[Local administration of amphotericin B by VAC instillation: Therapeutic aid in the treatment of mucormycosis]. / Application locale d'amphotéricine B par VAC instillation : aide thérapeutique dans le traitement de la mucormycose.

Mucormycosis is a rare and serious fungal infection, occurring mainly in immunocompromised, diabetic, polytrauma or burn patients. Current standard treatments include iterative carcinological surgical trimming, systemic treatment with liposomal amphotericin B and second-line Posaconazole or Isavuconazole. We report the case of a 37-year-old female patient with no previous medical history who developed a disseminated mucormycosis, with an estimated 25 % loss of skin substance and major decay of the chest wall. In addition to standard treatment, local instillations of amphotericin B using the VAC Veraflow® system were performed. We believe that local instillations of amphotericin B by VAC could improve the functional prognosis of patients with skin involvement.

Induction of anti-tobacco mosaic virus antibodies in mice by rabbit antiidiotypic antibodies.

Conventional rabbit antiidiotypic antibodies were raised against a private rabbit anti-tobacco mosaic virus (TMV) idiotype. These rabbit antiidiotypic antibodies were covalently coupled to lipopolysaccharide and then injected into BALB/c mice. As compared with controls, these mice, which have never seen the antigen, synthesized anti-TMV antibodies that are strongly idiotypically cross-reactive with the starting rabbit idiotype. Monoclonal anti-TMV antibodies were prepared from these mice. Furthermore, xenogeneic or syngeneic antiidiotypic antibodies, raised against these monoclonal anti-TMV antibodies, recognized specifically the rabbit idiotype. Rabbit antiidiotypic antibodies alone can induce the same effects, but the concentration of anti-TMV antibodies is lower.

Immunoregulatory role of maternal idiotypes. Ontogeny of immune networks.

Specific idiotypie can be induced in randomly chosen rabbits by preimmunization with anti-idiotypic antibodies (Ab2). Rabbits that synthesize anti-anti-idiotypic antibodies (Ab3) when injected with antigen produce antibodies that display idiotypic specificities that are also found on the starting idiotype. When female rabbits actively producing Ab3 are crossed with naive males, a significant proportion of the offsprings (approximately 40%) produce antibodies that were idiotypically cross-reactive with the starting idiotype, as compared to 3% of the controls. This conclusion was obtained using 5 female rabbits and their 32 surviving offspring. Maternal idiotypes have therefore strong immunoregulatory properties and influence the emergence of the available idiotypic repertoire.

B lymphocytes regulate dendritic cell (DC) function in vivo: increased interleukin 12 production by DCs from B cell-deficient mice results in T helper cell type 1 deviation.

Increasing evidence indicates that dendritic cells (DCs) are the antigen-presenting cells of the primary immune response. However, several reports suggest that B lymphocytes could be required for optimal T cell sensitization. We compared the immune responses of wild-type and B cell-deficient (muMT) mice, induced by antigen emulsified in adjuvant or pulsed on splenic dendritic cells. Our data show that lymph node cells from both control and muMT animals were primed, but each released distinct cytokine profiles. Lymph node T cells from control animals secreted interferon (IFN)-gamma, interleukin (IL)-2, and IL-4, whereas those from muMT mice produced IFN-gamma and IL-2 but no IL-4. To test whether B cells may influence the T helper cell type 1 (Th1)/Th2 balance by affecting the function of DCs, we immunized mice by transferring antigen-pulsed DCs from wild-type or mutant mice. Injection of control DCs induced the secretion of IL-4, IFN-gamma, and IL-2, whereas administration of DCs from muMT animals failed to sensitize cells to produce IL-4. Analysis of IL-12 production revealed that DCs from muMT mice produce higher levels of IL-12p70 than do DCs from wild-type animals. These data suggest that B lymphocytes regulate the capacity of DCs to promote IL-4 secretion, possibly by downregulating their secretion of IL-12, thereby favoring the induction of a nonpolarized immune response.

Idiotypic analysis of potential and available repertoires in the arsonate system.

