Not only for caregivers: intranasal glucagon for severe hypoglycaemia in a simulation study.

AIMS: To evaluate: (i) the propensity of paediatrics and emergency medicine residents to select different therapeutic options and (ii) the speed and administration success in a high-fidelity simulation of severe hypoglycaemia in a child with type 1 diabetes (T1DM). METHODS: In this single-centre high-fidelity simulation study, 51 paediatrics or emergency medicine residents were exposed to a scenario of severe hypoglycaemia in a T1DM child attending an ambulatory setting, before and after a training on the preparation and administration of both injectable and IN glucagon. Time for drug delivery and its effectiveness were collected. RESULTS: Before training, 45.1% of participants chose to administer injectable glucagon, 43.1% intravenous glucose solution, 5.9% intranasal (IN) glucagon, and 5.9% took no action. Administration was successful in 74% of injectable glucagon, 33.3% intravenous glucose solution, and 22.7% IN glucagon. After training, 58.8% of participants chose IN and 41.2% injectable glucagon, with 100% of successful administrations for IN glucagon and 90.5% for injectable glucagon. Time to successful administration was shorter for IN than injectable glucagon (23 ± 10 vs. 38 ± 7 s, p < 0.0001). CONCLUSIONS: IN glucagon is an easy and effective option for severe hypoglycaemia treatment, with an almost zero possibility of failure provided that adequate training is imparted.

Empowering rare disease patients through patient education: the new BehçeTalk programme.

BACKGROUND: Educating patients and caregivers on their disease can improve their knowledge and promote the active involvement in the therapeutic decision-making process. Naturally, patient education programmes are critically important in rare systemic autoimmune diseases, where relevant knowledge and expertise still remain scattered. Behçet's disease (BD) represents a challenging rare condition, characterized by a variable spectrum of disease profile and a relapsing course. RESULTS: Recently, BehçeTalk, an educational programme tailored for BD patients, families and caregivers with, was launched. BehçeTalk, entirely co-designed with BD patients, is offering educational on-line webinars on different aspects of the disease, as well support groups for patients and caregivers coordinated by a psychologist with specific expertise in BD. CONCLUSIONS: The therapeutical management of BD is often challenging and frequently includes off-label treatments. Considering the specificities of BD, providing a specific education on the disease to patients will lead to empower them in being part of the decision-making processes, in the self-management and in improving their quality of life.

B Cell Synovitis and Clinical Phenotypes in Rheumatoid Arthritis: Relationship to Disease Stages and Drug Exposure.

OBJECTIVE: To define the relationship of synovial B cells to clinical phenotypes at different stages of disease evolution and drug exposure in rheumatoid arthritis (RA). METHODS: Synovial biopsy specimens and demographic and clinical data were collected from 2 RA cohorts (n = 329), one of patients with untreated early RA (n = 165) and one of patients with established RA with an inadequate response to tumor necrosis factor inhibitors (TNFi-IR; n = 164). Synovial tissue was subjected to hematoxylin and eosin and immunohistochemical staining and semiquantitative assessment for the degree of synovitis (on a scale of 0-9) and of CD20+ B cell infiltrate (on a scale of 0-4). B cell scores were validated by digital image analysis and B cell lineage-specific transcript analysis (RNA-Seq) in the early RA (n = 91) and TNFi-IR (n = 127) cohorts. Semiquantitative CD20 scores were used to classify patients as B cell rich (≥2) or B cell poor (<2). RESULTS: Semiquantitative B cell scores correlated with digital image analysis quantitative measurements and B cell lineage-specific transcripts. B cell-rich synovitis was present in 35% of patients in the early RA cohort and 47.7% of patients in the TNFi-IR cohort (P = 0.025). B cell-rich patients showed higher levels of disease activity and seropositivity for rheumatoid factor and anti-citrullinated protein antibody in early RA but not in established RA, while significantly higher histologic synovitis scores in B cell-rich patients were demonstrated in both cohorts. CONCLUSION: We describe a robust semiquantitative histologic B cell score that closely replicates the quantification of B cells by digital or molecular analyses. Our findings indicate an ongoing B cell-rich synovitis, which does not seem to be captured by standard clinimetric assessment, in a larger proportion of patients with established RA than early RA.

