developed in two dogs injected with lymphocytes from BCG immunized donors. The disease was characterized by bone marrow depression, ulcerative enteritis, necrotizing cholangiohepatitis, thymic atrophy, pancreatitis, lymphadenopathy, inflammation of mucous membranes and weight loss. In one of the two dogs repopulation of bone marrow and lymphoid tissue by donor cells was demonstrated by cytogenetics. The development of GVHD was considered unusual because both animals received on immunosuppressive treatment and both responded well to PHA in lymphocyte transformation assays indicating they were immunocompetent. It was hypothesized that stimulation of donor lymphocytes by BCG enhanced their ability to induce a graft-versus-host reaction.

Production of mesenchymal tumors in nude mice by Ph1 negative fibroblasts obtained from a Ph 1 positive CML patient: a preliminary report.

An experimental model system is presented for the investigation in humans of the role of hematopoietic stromal elements in the regulation of hematopoiesis as well as in the pathogenesis of myelofibrosis in myeloproliferative disorders. The model is based on the simultaneous application of three experimental techniques: (1) growth of bone-marrow derived fibroblastic colonies in vitro, (2) cytogenetic demonstration of marker chromosomes associated with hematopoietic malignancies, and (3) the transplantation of isolated stromal elements into athymic (nude) mice. Using this model, we describe the induction of mesenchymal tumors in nude mice by Ph1 negative fibroblasts obtained from the bone marrow of a patient with a Ph1 positive chronic myelogenous leukemia. Mesenchymal tumors also were induced in nude mice with bone marrow-derived fibroblasts from a patient with aplastic anemia, who was successfully treated with bone marrow transplantation, and from a normal human volunteer. Morphologic, cytogenetic and electron microscopic studies of bone marrow mesenchymal elements in culture and of tumors induced in nude mice from the CML patient indicate the cells composing the tumor are of human origin and are negative for the Ph1 chromosome. The results provide the first in vivo morphological and cytogenetic support using human materials, of the hypothesized relationship of progenitors of in vitro fibroblastic colonies to marrow stromal elements.

Growth of canine T-lymphocyte colonies in vitro.

Canine lymphocytes from peripheral blood, lymph nodes, thymus and bone marrow were stimulated with phytohemagglutinin-P (PHA) or concanavalin-A (CON-A) to form colonies in methylcellulose. Lymphocytes exposed to mitogens in liquid phase formed clumps the size of colonies. Lymphocyte clumping was eliminated by plating cells directly into methylcellulose, but high concentrations of mitogens (CON-A or PHA is greater than 10 mg/10(6) lymphocytes) were required in order to get subsequent colony formation. Thus, in contrast to published reports, exposure of lymphocytes to mitogen prior to plating was not required for cloning of canine peripheral blood lymphocytes. Colonies from thymus, lymph node, or peripheral blood consisted predominantly of T lymphocytes, whereas cultures from bone marrow also produced colonies with macrophage morphology and surface-adherent colonies with mesenchymal morphology.

Stimulation of normal rat bone marrow fibroblast proliferation by sera from leukemic Fischer rats.

Fibroblast hyperplasia and accumulation of fibrous material in the bone marrow of patients with idiopathic (primary) or secondary myelofibrosis (MF) is believed to result from a reaction by marrow fibroblasts to an altered marrow microenvironment, the alteration being potentiated by abnormal hemic cells. We investigated the hypothesis that humoral factors might contribute to fibroblast overgrowth in MF by using an animal model, aged Fischer rats, where MF frequently occurs with leukemia. Sera from leukemic rats and leukemic cell conditioned media (CM) were assayed for enhancement of normal rat marrow fibroblast proliferation in a culture system where fibroblasts form discrete, adherent colonies. Our results demonstrated that: leukemic sera induced a 170% increase in total fibroblast colony numbers and a 325% increase in colonies containing more than 80 cells, stimulation of fibroblast growth was leukemia related since sera from rats with transplanted leukemia enhanced marrow fibroblast proliferation, leukemic cell CM did not contain a growth factor for marrow fibroblasts, sera from leukemic rats and 2-mercaptoethanol were additive in enhancing marrow fibroblast proliferation and probably act by different mechanisms, and leukemic rat sera was less effective as a colony-stimulating factor than normal rat sera, a condition mimicked when leukemic and normal spleen CM were compared. This is the first time that a serum component has been implicated in the pathogenesis of MF; our work may contribute to understanding the mechanism involved when MF occurs as a complication of leukemia.