We have shown that, by suitable idiotypic manipulation, BALB/c mice can express the major cross-reactive idiotype (CRI) of A/J mice in response to azophenylarsonate (Ars). In order to know if the CRIA idiotype is present in the potential repertoire of BALB/c before any intentional selection, we used polyclonal activation in vitro and limiting dilution analysis. The readout was done with two monoclonal anti-CRIA antibodies that recognize distinct idiotopes on a CRIA+ A/J germline-encoded monoclonal antibody. We studied the frequency of CRIA+ lipopolysaccharide (LPS)-reactive cells in the spleens of nonimmune and immune A/J mice and in the spleens of naive and manipulated (i.e., producing CRIA+ antibodies) BALB/c mice. A/J and BALB/c naive individuals presented very high frequencies of Ars-specific B cells while the frequency of CRIA+ B cells was only a minor subset (0.5%) of the total Ars-specific subset in the two strains. When A/J mice were immunized with Ars-keyhole limpet hemocyanin, a clear preferential expansion of the CRIA+ minor subset of A/J mice was observed (100x). No such enhancement was observed in BALB/c mice similarly treated. Manipulated BALB/c mice presented a higher frequency of CRIA+ anti-Ars B cells than naive or antigen-immunized BALB/c individuals.

Complete amino acid sequence of heavy chain variable regions derived from two monoclonal anti-p-azophenylarsonate antibodies of BALB/c mice expressing the major cross-reactive idiotype of the A/J strain.

The primary structure of A/J anti-p-azophenylarsonate (anti-Ars) antibodies expressing the major A-strain cross-reactive idiotype (CRIA) has provided important insights into issues of antibody diversity and the molecular basis of idiotypy in this important model system. Until recently, this idiotype was thought to be rarely, if ever, expressed in BALB/c mice. Indeed, it has been reported that BALB/c mice lack the heavy chain variable segment (VH) gene that is utilized by the entire family of anti-Ars antibodies expressing the A/J CRI. Recently, however, it has been possible to elicit CRIA+, Ars binding antibodies in the BALB/c strain by immunizing first with anti-CRI and then with antigen. Such BALB/c, CRIA+ anti-Ars antibodies can be induced occasionally with antigen alone. VH region amino acid sequences are described for two CRIA+ hybridoma products derived from BALB/c mice. While remarkably similar to each other, their VH segments (1-98) differ from the VH segments of A/J CRIA+, anti-Ars antibodies in over 40 positions. Rather than the usual JH2 gene segment used by most A/J CRIA+ anti-Ars antibodies, one BALB/c CRIA+ hybridoma utilizes a JH1 gene segment, while the other uses a JH4. However, the D segments of both of the BALB/c antibodies are remarkably homologous to the D segments of several A/J CRIA+ antibodies sequenced previously, as are the amino terminal amino acid sequences of their light chains. These data imply that BALB/c mice express the A/J CRIA by producing antibodies with very similar, if not identical, light chain and heavy chain D segments, but in the context of different VH and JH gene segments than their A/J counterparts. The results document that molecules that share serologic specificities can have vastly different primary structures.

Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo.

Dendritic cells (DC) are described as "nature's adjuvant," since they have the capacity to sensitize T cells in vivo upon first encounter with the antigen. The potent accessory properties of DC appear to develop sequentially. In particular, the ability to process antigens and to sensitize native T cells develops in sequence, a process termed "maturation" that is well described in vitro. Here, we obtain evidence for maturation in vivo in response to the bacterial product lipopolysaccharide (LPS). Before LPS treatment, many DC are found at the margin between the red and white pulp. These cells lack the M342 and DEC-205 markers, but process soluble proteins effectively. 6 h after LPS, DC with the M342 and DEC-205 markers are found in increased numbers in the T cell areas. These cells have a reduced capacity to process proteins, but show increases in the B7 costimulator and T cell stimulatory capacity. 48 h after LPS, the number of DC in the spleen is reduced markedly. We interpret these findings to mean that LPS can cause DC in the marginal zone to mature and to migrate into and then out of the T cell areas.

Antigen-pulsed dendritic cells can efficiently induce an antibody response in vivo.

The aim of this study was to develop an immunization procedure avoiding external adjuvant. Data are presented showing that syngeneic dendritic cells (DC), which have been pulsed in vitro with antigen, induce a strong antibody response in mice. By contrast, antigen (Ag)-pulsed low-density B cells, although equally able to induce interleukin 2 secretion by an Ag-specific T cell hybridoma in vitro, only weakly prime the mice in vivo. Moreover, we show that the injection of Ag-pulsed DC induces the synthesis of isotypes similar to the immunoglobulin classes detected after immunization with the same Ag in complete Freund's adjuvant. Importantly, high amounts of IgG2a antibodies are produced, suggesting that T helper type 1 cells are activated. Collectively, these data indicate that DC can initiate a primary humoral response and that they may be used as physiological adjuvant in vivo.

The injection of deaggregated gamma globulins in adult mice induces antigen-specific unresponsiveness of T helper type 1 but not type 2 lymphocytes.