Histopathology of the synovial tissue: perspectives for biomarker development in chronic inflammatory arthritides.

The histopathological and molecular analysis of the synovial tissue has contributed to fundamental advances in our comprehension of arthritis pathogenesis and of the mechanisms of action of currently available treatments. On the other hand, its exploitation in clinical practice for diagnostic or prognostic purposes as well as for the prediction of treatment response to specific disease-modifying anti-rheumatic drugs is still limited. In this review, we present an overview of recent advances in the field of synovial tissue research with specific reference to the methods for synovial tissue collection, approaches to synovial tissue analysis and current perspectives for the exploitation of synovial tissue-derived biomarkers in chronic inflammatory arthritides.

Modulatory effects of alpha-lipoic acid (ALA) administration on insulin sensitivity in obese PCOS patients.

PURPOSE: To evaluate the efficacy of alpha-lipoic acid (ALA) administration on hormonal and metabolic parameters of obese PCOS patients. METHODS: A group of 32 obese PCOS patients were selected after informed consent. 20 patients referred to have first grade relatives with diabetes type I or II. Hormonal and metabolic parameters as well as OGTT were evaluated before and after 12 weeks of ALA integrative administration (400 mg per os every day). RESULTS: ALA administration significantly decreased insulin, glucose, BMI and HOMA index. Hyperinsulinemia and insulin response to OGTT decreased both as maximal response (Δmax) and as AUC. PCOS with diabetes relatives showed the decrease also of triglyceride and GOT. Interestingly in all PCOS no changes occurred on all hormonal parameters involved in reproduction such as LH, FSH, and androstenedione. CONCLUSIONS: ALA integrative administration at a low dosage as 400 mg daily improved the metabolic impairment of all PCOS patients especially in those PCOS with familiar diabetes who have a higher grade of risk of NAFLD and predisposition to diabetes.

Post-prandial effects of hazelnut-enriched high fat meal on LDL oxidative status, oxidative and inflammatory gene expression of healthy subjects: a randomized trial.

OBJECTIVE: Postprandial oxidative stress is characterized by an increased susceptibility of the organism towards oxidative damage after consumption of a meal rich in lipids and/or carbohydrates. Micronutrients modulate the immune system and exert a protective action by reducing low-density lipoproteins oxidation (ox-LDL) via induction of antioxidant enzymes. SUBJECTS AND METHODS: The clinical study was a randomized and cross-over trial, conducted through the CONSORT flowchart. We evaluated the gene expression of 103 genes related to oxidative stress (HOSp) and human inflammasome pathways (HIp), and ox-LDL level at fasting and after 40 g raw "Tonda Gentile delle Langhe" hazelnut consumption, in association with a McDonald's® Meal (McDM) in 22 healthy human volunteers. RESULTS: Ox-LDL levels significantly increased comparing no dietary treatment (NDT) vs. McDM, and decreased comparing McDM vs. McDM + H (p<0.05). Percentage of significant genes expressed after each dietary treatment were the follows: (A) NDT vs. McDM: 3.88% HIp and 17.48% HOSp; (B) NDT vs. McDM + H: 17.48% HIp and 23.30% HOSp; (C) McDM vs. McDM + H: 17.48% HIp and 33.98% HOSp. CONCLUSIONS: Hazelnut consumption reduced post prandial risk factors of atherosclerosis, such as ox-LDL, and the expression of inflammation and oxidative stress related genes. Chronic studies on larger population are necessary before definitive conclusions.