Stem cell intervention ameliorates epigallocatechin-3-gallate/lipopolysaccharide-induced hepatotoxicity in mice.

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor ß1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Increased clonogenic (CFU-C, PFU-C) populations from bone marrow and spleen of nude mice.

Clonogenic populations from bone marrow and spleen of nude mice and their normal littermates were enumerated in vitro using a methylcellulose supported culture system. This technique allows for the simultaneous quantitation of progenitors of granulocyte-monocyte pathways (colony forming units in culture, CFU-C) and for progenitors of "mesenchymal" elements (plaque-forming units in culture or PFU-C). These populations were distinguished in culture by their growth, characteristics and morphology. CFU-C gave rise to suspended colonies of granulocyte-monocyte composition while PFU-C formed surface-adherent colonies of mesenchymal morphological features (fibroblastic and reticuloendothelial morphology). Significant elevations in the relative and absolute numbers of CFU-C and PFU-C were observed in the bone marrow and spleen of 6 wk old nu/nu mice relative to heterozygous littermates. The results are discussed in terms of non-T cells components involved in cell-medited immunity against neoplastic development.

Increased in vitro radioresistance of bone marrow fibroblastic progenitors (CFU-F) from patients with acute non-lymphocytic leukemia.

The proliferative potential following in vitro irradiation of bone marrow fibroblastic progenitors (CFU-F) derived from four patients with acute nonlymphocytic leukemia (ANLL) and seven nonleukemic subjects was compared. The CFU-F from the ANLL patients were significantly more radioresistant than the CFU-F from the nonleukemic subjects. The increased radioresistance in ANLL patients was evident in both the mean slope of the survival curve (control = -0.385, ANLL = -0.256) and in the Do values (control = 2.68 Gy, ANLL = 4.61 Gy). Thus CFU-F derived from ANLL patients differ from those derived from nonleukemics in both radioresistance and in granulopoietic effects as suggested from previous studies.

Effect of continuous, whole-body gamma irradiation upon canine lymphohematopoietic (CFU-GM, CFU-L) progenitors and a possible hematopoietic regulatory population.

Clonogenic assays for granulocytes-macrophages (CFU-GM) in bone marrow and for T lymphocytes (CFU-L) in peripheral blood were performed on dogs continuously exposed to 60Co irradiation (0.02, 0.04, or 0.11 Gy/day). When decreased numbers of CFU-GM were observed they correlated well with the clinical status of the dogs but were not generally associated with increasing cumulative doses of absorbed irradiation. In clinically normal, irradiated animals, decreased CFU-GM values and myeloid-erythroid ratios were observed, suggesting that chronic irradiation may affect the granulocytic series well before decreased peripheral blood values are seen. In hypocellular dogs the number of CFU-GM were significantly decreased compared to values obtained from control or clinically normal irradiated dogs, while virtually no CFU-GM were observed in the leukemic dogs. Only the CFU-GM values of the hypocellular group showed an association, e.g., a suggestion of an abortive regenerative effort, with increasing absorbed dose. Proliferative capacity of T lymphocytes (CFU-L) was not affected by either increasing absorbed irradiation or the presence of leukemia. D0 values were determined on marrow fibroblastic cells to ascertain whether a radioresistant subpopulation of stromal elements would result from continuous in vivo irradiation. No correlation was found between absorbed dose and increased D0 values. However, seven of eight dogs which developed acute nonlymphocytic leukemia displayed marrow fibroblastic cells with elevated D0 values. These radioresistant marrow fibroblastic cells were assayed for their ability to support normal granulopoiesis and found to be not significantly different from control fibroblasts.