Injection of adult mice with high doses of monomeric human gamma globulins (dHGG) has been previously shown to produce a state of peripheral tolerance in both B and T cells. To gain insight into the mechanism of induction and maintenance of adult tolerance in this model, we have analyzed the pattern of lymphokines produced by control and tolerant animals in response to the tolerogen. The data presented indicate that HGG-specific, interleukin 2 (IL-2)- and interferon gamma (IFN-gamma)-producing T cells (thus referred to as T helper type 1 [Th1] cells) are rendered unresponsive after in vivo administration of soluble HGG. In contrast, antigenic stimulation of T cells isolated from tolerant adult mice leads to increased production of IL-4 in vitro. In vivo challenge of dHGG-treated adult animals with hapten-coupled HGG (p-azophenylarsonate [ARS]-HGG) induced a significant ARS-specific antibody response, suggesting that tolerance induction in this model does not completely abrogate tolerogen-specific Th activity in vivo. In agreement with the in vitro data, hapten-specific antibody response of tolerant animals is characterized by a selective deficiency in the IFN-gamma-dependent IgG2a subclass. Injection of immunogenic forms of HGG into tolerant animals also produced an IL-4-dependent increase in total serum IgE levels, indicative of an increased activity of HGG-specific Th2 cells in these animals. The finding that tolerance induction differentially affects Th subpopulations suggests that crossregulation among lymphocyte subsets may play a role in the induction and/or maintenance of acquired tolerance in adults.

Glucocorticoids attenuate T cell receptor signaling.

Glucocorticoids (GCs) affect peripheral immune responses by inhibiting T cell immunity at several stages of the activation cascade, causing impaired cytokine production and effector function. The recent demonstration that the thymic epithelium and possibly thymocytes themselves produce steroids suggests that endogenous GCs also play a role in the control of T cell development. As both peripheral responsiveness and thymic differentiation appear to be regulated by the quantity and quality of intracellular signals issued by antigen-major histocompatibility complex-engaged T cell receptor (TCR) complexes, we investigated the effects of GCs on the signaling properties of T cells stimulated by anti-CD3 monoclonal antibodies or agonist peptides. We demonstrate in this work that dexamethasone, a synthetic GC, inhibits the early signaling events initiated upon TCR ligation, such as tyrosine phosphorylation of several TCR-associated substrates including the zeta chain, the ZAP70 kinase, and the transmembrane adapter molecule linker for activation of T cells. Hypophosphorylation was not a consequence of reduced kinase activity of src protein tyrosine kinases, but was correlated with an altered- membrane compartmentalization of these molecules. These observations indicate that in addition to their well-described ability to interfere with the transcription of molecules involved in peripheral responses, GCs inhibit T cell activation by affecting the early phosphorylating events induced after TCR ligation.

Idiotypic regulation of the immune system. Common idiotypic specificities between idiotypes and antibodies raised against anti-idiotypic antibodies in rabbits.

Anti-idiotypic antibodies (Ab2) were raised in allotype-matched rabbits against anti-carbohydrate or anti-tobacco mosaic virus antibodies (Ab1). Several Ab2 were purified and injected into a third series of rabbits III which synthesized antiantiidiotypic antibodies (Ab3). Antigen was then given for the first time in those rabbits who had synthesized Ab3. The specific antibody synthesized in rabbits III was called Ab1'. Anti-idiotypic antibodies were raised against purified Ab3 antibodies (Ab4). In most cases, Ab1' antibodies are sharing idiotypic specificities with Ab1. Ab3 did not react with antigen but shared idiotopes with Ab1 and Ab1' because Ab4 antibodies, which are anti-idiotypes to Ab3 do recognize specifically Ab1 and Ab1' antibodies belonging to the same chain of immunization. It seems therefore that Ab3 looks idiotypically like Ab1 and Ab4 displays the same behaviour as Ab2. A general view of the functioning of the immune system is presented.

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo.

Cells of the dendritic family display some unique properties that confer to them the capacity to sensitize naive T cells in vitro and in vivo. In the mouse, two subclasses of dendritic cells (DCs) have been described that differ by their CD8alpha expression and their localization in lymphoid organs. The physiologic function of both cell populations remains obscure. Studies conducted in vitro have suggested that CD8alpha+ DCs could play a role in the regulation of immune responses, whereas conventional CD8alpha- DCs would be more stimulatory. We report here that both subclasses of DCs efficiently prime antigen-specific T cells in vivo, and direct the development of distinct T helper (Th) populations. Antigen-pulsed CD8alpha+ and CD8alpha- DCs are separated after overnight culture in recombinant granulocyte/macrophage colony-stimulating factor and injected into the footpads of syngeneic mice. Administration of CD8alpha- DCs induces a Th2-type response, whereas injection of CD8alpha+ DCs leads to Th1 differentiation. We further show that interleukin 12 plays a critical role in Th1 development by CD8alpha+ DCs. These findings suggest that the nature of the DC that presents the antigen to naive T cells may dictate the class selection of the adaptative immune response.