B cell autoimmunity and bone damage in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic immune-inflammatory disease associated with significant bone damage. Pathological bone remodeling in RA is primarily driven by persistent inflammation. Indeed, pro-inflammatory cytokines stimulate the differentiation of bone-resorbing osteoclasts and, in parallel, suppress osteoblast function, resulting in net loss of bone. Abating disease activity thus remains the major goal of any treatment strategy in patients with RA. Autoantibody-positive patients, however, often show a rapidly progressive destructive course of the disease, disproportionate to the level of inflammation. The epidemiological association between RA-specific autoantibodies, in particular anti-citrullinated protein autoantibodies, and poor structural outcomes has recently found mechanistic explanation in the multiple roles that B cells play in bone remodeling. In this review, we will summarize the substantial progress that has been made in deciphering how B cells and autoantibodies negatively impact on bone in the course of RA, through both inflammation-dependent and independent mechanisms.

[Psychic disorders in emergency room: a qualitative analysis of psychological intervention]. / Disturbi psichici in pronto soccorso: una analisi qualitativa dell'intervento psicologico.

OBJECTIVES: Our study aims to analyze typologies of psychological intervention that respond to spontaneous request of Emergency Room's users and care providers, and their distribution in relation to observed psychic disorders. MATERIALS AND METHODS: 364 Subjects (134 males and 230 females), mean age 41.55 (± 22.38) reaching Emergency room were involved in this study. Data from an observation form were related to patients' triage code, their provisional diagnosis, the request of psychiatric advice and emergency outcome. Non-parametric variables were analyzed by Chi Square method, while parametric ones by ANOVA method. RESULTS: Patients were the more frequent users of psychological intervention, while relatives used it in lesser proportion. Anxiety Disorder was the most frequent psychiatric diagnosis associated to psychological consulting. The patient's triage code was not significantly related to frequency of consulting. The type of intervention that was most often choosen has been supportive. As to outcome, the majority of patients who consulted psychologists was discharged, while a low percentage was admitted, particularly in psychiatric wards. CONCLUSIONS: Psychological consulting appears related to a wider and more varied range of urgent situations than psychiatric consulting. Therefore, psychological intervention seems to be useful both to relieve hic et nunc psychological discomfort, and to help and direct sicker patients to formulate a long-term treatment plan.

Comparative study of heart rate variability between healthy human subjects and healthy dogs, rabbits and calves.

Several studies have been performed to assess heart rate variability (HRV) in several species such as humans, dogs, pigs, calves, rabbits and rats. However, haemodynamic parameters are totally different in each animal, and optimal animal models for studying HRV corresponding to human HRV are still unclear. The purpose of this study was to assess HRV in human subjects and to compare those HRV data with canine, bovine and rabbit HRV data. The heart rate in the human subjects (62.8 +/- 7.4 bpm) was significantly lower than that in dogs (124.2 +/- 18.8 bpm, P < 0.001), calves (73.4 +/- 10.5 bpm, P < 0.05), and rabbits (217.3 +/- 21.5 bpm, P < 0.001). The low-frequency waves (LF) (57.9 +/- 65.8 ms(2)/Hz) and high-frequency waves (HF) (33.8 +/- 49.1 ms(2)/Hz) in rabbits were significantly lower than human LF (1216.3 +/- 1220.7 ms(2)/Hz, P < 0.05) and HF (570.9 +/- 581.3 ms(2)/Hz, P < 0.05). Dogs and calves showed similar LF (991.1 +/- 646.1 ms(2)/Hz and 547.0 +/- 256.9 ms(2)/Hz, respectively), HF (702.1 +/- 394.1 ms(2)/Hz and 601.0 +/- 666.6 ms(2)/Hz, respectively) and LF/HF (2.0 +/- 1.3 and 2.5 +/- 1.9, respectively) when compared with the human data. The present study shows that dogs and calves revealed similar HRV values as those which relate to humans. Large deviation of the HRV values in rabbits compared with humans might be considered when conducting animal studies using those animals to reflect human clinical situations.

Association of CXCL13 and CCL21 expression with the progressive organization of lymphoid-like structures in Sjögren's syndrome.