The os acromiale: another cause of impingement.

Impingement of the shoulder is a relatively common clinical entity. The os acromiale anomaly is an uncommon one (1-8%) but can be an important cause of the impingement syndrome. The most common place of nonfusion is between the meso- and meta-acromion. The key to diagnosis is a history and physical examination compatible with the impingement syndrome and appropriate radiologic studies (i.e., an axillary view or profile view or computed tomographic scan if necessary). After diagnosis, the initial treatment is conservative with rest, ice, nonsteroidal anti-inflammatory drugs (NSAIDs), injections of corticosteroids in the subacromial space, and most importantly, an appropriate rehabilitation program. If unsuccessful, treatment should be planned based on the size of the unfused fragments. Small fragments (< 4 cm) may be removed by either arthroscopic or open means. Larger fragments may require an attempt at bone grafting and fixation since their removal may result in loss of strength of the deltoid.

The adolescent "swimmer's back".

Three patients with backache, aggravated by swimming the butterfly stroke, were subsequently diagnosed as having Scheuermann's kyphosis. These patients were treated with conventional methods. However, they were allowed additional time out of the brace to participate in swimming and were encouraged to do so, but they withheld from the butterfly. An average of 27% correction of curvature was seen with an average follow-up of 1.6 years. Symptoms subsided in all cases. We feel that the psychological and emotional well-being of the athlete can likewise be enhanced by continual participation without compromising the overall result.

Valgus extension overload in the pitching elbow.

Five baseball pitchers, three college and two professional, with an average age of 24 years, exhibited pain between the acceleration phase and follow-through phase of the pitching motion. This caused the players to be unable to continue at the level of competition necessary to play. A significant osteophyte on the posteromedial aspect of the olecranon process was identified in all pitchers. This caused impingement with the articular wall of the olecranon fossa and often created an area of chondromalacia. The more commonly identified posterior osteophyte was present in all cases. However, if just this posterior osteophyte is removed, the described lesion will be missed, with resultant persistent disability. Surgical excision of the posteromedial osteophyte through a relatively atraumatic posterolateral approach allowed early return of function without morbidity. With an average follow up of 1 year, all of the pitchers returned for one full season at maximum effectiveness.

Comparison of whole blood and purified canine lymphocytes in a lymphocyte-stimulation microassay.

The optimum mitogen concentration and time required for using whole blood from dogs in a microassay were determined, and this test then was compared with a standard lymphocyte-stimulation microtest, using gradient-isolated lymphocytes, 2 different mitogens (phytohemagglutinin and concanavalin A), and 2 different culture media. Statistical analysis of the data from 10 dogs showed that whole blood was significantly more reactive than were gradient-isolated lymphocytes (P less than 0.05). Waymouth's medium was significantly better than RPMI 1640 (P less than 0.001), and concanavalin A was significantly more mitogenic than phytohemagglutinin (P less than 0.001). The interaction between lymphocyte source and mitogens was the only one of the various interactions that was significant at P less than 0.05.

Waterskiing's Impact on Knees.

In brief Knee dislocation is a rare but brief potentially catastrophic water-skiing injury. Our patient sustained a fracture-dislocation of the knee and, because of vascular damage, required amputation of his lower leg. A subsequent survey of approximately 150 orthopedic surgeons revealed that, among their patients, the majority of serious knee injuries related to waterskiing resulted from noncontact falls into the water. Dislocation was the most serious consequence recorded, while damage to the tibial collateral ligament occurred most frequently.

Diagnosing Posterolateral Rotatory Knee Instability.

In brief A recent case of posterolateral rotatory knee instability in a recreational athlete provides an opportunity to review diagnosis and treatment of this often unrecognized condition. Two simple clinical tests, the external rotational recurvatum test and the posterolateral drawer test, can identify this injury to the arcuate ligament complex. Treatment consists of bracing and physical therapy and, in some cases, surgery.