Evidence for a circadian rhythm of insulin sensitivity in patients with NIDDM caused by cyclic changes in hepatic glucose production.

Diurnal variation in insulin sensitivity in patients with NIDDM has long been suspected but has been difficult to document mainly because of the interdependence of changes in glucose and insulin. Stable serum insulin levels during hyperglycemic clamping in patients with NIDDM in the present study provided the opportunity to examine changes in insulin sensitivity unaffected by changes in blood glucose and insulin concentrations. Six patients with NIDDM (four men and two women, BMI 33.9 +/- 2.5) underwent hyperglycemic (11.1 mmol/l, approximately 200 mg/dl) clamping for 72 h. Measured were serum insulin, free fatty acid (FFA), cortisol, and growth hormone concentrations and rates of insulin secretion, insulin clearance, and glucose infusion rate (GIR) needed to maintain hyperglycemia. In addition, five patients (three men and two women, BMI 32.6 +/- 0.6) underwent hyperglycemic clamping for 24 h with hourly determinations of hepatic glucose production (HGP) and glucose disappearance rates (GRd). GIR, reflecting insulin sensitivity, changed rhythmically with a cycle duration of 22.9 +/- 1.4 h and an amplitude of 47.8 +/- 11.2%. GIR was lowest at 8:31 a.m. (+/- 52 min) and highest at 7:04 p.m. (+/- 58 min). Circadian changes in GIR were completely accounted for by changes in HGP, while GRd remained unchanged. Plasma levels of FFAs and cortisol also exhibited circadian fluctuations, and their blood levels correlated negatively with GIR (r = -0.72 and -0.64, respectively). We concluded that insulin sensitivity in patients with NIDDM changed with circadian (approximately 24 h) rhythmicity (decreasing during the night and increasing during the day). These changes were unrelated to blood levels of glucose and insulin, insulin clearance, exercise, food intake, and sleep. They were caused by circadian changes in HGP, which in turn were closely correlated with circadian changes in blood FFA and cortisol levels. We believe that recognition of these circadian changes has implications for the diagnosis and the treatment of patients with NIDDM.

Role of CD8alpha+ and CD8alpha- dendritic cells in the induction of primary immune responses in vivo.

Data from adoptive transfer of mature dendritic cells (DC) indicate that they are responsible for the induction of primary immunity. Two subclasses of DC have been recently identified in spleen that differ in their phenotype and in certain regulatory features. In vitro, both subsets have the capacity to activate naive T cells, although CD8a+ DC have been shown to induce T cell apoptosis and to stimulate lower levels of cytokines compared with CD8alpha- DC. The objective of this study was to analyze the function of these distinct DC types in vivo. Our results show that both subsets, pulsed extracorporeally with antigen and injected in the footpads of syngeneic mice, sensitize an antigen-specific T cell primary response. However, CD8alpha+ cells trigger the development of Thl-type cells, whereas CD8alpha- DC induce a Th2-type response. These observations suggest that the Th1/Th2 balance in vivo is regulated by the antigen-presenting-cells of the primary immune responses.

An additional hypervariable region encoded by V gene segments occurs in Tcr V beta at a location compatible with its involvement in Tcr active site--a general model for alloreactivity.

The number of V alpha and V beta sequences of T cell receptors now available allows a meaningful analysis of their variability profiles. Variability plots were derived using a modified form of Wu and Kabat's algorithm: variability is not computed as a proportion of the number of different residues occurring at a position, but rather proportionally to the physicochemical differences between the different residues. Results show that the classical hypervariable regions occurring in immunoglobulins also occur in T cell receptors at equivalent positions. Contrary to immunoglobulins the framework of Tcr V regions displays many relatively variable regions and positions. This phenomenon can be connected with the genetic organization of V genes of T cell receptors which seem to avoid any framework homogenization and the resulting gene conversion. More importantly an additional hypervariable region was detected in V beta but not in V alpha. This fourth hypervariable region is located between the second and the D hypervariable CDR. The predicted three-dimensional location of this additional hypervariable region is compatible with a possible role in antigen recognition and therefore also in positive and/or negative selection. Furthermore our data suggest that this fourth hypervariable region is involved in the recognition of superantigens like bacterial enterotoxins. Indeed this additional hypervariable region is not detected when variability is derived using an alignment of the V beta subgroups stimulated by one toxin of S. aureus. Finally we propose a new and simple molecular model to explain alloreactivity as crossreactivity between the universe of shapes (isomers of conformation) of different MHC haplotypes.