OBJECTIVE: Ectopic lymphoneogenesis can occur in the salivary glands of Sjögren's syndrome (SS) patients and is associated with local antigen-driven B cell responses, autoantibody formation, and potential lymphomatous transformation. CXCL13 and CCL21 have been identified in salivary glands, but their role in ectopic lymphoneogenesis in SS remains unclear. This study aimed to evaluate the microanatomic association between CXCL13 and CCL21 expression and the acquisition of lymphoid features in periductal foci. METHODS: Salivary glands from 37 SS patients and 9 chronic sialadenitis patients were analyzed by immunohistochemistry for T cell/B cell segregation, CD21+ follicular dendritic cell networks, and peripheral lymph node addressin (PNAd)-positive high endothelial venules (HEVs) in relationship to the size of the aggregates and the expression of CXCL13 and CCL21 within infiltrating cells, epithelium, and endothelium. RESULTS: Grade 1 aggregates (10-50 lymphocytes) demonstrated predominance of nonorganized CD3+ cells, while grade 2 (>50 lymphocytes) and grade 3 (>50 with germinal centers) showed a progressive increase in CD20+ B cells and T cell/B cell segregation. This higher degree of lymphoid organization was significantly related to an increased expression of CXCL13 within infiltrating cells and PNAd+ HEV-associated CCL21-producing cells. Conversely, no association between lymphoid organization and lymphoid chemokine expression by epithelial cells was observed. CONCLUSION: The acquisition of lymphoid features by inflammatory foci in SS is critically associated with the enlargement of the inflammatory foci and with the expression of CXCL13 and CCL21 within the infiltrate, but is not associated with their expression by epithelial cells. These data strongly support an active participation of CXCL13 and CCL21 in regulating the progressive organization and maintenance of periductal foci.

Role of chemokines and chemokine receptors in regulating specific leukocyte trafficking in the immune/inflammatory response.

Antigen recognition, lymphocyte priming and effector responses in inflamed tissues depend on a coordinated and sequential series of events that take place in different anatomical compartments. The integration of these processes is favoured by the dynamic capacity of leukocytes to recirculate between the bloodstream and specific organs and to navigate inside the tissues in a programmed fashion, regulated by a complex interaction of cell adhesion molecules and soluble chemoattractants, in particular chemokines. In this review we discuss the role of chemokines and chemokine receptors in regulating leukocyte trafficking in different anatomical sites in the context of distinct functional phases of the immune/inflammatory response.

Current concepts and new developments in the treatment of psoriatic arthritis.

OBJECTIVES: Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy characterized by the association of arthritis with psoriasis. Many agents have been proposed for the treatment of PsA, but their use is based more on clinical experience than on sound scientific evidence. METHODS: We reviewed MedLine up to November 2002, searching for 'psoriatic arthritis', 'drug therapy, 'controlled trials' and 'outcomes' and all possible acronyms for these terms. All relevant papers were then examined in detail. RESULTS: PsA is a condition that runs a variable clinical course. Mild forms can usually be controlled by non-steroidal anti-inflammatory drugs (NSAIDs). Intra-articular glucocorticoid injections are indicated in patients with persistent mono- or oligoarthritis. Patients with severe and progressive articular disease not responsive to NSAIDs should be treated with disease-modifying anti-rheumatic drugs (DMARDs) to prevent joint damage and disability. Currently, methotrexate and sulphasalazine are considered the DMARDs of choice, but the evidence for the use of methotrexate in PsA is still largely empirical, while the clinical benefit induced by sulphasalazine appears to be modest. Other DMARDs proposed for the treatment of PsA include cyclosporin, gold salts and, more recently, leflunomide. However, none of the DMARDs available to date are effective in the treatment of psoriatic pelvispondylitis; in addition, a number of patients with severe peripheral arthritis fail to respond to standard DMARDs. Recently, tumour necrosis factor alpha inhibitors have proved effective in many PsA patients with pelvispondylitis or recalcitrant peripheral synovitis. CONCLUSIONS: None of the current treatments for PsA is curative, but significant clinical amelioration can be achieved in the vast majority of patients. Identification and prompt treatment of patients with severe articular disease is crucial for the achievement of a satisfactory clinical response and the improvement of the long-term outcome.

[Role of cell migration in the pathogenesis of rheumatoid arthritis: in vivo studies in SCID mice transplanted with human synovial membrane]. / Ruolo della migrazione cellulare nella patogenesi dell'artrite reumatoide: studi in vivo in topi SCID trapiantati con membrana sinoviale umana.

OBJECTIVE: Adhesion mechanisms play a central role in the recruitment of leukocytes which characteristically infiltrate rheumatoid synovium. Therefore, we adapted an animal model, in which human rheumatoid synovium was transplanted into severe combined immunodeficient (SCID) mice, to study the effects of Tumor Necrosis Factor-alpha (TNF-alpha) in modulatine leukocyte migration and to investigate the chemotactic potential of Stromal Derived Factor-1 alpha (SDF-1 alpha). MATERIALS AND METHODS: Human synovium samples, obtained from patients undergoing joint replacement, were divided into two parts. One was analysed by immunohistology and the other was implanted subcutaneously into SCID mice under general anaesthesia. Four weeks post-transplantation, grafts were injected with optimal dose of SDF-1, TNF-alpha or saline (negative control). At the same time, animals were injected iv with fluorescently labelled cells. 48 hours later mice were sacrificed and grafts removed for cryo-hystology. The number of cells migrating to the grafts was determined by UV-microscopy and the results expressed as cells per high power field. RESULTS AND CONCLUSIONS: In these studies we provide the evidence that: 1) the animal model, in which human tissues are grafted into SCID mice, can be used to study cell migration under controlled experimental conditions; 2) direct intragraft injection of TNF-alpha increases lymphocytes migration and up-regulates the expression of human adhesion molecules (CAMs) and 3) SDF-1 alphainjected intragraft increases the migration of the pro-myelo-monocytic U937 cells to synovial transplants, even more efficiently than TNF-alpha, but without modifications of CAMs' expression.

[Role of chemokines in the pathogenesis of chronic synovitis during rheumatoid arthritis]. / Ruolo delle chemochine nella patogenesi della sinovite cronica in corso di artrite reumatoide.

Chemokines play a central role in the pathogenesis of rheumatoid arthritis (RA) synovitis which is characterised by new blood vessel formation, thickening of the lining layer and infiltration of immune cells. The inflammatory infiltrate is generated by a series of events which include the recruitment of leukocytes from the blood stream into the tissue, their local retention and activation to effector cells. All these processes are finely regulated by the interplay of different cell adhesion molecules (CAMs) and chemoattractant factors called chemokines (CK). CK are a superfamily of small proteins that play a crucial role in immune and inflammatory reactions. These chemoattractant cytokines share structural similarities including four conserved cysteine residues which form disulphide bonds in the tertiary structure of the proteins. CK mediate their effects by binding specific receptors (CK-R) characterised by a domain structure which spans the cell membrane seven times and signal through heterotrimeric GPT-binding proteins. Activation of the CK network results in an amplification of the inflammatory cascade and consequently in the progressive destruction of RA joints. The recognition of the central role of CK in inflammation has paved the way to the development of new agents capable of interfering with CK and CK-R. This review will focus in particular on the role of CK in regulating leukocyte trafficking in RA joints.

Stromal cell-derived factor 1 (CXCL12) induces monocyte migration into human synovium transplanted onto SCID Mice.