In vivo induction of A/J anti-ARS responses with different ranges of affinities: correlation between affinity and CRIA idiotype dominance.

The anti-ARS immune response of A/J mice is characterized by the reproducible and dominant selection of CRIA bearing antibodies. In this report, we have investigated the role of affinity for the antigen in the selection of antibody repertoire during an immune response. A/J anti-ARS responses with different ranges of affinities for arsonate were elicited by the injection of differently arsanylated carrier proteins. The selection of higher affinity A/J anti-ARS responses was shown to be associated with the induction of higher levels of CRIA bearing anti-ARS antibodies. A detailed idiotopic analysis also showed a more precocious selection of the CRIA "canonical combination" in the higher affinity anti-ARS responses. These results strongly suggest an important role for affinity and clonal selection in the dominant expression of the CRIA idiotype in the A/J anti-ARS response.

Idiotypic manipulations: hierarchy in idiotype expression in rabbits immunized against Micrococcus luteus.

Idiotypic cross-reactions were analyzed among three series of anti-peptidoglycan antibodies of the Micrococcus luteus system. The reference idiotype Ab1 was an antibody fraction isolated from an isoelectric focusing preparative column. Cross-reactive idiotypes, Ab1', were induced through the immunization chain (Ab1-Ab2-Ab3). Idiotypic antibodies of Ab1-F1 type were obtained from offspring of female rabbits, actively producing Ab3 during pregnancy. Finally, Ab1 CRI were cross-reactive idiotypes with Ab1 found in a random population of rabbits immunized with M. luteus. Three idiotopes could be characterized within Ab1 antibody. Ab1' usually expressed two of these idiotopes, but never the third specificity which is "private" to Ab1. Ab1-F1 shared one or two idiotopes with Ab1 and Ab1' antibodies. Only one common idiotope appeared to be present on Ab1 CRI. Finally, this idiotope, IdX, could be detected by radioimmunoassay in 20% of rabbits immunized with micrococcal vaccine. It appears that a recurrent idiotype of anti-peptidoglycan antibodies can be preferentially amplified through idiotypic manipulations. On the other side, cascade immunizations lead to the expression on Ab1' and on some Ab1-F1 of a second idiotypic specificity, shared with Ab1. This hierarchy of idiotype expression may well be important in the regulation of antibody synthesis through idiotypes.

Idiotypic studies of monoclonal anti-tobacco mosaic virus antibodies from one mouse.

Monoclonal antibodies have been prepared from one BALB/c mouse immunized with tobacco mosaic virus. The monoclonal antibodies are distributed into three subgroups recognizing different epitopes on tobacco mosaic virus subunits. The idiotypic specificities of these monoclonal antibodies have been studied using syngeneic antiidiotypic sera. A sharing of idiotypic specificities has been observed between members of each subset. These idiotypes are not recurrent in BALB/c mice immunized with tobacco mosaic virus.

The influence of V kappa gene polymorphism on the induction of silent idiotypes in the arsonate system.

It has been previously shown that it is possible to modify the expressed repertoire of a given individual using idiotypic manipulation. For example, A/J mice respond to arsonate challenge by synthesizing a dominant idiotype, CRIA, whereas BALB/c mice do not. However, after treatment with rabbit polyclonal anti-CRIA antibodies (Ab2 or anti-idiotypic antibodies) and arsonate, BALB/c mice are able to synthesize a CRIA-like idiotype. To determine whether this modification of repertoire is dependent on the immunoglobulin loci (Ig-h, kappa), we have analyzed the anti-arsonate response after anti-idiotypic treatment of three strains of mice (C58, C.C58, AKR), chosen because they are among a small group of strains which express Kappa V regions not seen in other strains. There are also L chains lacking in these strains which are expressed in other mice. The C58 and C.C58 strains share the same Ig-h locus (Ig-ha) with BALB/c mice but C.C58 are congenic mice, that express the kappa loci on a BALB/c genetic background. AKR mice express the Ig-hd haplotype. AKR, C58 and C.C58 do not produce CRIA positive antibodies in response to arsonate; a defect which has been previously mapped to the kappa locus. These three strains of mice (C58, C.C58 and AKR) were treated with rabbit anti-CRIA and boosted with Ars-KLH. The results show that after such treatment, the C.C58 mice were able to express CRIA-like antibodies which are serologically identical to those of BALB/c.