OBJECTIVE: The mechanisms by which monocyte/macrophage cells migrate to the joint involve a series of integrated adhesion and signaling events in which chemokines and their receptors are strongly implicated. This study was undertaken to investigate the hypothesis that stromal cell-derived factor 1 (SDF-1), a CXC chemokine (CXCL12), plays a critical role in monocyte/macrophage localization to synovium. METHODS: SDF-1 and CXC receptor 4 (CXCR4) expression in rheumatoid arthritis (RA) and osteoarthritis synovium and graft SDF-1, tumor necrosis factor alpha (TNF alpha), and human and murine vascular markers were examined by immunohistochemistry and double-immunofluorescence. The functional capacity of SDF-1 to modulate monocyte migration into joints was investigated by examining the localization of pro-myelomonocytic U937 cells into synovial tissue transplanted into SCID mice. SDF-1, TNF alpha, or saline was injected into graft sites and response determined by the number of fluorescently labeled U937 cells (injected intravenously) detected in grafts by ultraviolet microscopy. RESULTS: SDF-1 and CXCR4 were highly expressed in CD68+ cells in the RA synovium. SDF-1 induced U937 cell migration in vitro and in vivo in a dose-dependent manner and, in vivo, SDF-1 was more effective than TNF alpha. In contrast to TNF alpha, SDF-1 did not induce intracellular adhesion molecule 1 in transplant microvasculature. Furthermore, intragraft injection of SDF-1 did not up-regulate TNF alpha, or vice versa. CONCLUSION: This study demonstrates, for the first time, that SDF-1 is functional in vivo when injected into synovial grafts. In addition, SDF-1 is more potent than TNF alpha, and its mechanisms of action appear to be autonomous. Therefore, SDF-1 may be an important TNF-independent molecule involved in the migration to and retention of inflammatory effector cells in the joint.

[Precut sphincterotomy in impacted choledocholithiasis at the Vater's ampulla]. / Esfinterotomía de precorte en coledocolitiasis impactada en el ampula de vater.

BACKGROUND: Endoscopic sphincterotomy is an established treatment for common bile duct stones. Stone impaction at the ampulla makes deep cannulation and standard sphincterotomy more difficult. The use of precut papillotomy may facilitate stone extraction, although risks may be greater. AIM: To evaluate precut papillotomy in impacted common bile duct stone at the ampulla of Vater. PATIENTS AND METHODS: Between October 1990 and September 1995, 27 of 345 patients with common bile duct stones underwent needle knife precut papillotomy after conventional sphincterotomy failed due to impacted ampullary stone. This facilitated deep cannulation and subsequent standard sphincterotomy in 12 patients. RESULTS: Eleven patients had spontaneous expulsion of the stone when precut papillotomy was extended. Oedema or bleeding precluded stone extraction in 3 patients, and these stones were removed at a second endoscopic retrograde cholangiopancreatography (ERCP) session. Mild bleeding occurred in one patient and hemotransfusion was necessary. There was no perforation or pancreatitis following the procedure. CONCLUSIONS: Precut papillotomy is effective in the treatment of impacted common bile duct stone at the ampulla of Vater. It's a technique that should be performed only by an experienced endoscopist and does not increase the complications risk.

Time course of adaptation to a high-fat diet in obesity-resistant and obesity-prone rats.

The purpose of the present study was to characterize the time course of adaptation (i.e., circulating metabolites and hormones, fat pad mass, lipoprotein lipase) to a high-fat diet in obesity-prone (OP) and obesity-resistant (OR) male Wistar rats. Delineation of OP and OR was based on body weight gain (upper tertile for OP; lower tertile for OR) after 1 wk on a high-fat diet (60% of kcal from corn oil). Rats were killed after 1, 2, or 5 wk of the dietary period. Increased body weight and percent body fat in OP rats at 1 wk could not be accounted for by increased retroperitoneal or epididymal fat pad weight. Plasma nonesterified fatty acids and triglycerides, as well as blood concentrations of glucose, lactate, and glycerol, were similar throughout the study. Plasma insulin was significantly greater in OP vs. OR rats and low-fat diet (LFD; 20% of kcal from corn oil) controls at 5 wk only, and blood beta-hydroxybutyrate (mM) was significantly higher in OR compared with OP and LFD rats at 1, 2, and 5 wk. Lipoprotein lipase mRNA and activity were significantly greater in epididymal fat pad and significantly lower in gastrocnemius muscle of OP vs. OR rats at 1 wk. Results suggest that early (i.e., 1 wk) differences in body weight and fat weight between OP and OR rats are not due to fat deposition in retroperitoneal or epididymal fat depots, and tissue-specific changes in LPL (increase in epididymal fat pad and decrease in gastrocnemius muscle) that occur in OP compared with OR rats after 1 wk on a high-fat diet provide a metabolic environment favoring fat storage.

A one-step monoclonal antibody typing procedure that simplifies HLA class I and class II typing.

We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step microcytotoxicity test. The test is performed by adding 1 microliter of cells to 1 microliter of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.

Diversity of T-cell receptor alpha gene transcripts in the newborn and adult periphery.

Recent studies have demonstrated that the diversity of T-cell receptor alpha (Tcra) gene expression may be confined by a developmental program for gene rearrangement. To examine the effect of age on Tcra gene usage in peripheral tissues, a comparison of Tcr transcripts from newborn and adult mouse splenocytes was made. RNA was first isolated from the spleens of newborn (within five days from birth) and adult B10.BR mice. The polymerase chain reaction was then used to assess the presence of Tcra-V1, Tcra-V2, and Tcra-V3 gene sequences within the two RNA pools. The Tcra-V2 transcript was frequent in both newborn and adult populations and was therefore selected for sequencing analyses, by which V-gene family member and J gene usage could be delineated. Forty-one sequences were obtained, demonstrating Tcra-V2 gene family structure in the B10.BR mouse. Six family members were identified, of which four were new. Although there were differences in gene usage between newborn and adult animals, some junctional diversity added to the repertoire of both populations. A striking feature of V-J joining, as illustrated by this study, was the restriction of combinations based on the J gene location within the Tcra locus. The Tcra-V2 gene of dominant expression in the newborn (B10.BR.6) rearranged exclusively with the 30 most 5' Tcra-J genes. The Tcra-V2 gene of dominant expression at the adult stage (B10.BR.1) rearranged exclusively with the 21 most 3' Tcra-J genes in the locus. Thus, V-J combinatorial diversity was restricted in both newborn and adult mice, yielding a trend from 5'-3' Tcra-J gene usage with age. Inherent restrictions in V-J combinations should now be considered with regard to antigen responsiveness, particularly in the young animal. Qualitative restrictions in Tcr repertoire, compounding low T-cell numbers in peripheral tissues, may well contribute to functional voids and immunodeficiencies in early life.

Environmental and allele-specific influences on T cell receptor gene rearrangement: skewed alpha, delta, and gamma gene rearrangement patterns in chimeric mice.

This study was performed as a means to examine the effects of environment and stem cell origin on the molding of T cell receptor gene rearrangements during T cell development. Lethally irradiated adult mice were first reconstituted with (C57BL/6 x DBA/2)F1 stem cells, which derived either from a fetal liver or adult bone marrow source. The gene rearrangements in the irradiated thymi were then compared to those in normal fetal or adult thymi by hybridoma analysis. Results showed a general absence of gene rearrangements typical of the normal fetal thymus in both sets of chimeras. alpha and gamma gene rearrangements in chimeric mice matched normal adult patterns. However, chimeric thymocytes were unique in an unusually low frequency of rearrangements at the delta locus. The use of the bone marrow vs. fetal liver as stem cell sources in chimeric mice did not affect the patterns, indicating that environmental factors played a major role in the molding of gene rearrangement at multiple T cell receptor loci. Interestingly, the chromosomal sequence added an additional influence as homologous chromosomes showed allele-specific rearrangement patterns. We now question whether the unique patterns of rearrangement in irradiated adult thymi may substantially affect developing T cell populations in chimeric animals, particularly with regard to the gamma/delta T cell subset. Further analyses are warranted, both in experimental and clinical settings, regarding ultimate potentials for T cell diversity and function in transplantation recipients.